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    • 专利摘要:
    Composition for the treatment of diseases associated with lysosomal disorders. Pharmaceutical composition comprising a pharmacological chaperone (cf) and coenzyme q10 (coq) and its use for the treatment of a disease caused by mutations in the gba1 gene, particularly for the treatment of gaucher's disease . (Machine-translation by Google Translate, not legally binding)
    公开号:ES2597728A1
    申请号:ES201530475
    申请日:2015-04-10
    公开日:2017-01-20
    发明作者:José Manuel García Fernández；Carmen Ortiz Mellet；José Antonio SÁNCHEZ ALCÁZAR；Mario DE LA MATA FERNÁNDEZ
    申请人:Consejo Superior de Investigaciones Cientificas CSIC；Universidad de Sevilla；Universidad Pablo de Olavide；
    IPC主号:
    专利说明:

    The present invention relates to a pharmaceutical composition comprising a pharmacological chaperone (CF) and coenzyme 0 10 (CoO). Likewise, the present invention relates to the method of preparing said composition and its use for the treatment of a lysosomal storage disease associated with the
    10 mutation of lysosomal acid ~ -glucocerebrosidase (GBA1 gene), particularly for the treatment of Gaucher disease, and more particularly for the treatment of Gaucher disease with neuropathic phenotype (Gaucher disease type II or type 111).
    15 STATE OF THE TECHNIQUE
    Smooth storage disorders describe a heterogeneous group of rare inherited diseases with loss of function of lysosomal enzymes. As a result, an abnormal metabolism of several substrates is generated that is not
    20 gradually degrade and accumulate in lysosomes, affecting their function and that of other organelles such as mitochondria, autophagic imbalance and inflammation, and which results in phenotypes that include visceromegaly, neurological pathologies, skeletal injuries and premature death.
    25 Currently, some of these pathologies only have symptomatic therapies that follow two therapeutic strategies: substrate reduction therapy (TRS), which inhibits the enzymes involved in the production of the substrate that accumulates, and enzyme replacement therapy (TER) , which exogenously administers the active recombinant enzyme that is found to be defective.
    Gaucher's disease is the most predominant lysosomal storage disorder. It is caused by mutations in the GBA1 gene that result in a defective enzyme ~ -glucocerebrosidase or insufficient activity. Many of these mutations lead to significant defects in the folding of protein 35 during translation in the endoplasmic reticulum (ER), resulting in a reduction in the transport of the enzyme to the lysosome (degradation mediated by the
    cellular quality control machinery). The decrease in its catalytic activity causes the accumulation of glucosylceramide and glucosyl sphingolipids in macrophage lysosomes and visceral organs. Gaucher's disease is subdivided into 3 types based on age that begins to manifest the disease and the involvement of the central nervous system (CNS). Gaucher patients without CNS manifestations are classified as type 1, more common; while those patients with neurological manifestations are classified in types 11 and III (Grabowski, G.A., Gaucher disease: gene frequencies and genotype / phenotype correlations. Genet Test,
    1997. 1 (1): p. 5-12)
    10 For lysosomal disorder of Gaucher's disease, TER is a high economic cost and is not very effective for cases that show involvement of the central nervous system 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.
    The TRS drugs have greater potential to penetrate the CNS and produce benefits at the neuronal level. This is the case of N-butyl-1-deoxynojirimycin (NBDNJ, Miglustat, Zavesca®), which acts as a weak glucosylceramide inhibitor
    20 synthase, reducing the biosynthesis of glucosylceramide. Miglustat has been approved only for use in Gaucher type 1 patients, of mild to moderate severity, but not for patients of type 11 and 111. In addition, many patients treated with Miglustat have experienced side effects that include diarrhea, loss of weight and tremors.
    25 Some inhibitors of glycosidases enzymes involved in lysosomal storage diseases are able to bind to the active site and stabilize the appropriate folding, being able to act as "pharmacological chaperones» (CF) that facilitate the transport of the catalytically active form to lysosomes. Of this
    Thus, the development of compounds with pharmacological chaperone activity has been postulated as a possible therapeutic strategy for the treatment of lysosomal storage diseases, of particular interest for those clinical manifestations of the disease that involve the central nervous system. For this, it is essential that increases in the activity of the enzyme are achieved
    35 mutated, at the origin of the disease, sufficiently high, which can hardly be achieved with the use of pharmacological chaperones alone.
    One of the most prevalent mutations in Gaucher's disease is the L444P variant, which results in an incorrect folding in the ER and failures in its transport to the lysosome. Homozygous patients for the L444P mutation have severe neurological forms of the disease. The mutation is a 1448T> C 5 base change in the GBA1 gene that results in the substitution of the amino acid lysine for proline at position 444 in the polypeptide chain, located in one of the non-catalytic domains of the enzyme glucocerebrosidase (GBA) . This mutation is especially refractory to the available treatments, including TCF, so there is an urgent need to develop therapeutic strategies that are useful, in particular, in patients with this genotype. Recently, it has been reported that b2-cyclic spino iminoazúres derived from L-idonojirimycin behave as CFs in human Gaucher fibroblasts homozygous for the L444P mutation, increasing glucocerebrosidase activity and lysosome enzyme traffic. One of the most promising candidates within this family of CFs is the N- [N '- (4
    15 adamantan-1-ylcarboxamidobutyl) thiocarbamoyl] -1,6-anhydro-L-donoji rimycin (NAd 8TAlJ) (Alfonso, P., et al., Bicyclic derivatives of L-idonojirimycin as pharmacoJogicaJ chaperones for neuronopathic forms of Gaucher disease. Chembiochem, 2013. 14 (8): p.943-9).
    20 A lower production cost, the possibility of oral administration and the ability to cross the blood brain barrier are advantages of this therapy for Gaucher's disease, especially for types II and 111.
    Despite its potential, research in pharmacological chaperone therapy
    25 (TCF) has not yet led to new drugs. This ultimate objective would be greatly facilitated by the development of strategies that enhance the action of pharmacological chaperones, for example by co-formulation with other active compounds.
    30 There is therefore a need to identify molecules that allow correcting or improving the pathologies associated with lysosomal storage diseases, particularly Gaucher disease, and acting in synergy with pharmacological chaperones, so that formulations containing both components produce a superior therapeutic benefit.

    DESCRIPTION OF THE INVENTION
    In the present invention it is described that the combination of a pharmacological chaperone and coenzyme 0 10 (CoO) produces a benefit in patient cells
    5 of Gaucher greater than the sum of those that would produce the individual treatments,representing an improved therapeutic option for the treatment of the diseaseof Gaucher, especially for the neuropathic types of it.
    Thus, one aspect of the present invention relates to a pharmaceutical composition 10 comprising a pharmacological chaperone (CF) and Coenzyme 0 10 (Coa).
    Throughout the present invention, the term "pharmacological chaperone" refers to a compound capable of binding to the human p-glucocerebrosidase enzyme, which is mutated and is dysfunctional in patients suffering from 15 Gaucher disease, and driving to an increase in your medically relevant activity. As examples of pharmacological chaperones currently under study, iminoazúres derivatives such as 1-deoxinojirimycin, isofagomine, iminoazúres sp2 derivatives such as NAdB-AIJ and other compounds not related to iminoazúres such as trans-4- (2) -amino-3,5
    Dibromobenzylamino) cyclohexanol (ambroxol).
    Coenzyme 0 10, also known as ubiquinone, ubidecarenone or coenzyme Q, is a 1,4-benzoquinone that can exist in three redox states: fully oxidized (ubiquinone), semiquinone (ubisemiquinone), and totally reduced (ubiquinol). In the following, the term coenzyme 0 10 (Coa) refers to any of these redox forms or states. CoO is an antioxidant and energy transporter considered as a potential treatment for neurodegenerative diseases, such as Parkinson's disease (Ebadi, M., et aL, Ubiquinone (coenzyme q10) and mitochondria in oxidative stress of parkinson's disease. Biol Signals Recept , 2001.
    30 10 (3-4): p. 224-53) or Sanfilippo disease (Malalonga, L., al., Treatment effect of coenzyme Q and an antioxidant cocktail in fibroblasts of patients with Sanfilippo disease. J Inherit Metab Dis, 2014). Coenzyme 0 10 acts on mitochondrial dysfunction and autophagic imbalance.
    In the pharmaceutical composition of the invention, pharmacological chaperone and coenzyme 0 10 can be combined in any relative proportion.
    In another embodiment the invention relates to the pharmaceutical composition as defined above, where the CF: CoQ molar ratio is between 1:10 and 10: 1, and preferably where the CF: CoQ molar ratio is 1: one.
    In another embodiment the invention relates to the pharmaceutical composition as it isdefined above, where the CF is an iminoazúcar sp2, and preferably theiminioazúcar sp2 is a derivative of L-idonojirimycin.
    The sp2 iminoazúres derived from L-idonojirimicina to which this invention refers are characterized by having a high selectivity towards ~ glucocerebrosidase.
    In another embodiment the invention relates to the pharmaceutical composition as defined above where the CF is N- [N '- (4-adamantan-115 ylcarboxamidobutyl) thiocarbamoyl] -1,6-anhydrous-L-donojirimycin ( NAdBT -AIJ).
    In another embodiment the invention relates to the pharmaceutical composition as it is
    defined above, which also includes one or more active ingredients
    additional, and preferably also comprising one or more active ingredients
    Additional 20 selected from a recombinant p-glucocerebrosidase enzyme, a second CF, a glucosylceramide synthase inhibitor and / or a proteostasis regulator.
    In another embodiment the invention relates to the pharmaceutical composition as it is
    25 defined above, which further comprises a recombinant p-glucocerebrosidase enzyme, and preferably where the recombinant p-glucocerebrosidase enzyme is selected from imiglucerase, sailglucerase alpha or taliglucerase alpha.
    In another embodiment the invention relates to the pharmaceutical composition as it is
    30 defined above, which further comprises a second CF, preferably where the second CF is an iminoazúcar sp2 derived from L-idonojirimine, isofagomin or ambroxol.
    In another embodiment the invention relates to the pharmaceutical composition as defined above, which further comprises a glucosylceramide inhibitor.
    synthase, and preferably where the glucosylceramide synthase inhibitor is
    selects from (2R, 3R, 4R, 5S) -1-butyl-2- (hydroxymethyl) piperidin-3,4,5-triol and N
    [(1 R, 2R) -1- (2,3-dihydro-1,4-benzodioxin-6-yl) -1-hydroxy-3- (1-pyrrolidinyl) -2
    propanil] octanamide.
    In another embodiment the invention relates to the pharmaceutical composition as defined above, which further comprises a proteostasis regulator, and preferably where the proteostasis regulator is selected from 4-phenylbutyric acid or celastrol.
    In another embodiment the invention relates to the pharmaceutical composition as defined above, which further comprises one or more drug transport vehicles, preferably comprising one or more drug transport vehicles selected from cyclodextrin derivatives, liposomes, micelles or nanocapsules, more preferably where drug transport vehicles are selected from cyclodextrin derivatives, and more preferably where cyclodextrin derivatives are selected from a-cyclodextrin (aCD), 3-cyclodextrin (¡3CD), V-cyclodextrin ( VCD), ¡3-cyclodextrin per (2,3,6-tri-O-methylated) (TRIMEB), 3-cyclodextrin per (2,6, -di-O-methylated) (DIMEB), ¡3-cyclodextrin randomly
    20 polymethylated (RAMEB), hydroxypropylated 3-cyclodextrin (HP-3CD) and sulfobutylated 3-cyclodextrin.
    In another embodiment the invention relates to the pharmaceutical composition as defined above, where:
    25 the CF: CoQ molar ratio is between 1:10 and 10: 1, and preferably where the CF: CoQ molar ratio is 1: 1; CF is an iminoazúcar sp2, preferably an iminioazúcar sp2 derived from Lidonojirimicina; and more preferably N- [N '- (4-adamantan-1-carboxyamidobutyl) thiocarbamoyl] -1,6-anhydro-L-donojirimycin (NAd BT-AIJ);
    30 and further comprising one or more active ingredients, preferably selected from: a recombinant f3-glucocerebrosidase enzyme, a second CF, a glucosylceramide synthase inhibitor and / or a proteostasis regulator.
    In another embodiment the invention relates to the pharmaceutical composition as defined above, where:
    the CF: CoQ molar ratio is between 1 10 and 10: 1, and preferably where the ratiomolar CF: CoQ is 1: 1;CF is an iminoazúcar sp2, preferably an iminioazúcar sp2 derived from Lidonojirimycin; and more preferably the N- [N '- (4-adamantan-1
    5-ylcarboxamidobutyl) thiocarbamoyl] -1,6-anhydro-L-idonojirimycin (NAd BT-AIJ); And which further comprises a recombinant l3-glucocerebrosidase enzyme, and preferably where the recombinant p-glucocerebrosidase enzyme is selected from imiglucerase, velaglucerase alpha or taliglucerase alpha.
    In another embodiment the invention relates to the pharmaceutical composition as defined above, where: the CF: CoQ molar ratio is between 1 10 and 10: 1, and preferably where the CF: CoQ molar ratio is 1 :one ; CF is an iminoazúcar sp2, preferably an iminioazúcar sp2 derived from L
    15 idonojirimycin; and more preferably N- [N '- (4-adamantan-1-carboxyamidobutyl) thiocarbamoyl] -1,6-anhydro-L-donojirimycin (NAd BT-AIJ); and which further comprises a second CF, preferably where the second CF is sealed from an iminoazúcar sp2 derived from L-idojirimine, isophagomine or ambroxol.
    In another embodiment the invention relates to the pharmaceutical composition as defined above, where: the CF: CoQ molar ratio is between 1 10 and 10: 1, and preferably where the CF: CoQ molar ratio is 1 :one ;
    CF is an iminoazúcar sp2, preferably an iminioazúcar sp2 derived from Lidonojirimicina; and more preferably N- [N '- (4-adamantan-1-carboxyamidobutyl) thiocarbamoyl] -1,6-anhydro-L-donojirimycin (NAd BT-AIJ); and which further comprises a glucosylceramide synthase inhibitor, and preferably where the glucosylceramide synthase inhibitor is selected from
    30 between (2R, 3R, 4R, 5S) -1-butyl-2- (hydroxymethyl) piperidin-3,4,5-tri01 and N - [(1 R, 2R) -1- (2,3 Dihydro-1, 4-be nzodioxy n-6-yl) -1-h idroxy-3- (1-pyrrolid ini I) -2 -propan i I] octa namid a.
    In another embodiment the invention relates to the pharmaceutical composition as defined above, where: the CF: CoQ molar ratio is between 1:10 and 10: 1, and preferably where the CF: CoQ molar ratio is 1: 1;
    CF is an iminoazúcar sp2, preferably an iminioazúcar sp2 derived from Lidonojirimicina; and more preferably N- [N '- (4-adamantan-1-carboxyamidobutyl) thiocarbamoyl] -1,6-anhydro-L-donojirimycin (NAd BT-AIJ); and which further comprises a proteostasis regulator, and preferably where the
    5 Proteostasis regulator is selected from 4-phenylbutyric acid or celastrol.
    In another embodiment the invention relates to the pharmaceutical composition as defined above, where:
    10 the CF: CoQ molar ratio is between 1:10 and 10: 1, and preferably where the CF: CoQ molar ratio is 1: 1; CF is an iminoazúcar sp2, preferably an iminioazúcar sp2 derived from Lidonojirimicina; and more preferably N- [N '- (4-adamantan-1-carboxyamidobutyl) thiocarbamoyl] -1,6-anhydro-L-donojirimycin (NAd BT-AIJ);
    15 which further comprises another active ingredient, preferably selected from a recombinant f3-glucocerebrosidase enzyme, a second CF, a glucosylceramide synthase inhibitor and a proteostasis regulator; and which also comprises one or more drug transport vehicles.
    In another embodiment the invention relates to the pharmaceutical composition as defined above, where: the CF: CoQ molar ratio is between 1:10 and 10: 1, and preferably where the CF: CoQ molar ratio is 1: 1; CF is an iminoazúcar sp2, preferably an iminioazúcar sp2 derived from L
    Idonojirimycin; and more preferably N- [N '- (4-adamantan-1-carboxyamidobutyl) thiocarbamoyl] -1,6-anhydro-L-donojirimycin (NAd BT-AIJ); which further comprises one or more active ingredients, preferably selected
    between a recombinant p-glucocerebrosidase enzyme, a second CF, a glucosylceramide synthase inhibitor and a proteostasis regulator; 30 and also comprising one or more vehicles transporting drugs selected from cyclodextrin derivatives.
    Another aspect of the present invention relates to the use of the pharmaceutical composition as defined above for the manufacture of a medicament for the treatment of a disease associated with lysosomal disorders, preferably for the treatment of a disease caused by
    mutations in the GBA1 gene, more preferably for the treatment of Gaucher disease, and even more preferably for the treatment of Gaucher disease type 1I or the treatment of Gaucher disease type 111.
    In another embodiment the invention relates to the use of the composition as mentioned above characterized in that the patient has at least one mutation that causes Gaucher pathology with implications of the central nervous system, and preferably where the patient has a mutation. 10 L444P / L444P.
    Thus, the present invention describes that, surprisingly, the combined treatment of pharmacological chaperones to recover the activity of the enzyme and Coenzyme 0 10, in any of its forms, to improve mitochondrial function and reduce autophageal stress, allows a benefit Therapeutic synergistic, superior to that which would be estimated by simple addition of the effects of chaperone and Coenzyme 0 10 • in trials conducted with Gaucher patient cells. Moreover, when chaperone is active in Gaucher patients with the L444P / L444P mutation, such as NAdB-AIJ, the administration of formulations that combine chaperone and
    20 Coenzyme 0 10 also produces a positive synergistic effect.
    Therefore, the present invention describes the use of a compound with activity as
    Pharmacological chaperone against mutant forms of the enzyme ~ -glucocerebrosidase
    human associated with Gaucher disease in combination with coa in
    25 any of its forms, for the treatment of Gaucher's disease in a human subject.
    As mentioned above, the present invention relates to a pharmaceutical composition comprising the components that have been defined.
    30 above and one or more pharmaceutically acceptable excipients. The excipients must be "acceptable" in the sense of being compatible with the other ingredients of the composition and of not being harmful to who takes said composition.
    The compounds of the present invention can be administered in the form of any pharmaceutical formulation, the nature of which, as is well known,
    It will depend on the nature of the active substance and its route of administration. In
    In principle, any route of administration can be used, for example oral, parenteral,
    Rectal
    5 Solid compositions for oral administration include tablets,granules and capsules. In any case, the manufacturing method is based ona simple mixture, dry granulation or wet granulation of the active ingredient withexcipients These excipients can be, for example, diluents such as lactose,microcrystalline cellulose, mannitol or calcium hydrogen phosphate; binding agents
    10 such as starch, gelatin or polyvinylpyrrolidone; disintegrants such as sodium carboxymethyl starch or croscarmellose sodium; and lubricating agents such as magnesium stearate, stearic acid or talc. The tablets may also be coated with suitable excipients and by known techniques in order to delay their disintegration and absorption in the gastrointestinal tract and thus
    15 achieve sustained action for a longer period of time, or simply to improve its organoleptic properties or its stability. The active ingredient can also be incorporated by coating on inert pellets by using natural or synthetic film-forming polymers. It is also possible to make soft gelatin capsules, in which the active substance is mixed with water or with
    Oily medium, for example coconut oil, liquid paraffin or olive oil.
    Powders and granules can be obtained for the preparation of oral suspensions by adding water, mixing the active ingredient with dispersing or wetting agents; suspending and preservative. You can also add others
    25 excipients, for example sweeteners, flavorings and dyes.
    Liquid forms for oral administration may include emulsions, solutions, suspensions, syrups and elixirs containing commonly used inert diluents, such as distilled water, ethanol, sorbitol, glycerol,
    30 polyethylene glycols (macrogoles) and propylene glycol. Such compositions may also contain adjuvants such as wetting, suspending, sweetening, flavoring, preservative and pH regulating agents.
    Injectable preparations, according to the present invention, for parenteral administration, comprise sterile solutions, suspensions or emulsions, in an aqueous or non-aqueous solvent such as propylene glycol, polyethylene glycol or
    vegetable oils. These compositions may also contain adjuvants, such as humectants, emulsifiers, dispersants and preservatives. They could be sterilized by any of the methods known or prepared as sterile solid compositions that will be dissolved in water or any other sterile injectable medium immediately before use. It is also possible to start from sterile raw materials and keep them in these conditions during the entire manufacturing process.
    For rectal administration, the active ingredient may preferably be formulated as a suppository in an oily base, such as for example vegetable oils or solid semi-synthetic glycerides, or in a hydrophilic base such as polyethylene glycols (macrogol).
    The dosage and frequency of the doses will vary depending on the nature and severity of the disease to be treated, the age, the general condition and the weight of the patient, as well as the particular compound administered and the route of administration, among other factors. . By way of example, an adequate dosage range ranges from about 0.01 mg / kg to about 100 mg / kg per day, which can be administered as a single dose or in several doses.
    The terms "one or more additional active ingredients" and "one or more drug transport vehicles" independently mean the possibility of one or more additional active ingredients or drug carriers, preferably one, two or three additional active ingredients or transporters of drugs, more preferably one or two additional active ingredients or drug carriers, and even more preferably an additional active ingredient
    or drug carrier.
    Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. The following examples and figures are provided by way of illustration, and are not intended to be limiting of the present invention.
    BRIEF DESCRIPTION OF THE FIGURES
    FIGURE 1. The mitochondrial function in Gaucher fibroblasts is recovered after treatment with Coenzyme 0 10 (CoO) and pharmacological chaperone (CF) NAdBT-AIJ.
    5 The control and pathological Gaucher dermal fibroblasts carrying the L444P mutation in homozygosis were cultured in the absence and presence of CoO (25IJM), CF NAdBT-AIJ (25 ~ M) or CoQ + CF (25 ~ M + 25 ~ M ) for 96 h.
    (A) Representative image of staining with MitoTracker Crop Network
    Gaucher fibroblasts. The magnification allows observing the presence of depolarized (a) and polarized (b) mitochondria.
    (B) Quantification of the number of depolarized mitochondria per cell.
    (C) The mitochondrial membrane potential (.6.lVm) in fibroblasts was measured by flow cytometry using the MitoTracker Red marker. The diminished potential in pathological cells is recovered with individual treatments but in a way
    15 more significant with the combined.
    (D) Oxidative stress derived from mitochondrial activity measured by flow cytometry using the MitoSox Red marker. Stress is partially reduced by individual treatments but more significantly by the combination. Data are mean ± standard deviation (SD) of three independent experiments. * p <0.01
    20 between control fibroblasts and Gaucher. ap <O.05 between the presence and absence of CoO. bp <O.05 between the presence and absence of CF. cp <O.05 between the presence and absence of CoO + CF. #p <O.05 between CoO + CF and CoO or CF treatment.
    FIGURE 2. Lysosomal activity and autophagic flow in Gaucher fibroblasts are
    25 improved after treatment with CoO and CF NAdBT-AIJ. The control and pathological Gaucher dermal fibroblasts carrying the L444P mutation in homozygosis were cultured in the absence and presence of CoQ (25 ~ M), CF NAdBT-AIJ (25 ~ M) or CoQ + CF (25 ~ M + 25 ~ M) for 96 h.
    (A) Lysosomal activity in fibroblasts was measured by flow cytometry
    30 using the LysoTracker Red marker. The increase in lysosome content in pathological cells is reduced with individual treatments but more significantly with the combination.
    (B) Magnification of the LC3 and mitochondrial cytochrome e autophagic marker staining
    which shows the presence of autophagosomes degrading mitochondria in fibroblasts 35 of patients. (a) Autophagosome / mitochondrial negative colocalization: Pearson's correlation coefficient = O, 0526; (b) Autophagosome / mitochondria positive colocalization: Pearsonn correlation coefficient = O, 8929.
    (C) The "punctatas" are quantified, decreasing significantly with the
    5 individual treatments but more significantly with the combination. Data are the mean ± SO of three independent experiments. "p <0.01 between control fibroblasts and Gaucher. 8p <0.05 between the presence and absence of coa. bp <O.05 between the presence and absence of CF. cp <O.05 between the presence and absence of CoO + CF. # p <O.05 between CoO + CF and CoO or CF treatment.
    10 FIGURE 3. Increase in enzymatic activity associated with an increase in the level of protein and proper traffic after treatment with CoO and CF NAdBT -AIJ. The control and pathological Gaucher dermal fibroblasts carrying the L444P mutation in homozygosis were cultured in the absence and presence of CoO (25IJM), CF 15 NAdBT-AIJ (25 ~ M) or CoQ + CF (25 ~ M + 25 ~ M ) for 96 h.
    (A) Measurement of glucocerebrosidase (GBA) activity that is increased especially after combined treatment. The data represent the% activity with respect to the control fibroblasts.
    (B) Glucocerebrosidase (GBA) protein expression levels determined by Western blot. a-tubulin is used as load control.
    (C) Densitometric analysis of the Western blot result. Data are mean ± SO of three independent experiments. * p <O.01 between control fibroblasts and Gaucher. "'p <O.05 between the presence and absence of CoO. bp <O.05 between the presence and absence of CF. cp <O.05 between the presence and absence of CoQ + CF. #p <O.05 between treatment
    25 CoQ + CF and CoQ or CF.
    FIGURE 4. Immunofluorescence images with lysosomal marker LAMP-2 and Glucocerebrosidase (GBA) to check the transport of the glucocerebrosidase enzyme to the lysosome after treatment. The positive colocalization of the LAMP-2 lysosomal marker and glucorerebrosidase (GBA) indicates the correct enzyme traffic. (a) Positive colocalization: Pearson's correlation coefficient = O, 9535;
    (b) Negative colocalization; (c) Positive colocalization: Pearson's correlation coefficient = O, 9076. The nuclei were stained with Hoechst staining.
    35 FIGURE 5IncreasefromactivityEnzymatic Glucocerebrosides (CBA)afterhe
    treatment withCoOYAmbroxol(ABX)ThefibroblastsdermalcontrolY
    14
    Pathological Gaucher with heterozygosis for the L444P and P415R mutations were cultured in the absence and presence of CoQ (25 ~ M), Ambroxol (50 ~ M) or CoQ + ABX (25IJM + 50IJM) for 96 h. The graph shows the measure of glucocerebrosidase (GBA) activity that is significantly increased after
    5 combined treatment. Data represented as% of activity with respect tocontrol fibroblasts * p <0.01 between control fibroblasts and Gaucher. Bp <0.05 betweenpresence and absence of CoQ. bp <0.05 between the presence and absence of CF. cp <0.05between the presence and absence of CoQ + CF. #p <0.05 between CoQ + CF and CoQ treatmentoCF.
    EXAMPLES
    The invention will now be illustrated by tests carried out by the inventors, which show the effectiveness of the product of the invention.
    Example 1. Effect of the combined CoQ and NAdBT-AIJ treatment, in relative 1: 1 molar ratio, in vitro in Gaucher cells with the l444P variant in homozygosis on mitochondrial dysfunction.
    Cultures of control dermal fibroblasts and patients with disease
    20 Gaucher carriers of the L444P mutation were analyzed to check the status and functioning of the mitochondrial network.
    First, a 20% reduction in mitochondrial respiratory chain activity was observed, specifically in the 11 + 111 complex, associated with a
    25 secondary deficiency in Coenzyme QlO levels. As a consequence of these anomalies, it was confirmed that the mitochondrial membrane potential (6lVm) is decreased in Gaucher fibroblasts with a high proportion of depolarized mitochondria isolated from the tubular mitochondrial network (Figure 1A and 1B) observed by immunofluorescence microscopy after staining with MitoTracker net. The essays
    Flow cytometry, using this same marker (Figure 1C), allows quantifying the reduction of mitochondrial membrane potential. The fibroblasts were cultured in the absence and presence of CoQ (25IJM), CF NAdBT-AIJ (25IJM) or CoQ + CF in relative 1: 1 mole ratio (25IJM + 25IJM) for 96 h. The mitochondrial membrane potential was recovered by individual treatments but from
    35 more significantly with the combined treatment.
    The energy production levels in the form of ATP are also reduced due to the reduction of mitochondrial membrane potential and the treatments also managed to increase them proportionally to the increase in mitochondrial potential.
    The association of mitochondrial dysfunction with an increase in the production of reactive oxygen species (ROS) is well demonstrated. Figure 1D depicts the measures of mitochondrial oxidative stress in fibroblasts obtained by flow cytometry assays using the MitoSOX Red marker. Production
    10 mitochondrial superoxide is increased 1.5 times confirmed oxidative stress in Gaucher fibroblasts. Supplementation with coa, NAdBT-AIJ or a combination of both in a 1: 1 relative mole ratio induces a considerable reduction of these levels.
    Example 2. Effect of the CoQ and NAdBT-AIJ combined treatment, in relative 1: 1 molar ratio, in vitro in Gaucher cells with L444P variant in homozygosis on autophagic stress.
    There is a relationship between high levels of oxidative stress and activation of autophagy.
    20 To determine whether autophagy is increased in Gaucher L444P fibroblasts, the amount of acid vacuoles was quantified by staining with LysoTracker and fluorescence microscopy and flow cytometry assays. Using this technique, a 1.4-fold increase in LysoTracker staining was observed in Gaucher fibroblasts compared to control fibroblasts (Figure 2A).
    25 The number of lysosomes in the cultures was quantified to elucidate whether autophagy in these fibroblasts decreases as a result of an improvement in mitochondrial function by CoQ supplementation and / or mutant glucocerebrosidase folding by treatment with NAdBT-AIJ chaperones. Figure 2A indicates that the
    30 lysosomal activity is reduced by individual treatment but more significantly by the combination of both.
    Analysis by polyacrylamide gel electrophoresis with dodecyl sulfate (SDSPAGE) -Western blot suggests an increase in autophagic markers such as Atg1235 5, beclin1 and LC3B in pathological fibroblasts versus controls. This autophagy was found to be selective of the mitochondria, observing the presence of
    Colocalization "punctatas" in immunofluorescence assays of autophagic markers (LC3B) with mitochondrial markers (cytochrome c) Figure 2B. LC3B staining was difficult to detect in control fibroblasts. In Gaucher fibroblasts the population of small round mitochondria shows a high
    5 colocalization of cytochrome c and LC3B (r = O.8929), while mitochondriatubular no (r = 0.0526).
    Supplementation with coa (25 IJM) or CF NAdBT-AIJ (25 IJM) partially reduced the number of "punctatas" LC3 / Cytochrome C. However, the combination of both 10 treatments in 1: 1 relative mola ratio was highly effective and drastically reduced these elements.
    Example 3. Effect of the CoQ and NAdBT -AIJ combined treatment, in 1: 1 relative molar ratio, in vitro in Gaucher cells with the L444P variant in
    15 homozygosis on the enzymatic activity glucocerebrosidase and its correct traffic to the lysosomes. Glucocerebrosidase activity in the fibroblasts of patients is reduced to levels below 10% compared to control cell values. Figure 3A shows how enzymatic activity is partially restored after
    20 improvement of mitochondrial function by CoQ (1.2 times) as well as by the correct folding induced by treatment with NAdBT-AIJ chaperones (2.4 times). The combined treatment of both compounds in a 1: 1 relative mole ratio achieves a greater improvement in activity levels (3.4 times).
    25 Deficiencies in enzymatic activity are associated with reduced levels of protein expression which, as with activity, are recovered with coa and CF treatment. The protein expression levels determined by Western blot are shown in Figure 3B. The expression of o-tubulin is used as load control. A densitometric analysis of the
    30 Western blot result.
    Example 4. Effect of the combined CoQ and NAdBT -AIJ treatment on glucocerebrosidase traffic_ The natural traffic of the glucocerebrosidase enzyme to the lysosome is also
    35 reestablished after co-treatment of coa and CF, as shown in Figure 4. It shows a fluorescence microscopy assay with antibodies against lysosomal LAMP-1 protein and glucocerebrosidase. The colocalization of both proteins confirms the presence of the enzyme in ellisosome. Fibroblasts that were ca-treated with CoQ and CF NAdBT-AIJ in relative 1: 1 mole ratio have a glucocerebrosidase staining profile with greater intensity and colocalization
    5 with the lysosomal LAMP-1 protein. In turn, the signal of the glucocerebrosidase that lays with the POI protein of the endoplasmic reticulum decreases, suggesting that the protein is transported correctly to the Golgi apparatus and to the lysosomes.
    10 Example 5. Effect of the combined CoQ and ambroxol (ABX) treatment, in relative 1: 1 molar ratio, in vitro of Gaucher cells with the variant l444P / P415R in heterozygosis on the glucocerebrosidase enzyme activity. Glucocerebrosidase enzyme activity in fibroblasts derived from patients
    15 Gaucher carriers of the L444P / P415R mutations in heterozygosis are also diminished compared to healthy control fibroblasts. The treatment with ambroxol (ABX), which acts as a pharmacological chaperone, at a concentration of 50 ... 1M, partially recovers the enzymatic activity (1.6 times more). To a lesser extent, but significantly, coa treatment also allows
    20 obtain greater glucocerebrosidase activity in cells derived from Gaucher patients (1.2 times more). The combined treatment of both compounds in a 1: 1 relative mole ratio achieves a significant improvement in activity levels (2.8 times).
    权利要求:
    Claims (11)
    [1]
    1. Pharmaceutical composition comprising a pharmacological chaperone (CF) and Coenzyme Q10 (CoQ).
    [2]
    2. The pharmaceutical composition according to claim 1, wherein the CF: CoQ molar ratio is between 1:10 and 10: 1.
    [3]
    3. The pharmaceutical composition according to any of claims 1 or 2, wherein the CF is an iminoazúcar sp2.
    [4]
    4. The pharmaceutical composition according to claim 3, wherein the iminioazúcar sp2 is a derivative of L-idonojirimycin.
    5. The pharmaceutical composition according to claim 4, wherein the CF is N- [N '- (4adamantan-1-ylcarboxamidobutyl) thiocarbamoyl] -1,6-anhydro-L-donoji rimicin (NAd BT
    AIJ).
    [6]
    6. The pharmaceutical composition according to any of claims 1 to 5, which further comprises one or more additional active ingredients.
    [7]
    7. The pharmaceutical composition according to claim 6, wherein the active ingredient
    additional is selected from a recombinant p-glucocerebrosidase enzyme, a second CF, a glucosylceramide synthase inhibitor and / or a proteostasis regulator.
    [8]
    8. The pharmaceutical composition according to any of claims 1 to 7, further comprising one or more drug transport vehicles.
    The pharmaceutical composition according to claim 8, wherein the drug transport vehicle is selected from derivatives of cyclodextrins, liposomes, micelles or nanocapsules.
    [10]
    10. The pharmaceutical composition according to any of claims 8 or 9, 35 wherein the drug transport vehicle is selected from cyclodextrin derivatives.
    [11 ]
    11. The pharmaceutical composition according to claim 10, wherein the cyclodextrin is selected from a-cyclodextrin (aCD), 3-cyclodextrin (¡3CD), and -cyclodextrin (yeD), ~ -cyclodextrin per (2,3 , 6-tri-O-methylated) (TRIMES), ~ -cyclodextrin per (2,6, -di
    5 O-methylated) (DIMES), ~ - randomly polymethylated cyclodextrin (RAM ES), ~hydroxypropylated cyclodextrin (HP-¡3CD) and sulfobutylated 3-cyclodextrin.
    [12]
    12. Use of the pharmaceutical composition according to any one of claims 1 to 11, for the manufacture of a medicament for the treatment of a disease caused by mutations in the GBA1 gene.
    [13]
    13. The use according to claim 12, for the treatment of Gaucher disease.
    14. The use according to claim 13, for the treatment of Gaucher disease type 11 or the treatment of Gaucher disease type 111.
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    同族专利:
    公开号 | 公开日
    WO2016162588A1|2016-10-13|
    ES2597728B1|2018-03-02|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    WO2020046132A1|2018-08-31|2020-03-05|Leiden University|Pharmacological chaperones for enzyme treatment therapy|
    NL2021840B1|2018-10-19|2020-05-13|Univ Leiden|Pharmacological Chaperones For Enzyme Treatment Therapy|
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