JP2019199413A - Agent for the prevention or treatment of gb3 accumulation-attributable disease - Google Patents

Abstract

To provide an agent for the prevention or treatment of Gb3 accumulation-attributable disease such as Fabry disease, and heart disease with abnormal heart stroke.SOLUTION: The present invention provides an agent for the prevention or treatment of Gb3 accumulation-attributable disease containing at least one selected from the group consisting of a chloroquine represented by the following formula and a chloroquine derivative.SELECTED DRAWING: None

Description

  The present invention relates to a preventive or therapeutic agent for diseases caused by Gb3 accumulation.

  It is known that various diseases develop due to accumulation of globotriaosylceramide (Gb3) in the body. For example, Fabry disease has various abnormalities such as cardiac dysfunction and renal dysfunction caused by accumulation of Gb3 in the body due to gene deficiency or decreased activity of an enzyme that degrades Gb3 (α-galactosidase (GLA)). It is a disease accompanied by. Further, it has been reported that blood Gb3 concentration may increase without GLA gene mutation and that the increase is associated with heart disease.

  As therapeutic agents for Fabry disease, recombinant GLA proteins such as Fabryzyme are known. However, because this is a recombinant protein, 1) it is expensive, 2) the anti-GLA antibody appears due to continuous administration, and the therapeutic effect is reduced, and 3) it is effective for patients with advanced disease. Problems such as low points are pointed out.

  Chloroquine and its derivatives (such as hydroxychloroquine) are known to have a therapeutic effect on malaria and systemic lupus erythematosus (Patent Document 1). However, it is not known that these have a therapeutic effect on diseases caused by Gb3 accumulation such as Fabry disease.

Special Publication 2009-504743

  An object of the present invention is to provide a prophylactic or therapeutic agent for Gb3 accumulation-induced diseases, which comprises a low molecular compound as an active ingredient.

  As a result of diligent research in view of the above problems, the present inventors have found that chloroquine and chloroquine derivatives are effective for the prevention or treatment of diseases caused by Gb3 accumulation. As a result of further research based on this knowledge, the present invention was completed.

That is, the present invention includes the following embodiments:
Item 1. A prophylactic or therapeutic agent for a disease caused by Gb3 accumulation, comprising at least one selected from the group consisting of chloroquine and chloroquine derivatives.
Item 2. Item 2. The prophylactic or therapeutic agent according to Item 1, wherein the chloroquine derivative is hydroxychloroquine.
Item 3. Item 3. The prophylactic or therapeutic agent according to Item 1 or 2, wherein the Gb3 accumulation-induced disease is at least one disease selected from the group consisting of Fabry disease and heart disease.
Item 4. Item 4. The preventive or therapeutic agent according to any one of Items 1 to 3, wherein the Gb3 accumulation-causing disease is Fabry disease.
Item 5. Item 5. The preventive or therapeutic agent according to any one of Items 1 to 4, wherein the Gb3 accumulation-induced disease is a disease accompanied by abnormal heart beat.

  According to the present invention, it is possible to provide a preventive or therapeutic agent for Gb3 accumulation-induced diseases, which comprises a low molecular compound as an active ingredient.

(A) The result of immunostaining (Example 1) of a Fabry disease model cardiomyocyte is shown. LAMP1 shows a lysosomal marker (LAMP1) stained image, mTOR shows an mTOR stained image, and Merge shows an image in which both images are superimposed. -DOX / + DOX was obtained by culturing Fabry disease model iPSC (Reference Example 2) in the presence of DOX (+ DOX) or in the absence (-DOX) and using cardiomyocytes obtained by myocardial differentiation (Reference Example 3). HCQ indicates the case where hydroxychloroquine is added to the medium. (B) The result of the pulsation evaluation of Fabry disease model cardiomyocytes (Reference Example 4, Example 1) is shown. The vertical axis represents the relaxation duration. In the horizontal axis, -D / + D indicates the myocardium obtained by culturing Fabry disease model iPSC (Reference Example 2) in the presence (+ D) or absence (-D) of DOX and differentiating myocardium (Reference Example 3). The case where cells are used is shown, HCQ shows the case where hydroxychloroquine is added to the medium, Fz shows the case where Fabryzyme is added to the medium, and Mock shows the case where no drug is added. (C) The results of cell area measurement (Reference Example 4, Example 1) of Fabry disease model cardiomyocytes are shown. The vertical axis represents the cell area. In the horizontal axis, -D / + D indicates the myocardium obtained by culturing Fabry disease model iPSC (Reference Example 2) in the presence (+ D) or absence (-D) of DOX and differentiating myocardium (Reference Example 3). The case where cells are used is shown, HCQ shows the case where hydroxychloroquine is added to the medium, and Mock shows the case where no drug is added. (D) The result of the pulsation evaluation (Example 1) of a cardiomyocyte derived from a Fabry disease patient is shown. The vertical axis represents the relaxation duration. In the horizontal axis, HCQ indicates the case where hydroxychloroquine is added to the medium (the value is the concentration in the medium (unit: μM)), Fz indicates the case where Fabryzyme is added to the medium, and Mock indicates the case where no drug is added. Indicates.

  In this specification, the expressions “containing” and “including” include the concepts of “containing”, “including”, “consisting essentially of”, and “consisting only of”.

  In one embodiment, the present invention contains at least one selected from the group consisting of chloroquine and chloroquine derivatives (in this specification, sometimes referred to as “active ingredient of the present invention”). The present invention relates to a prophylactic or therapeutic agent for sexual diseases (sometimes referred to herein as “the drug of the present invention”). This will be described below.

1． The active ingredient chloroquine is a compound represented by the following formula.

  The chloroquine derivative is not particularly limited, and a known chloroquine derivative can be adopted. Among the chloroquine derivatives, a derivative in which the alkyl group in the dialkyl (diethyl) amino group of chloroquine is the same or different and is an alkyl group having 1 to 4 carbon atoms (derivative 1), one in the dialkylamino group of chloroquine and derivative 1 Or a derivative (derivative 2) in which two alkyl groups are substituted with a hydroxy group, more preferably a derivative 2, more preferably a hydroxychloroquine:

Is mentioned.

  As an active ingredient of this invention, Preferably chloroquine, hydroxychloroquine, etc. are mentioned, More preferably, hydroxychloroquine is mentioned.

  The active ingredient of the present invention includes a salt form. The salt is not particularly limited as long as it is a pharmaceutically acceptable salt, and either an acidic salt or a basic salt can be employed. Examples of acid salts include inorganic acid salts such as hydrochloride, hydrobromide, sulfate, nitrate and phosphate; acetate, propionate, tartrate, fumarate, maleate, apple Organic acid salts such as acid salts, citrate salts, methanesulfonate salts, paratoluenesulfonate salts; and amino acid salts such as aspartate salts and glutamate salts. Examples of basic salts include alkali metal salts such as sodium salts and potassium salts; alkaline earth metal salts such as calcium salts and magnesium salts. As the salt, an acidic salt is preferable, an inorganic acid salt is more preferable, and a sulfate, phosphate, hydrochloride, and the like are more preferable.

  The active ingredient of the present invention includes a solvate form. The solvent is not particularly limited as long as it is pharmaceutically acceptable, and examples thereof include water, ethanol, glycerol, acetic acid and the like.

  The active ingredient of the present invention may be a single poison or a combination of two or more.

2． Use The active ingredient of the present invention is effective as a preventive or therapeutic agent for diseases caused by Gb3 accumulation.

  Examples of the Gb3 accumulation-causing disease include Fabry disease (preferably Fabry disease accompanied by heart disease) caused by Gb3 accumulation due to GLA gene mutation. In addition, it has been reported that blood Gb3 concentration may increase without GLA gene mutation and that the increase is associated with heart disease. Are also included. The Gb3 accumulation-causing disease is preferably a disease accompanied by abnormal heart beat.

  The drug of the present invention is not particularly limited as long as it contains the active ingredient of the present invention, and may consist only of the active ingredient of the present invention, or may further contain other components as necessary. May be. Other components are not particularly limited as long as they are pharmaceutically acceptable components. As other components, in addition to components having a pharmacological action, additives are also included. Examples of the additive include a base, a carrier, a solvent, a dispersant, an emulsifier, a buffer, a stabilizer, an excipient, a binder, a disintegrant, a lubricant, a thickener, a moisturizer, a colorant, a fragrance, Examples include chelating agents.

  Although the application target of the drug of the present invention is not particularly limited, examples of mammals include humans, monkeys, mice, rats, dogs, cats, rabbits, pigs, horses, cows, sheep, goats, deer and the like.

  The drug of the present invention can be used in any dosage form such as tablets (including orally disintegrating tablets, chewable tablets, effervescent tablets, troches, jelly-like drops, etc.), pills, granules, fine granules, powders, Oral dosage forms such as hard capsules, soft capsules, dry syrups, liquids (including drinks, suspensions, syrups), jellys, and injectable preparations (eg, intravenous infusion (eg, intravenous infusion) Formulations), intravenous injections, intramuscular injections, subcutaneous injections, intradermal injections), external preparations (eg, ointments, poultices, lotions), suppository inhalants, ophthalmic preparations, ophthalmic ointments, dots Parenteral preparation forms such as nasal drops, ear drops, and liposomes can be employed.

  The administration route of the drug of the present invention is not particularly limited as long as the desired effect is obtained, and enteral administration such as oral administration, tube feeding, enema administration, intravenous administration, transarterial administration, intramuscular administration, Examples include parenteral administration such as intracardiac administration, subcutaneous administration, intradermal administration, and intraperitoneal administration.

  The content of the active ingredient in the drug of the present invention depends on the use mode, application target, application target state, etc., and is not limited, but is, for example, 0.0001 to 100% by weight, preferably 0.001 to 50% by weight. %.

  The dosage in the case of administering the drug of the present invention to an animal is not particularly limited as long as it is an effective amount that exerts a medicinal effect, and is usually 0.1 to about 0.1 to 1 per day in the case of oral administration in general as the weight of the active ingredient. 1000 mg / kg body weight, preferably 0.5 to 500 mg / kg body weight per day, and 0.01 to 100 mg / kg body weight per day for parenteral administration, preferably 0.05 to 50 mg / kg body weight . The above dose can be appropriately increased or decreased depending on age, disease state, and symptoms.

  EXAMPLES The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

Reference example 1. Preparation of iPSCs from AFD patients Cells in blood collected from Anderson-Fabry Disease (AFD) patients, hematopoietic cytokines (10 ng / ml IL-3 (R & D system), 100 ng / ml IL-6) (R & D system), 300ng / ml SCF (R & D system), 300ng / ml TPO (R & D system), 300ng / ml Flt3L (308-FK, R & D)) medium containing StemSpan ACF (Stem Cell Technologies) The cells were selected and expanded by culturing for a week. Subsequently, the cultured cells were reprogrammed into iPS cells (iPSCs) using an episomal vector under feeder-free conditions. Specifically, Epstein-Barr nuclear antigen 1 (EBNA1) and four pCXLE episomal non-integrated plasmids encoding shRNAs against human OCT4, SOX2, KLF4, LIN28, L-MYC and p53 were transferred to 500,000 cells. (Human CD34 cell Nucleofector kit, program U-08, Lonza). Nucleofected cells were seeded onto wells of 6-well plates coated with iMatrix-511 silk (5 μl per well 1.5 ml medium) (Nippi, Tokyo, Japan). At this time, a hematopoietic medium containing Y-27632 (10 μM) (WAKO Pure Chemical Industries) was used as the medium. After nucleofection, 1.5 ml StemFit AK02N (Ajinomoto) was added to the culture twice every two days. Thereafter, all the medium was supplemented with fresh StemFit AK02N every other day until iPSC colonies appeared. In order to establish a stable cell line, 10 colonies were collected and cultured for 10 passages or more before analysis. GLA mutations in patient-specific iPSCs were confirmed by PCR amplification of genomic regions containing the mutation followed by Sanger sequencing.

Reference example 2. Production of Fabry disease model iPSC A genome editing system called CRISPR interference (CRISPRi) was used for human iPSC. In this system, dead Cas9 (dCas9) deficient in nuclease activity and KRAB, a transcriptional repression domain, are fused onto the target gene using a single guided RNA (gRNA) that directs the target gene to the transcription initiation region. Is a system that specifically suppresses transcriptional expression of a target gene. Using a human iPSC strain (CRIPSRi iPSC strain) that can suppress the expression of this dCas9-KRAB in a doxycycline (DOX) -dependent manner, gRNA targeting the GLA gene is introduced there, and the expression of the GLA gene is dependent on the addition of DOX. Cells that can be suppressed were made. Specifically, it was performed as follows.

<Reference Example 2-1. Human iPSC culture>
iPSCs were maintained in StemFit AK02N (Ajinomoto) medium on plates coated with iMatrix-511 silk (Nippi, Tokyo, Japan) and passaged every 5-7 days using Accutase (Innovative Cell Technologies). For 24 hours after each passage, ROCK inhibitor Y-27632 (10 μM) (Wako Pure Chemical Industries) was added to the medium.

<Reference Example 2-2. gRNA Design and Cloning to gRNA Expression Vector>
The region near 200 bp upstream of the transcription start site (TSS) of the GLA gene was used as the sgRNA design region. The location of TSS was determined using the UCSC genome browser (https://genome.ucsc.edu). The gRNA oligo was designed using the CRISPR design website (http://crispr.mit.edu), annealed and cloned into the gRNA expression vector pB-U6-CNKB.

<Reference Example 2-3. Selection of gRNA nucleofection and stable CRISPRi clone>
The sgRNA expression vector was transfected into a well-characterized CRIPSRi iPSC line with normal karyotype and differentiated into three germ layers in vitro and teratomas, followed by treatment of the transfected iPSC with DOX . Five to six days after the start of DOX treatment, RNA was collected from the cells and examined for GLA expression by RT-qPCR. Based on the expression level obtained, a cell line (GLA CRISPRi iPSC line) capable of efficiently suppressing the expression of GLA in a DOX-dependent manner was established.

Reference Example 3. Myocardial differentiation and purification from iPSC
StemFit AK02N containing Y-27632 and iMatrix-511 silk was seeded on a 12-well plate at 1 ml per well, and iPSCs were seeded there at a number of 40,000 to 120,000 cells. Thereafter, the cells were cultured for 3-4 days and cultured until the cell density reached 70-80%. Thereafter, the cells were treated with 6-12 μM CHIR99021 for 24 hours to initiate myocardial differentiation. After treatment with CHIR99021, treatment was performed for 2 days with 5 μM IWP2 for 48 hours. As the basal medium, RPMI1640 medium containing no insulin was used until the 6th day of differentiation, and RPMI1640 medium containing insulin was used until the 15th day thereafter. On the 15th day of differentiation, cells differentiated into myocardium were peeled off with 0.25% trypsin. After cell suspension in DMEM medium containing 10% FBS and 4.5 g / L glucose, the cells were cultured in this medium for 3 days. Myocardium differentiated from iPSC was purified and purified using lactic acid medium. To the glucose-free DMEM medium, 4 mM lactic acid, Glutamax, and non-essential amino acids were added to obtain a lactic acid medium. The medium was exchanged every other day for 5 days to purify the myocardium.

Reference example 4. Evaluation of Fabry disease model cardiomyocytes About Fabry disease model cardiomyocytes obtained by culturing Fabry disease model iPSC (Reference Example 2) in the presence of DOX and differentiating myocardium (Reference Example 3) The movement was quantified. Specifically, it was performed as follows.

  The heartbeat was measured using a video microscope called Sony SI8000. A video image of human iPSC-derived myocardial pulsations was taken for 10 seconds with a frame rate of 150 fps and a resolution of 2048x2048 pixels using a 10x lens. The Sony SI8000 analysis software analyzes the vector obtained from all myocardial contraction movements captured in 10 seconds, and then converts the average vector length per unit time into a time-motion speed diagram, thereby functional parameters. (Shrinkage speed, relaxation speed, contraction duration, relaxation duration, contraction distance, relaxation distance, contraction relaxation duration) were analyzed.

  As a result, it was confirmed that abnormalities were observed in the contraction / relaxation of cardiomyocytes in Gb3-accumulated cardiomyocytes (Fabry disease model cardiomyocytes) by suppressing DOX-induced GLA expression (FIG. 1b). .

  Further, the obtained cells were fluorescently stained with an antibody against actinin that specifically expresses myocardium, and then observed using a fluorescent microscope. There were a plurality of cardiomyocytes gathered on the plate to form a lump, and there were cardiomyocytes that were dissociated from the lump and existed alone. The cell area of cardiomyocytes present alone was analyzed using image analysis software Image J. As a result, it was clarified that the area of the myocardium was expanded by suppressing GLA expression (FIG. 1c).

Example 1. Evaluation of therapeutic effects of abnormalities caused by Gb3 accumulation The effects of hydroxychloroquine on Gb3 accumulation-induced abnormalities in cardiomyocytes were examined. Specifically, it was performed as follows.

<Example 1-1. Immunostaining>
Fabry disease model iPSC (Reference Example 2) is cultured in the presence or absence of DOX and added to the medium of cardiomyocytes obtained by cardiomyocyte differentiation (Reference Example 3) (concentration in the medium: 0.3 μM) After 4 to 5 days, lysosomal markers (LAMP1) and mTOR were immunostained. Specifically, it was performed as follows. Cardiomyocytes are fixed with cold 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton X-100 in phosphate buffered saline (PBS) for 10 minutes, and blocked with 2.5% skim milk in PBS for 30 minutes at room temperature did. Cells were reacted with anti-LAMP1 antibody and anti-mTOR antibody. After washing twice with PBS, cells were incubated briefly with 2.5% skim milk in PBS. Next, the cells were stained with a secondary antibody.

<Example 1-2. Evaluation of pulsation of Fabry model cardiomyocytes>
Beating of cardiomyocytes obtained by culturing Fabry disease model iPSC (Reference Example 2) in the absence of DOX and differentiating myocardium (Reference Example 3), and Fabry disease model iPSC (Reference Example 2) in the presence of DOX Hydroxychloroquine or Fabrizyme was added to cardiomyocytes obtained by culture and cardiomyocyte differentiation (medium concentration: Hydroxychloroquine 0.3 μM, Fabryzyme 5 μg / ml), and the pulsation after 4 to 5 days passed, Measurement was performed in the same manner as in Reference Example 4.

<Example 1-3. Cell area of Fabry model cardiomyocytes>
Cellular area of cardiomyocytes obtained by culturing Fabry disease model iPSC (Reference Example 2) in the absence of DOX and differentiating myocardium (Reference Example 3), and Fabry disease model iPSC (Reference Example 2) in the presence of DOX Hydroxychloroquine was added to the cardiomyocytes obtained by culture and myocardial differentiation (medium concentration: 0.3 μM hydroxychloroquine), and the myocardial cell area after 5 days was measured in the same manner as in Reference Example 4. .

<Example 1-4. Evaluation of pulsation of cardiomyocytes from Fabry disease patients>
Addition of hydroxychloroquine or Fabryzyme to cardiomyocytes obtained by myocardial differentiation (Reference Example 3) of Fabry disease patient-derived iPSC (Reference Example 3) (concentration in medium: hydroxychloroquine 0.1 to 1 μM, Fabryzyme 5 μg / ml) and the pulsation after 4 to 5 days was measured in the same manner as in Reference Example 4.

<Example 1-5. Result>
The results of immunostaining are shown in FIG. 1a, the results of cell area measurement are shown in FIG. 1c, and the results of pulsation evaluation are shown in FIGS. 1b and 1d. In cardiomyocytes in which GLA was suppressed in a DOX-induced manner, the localization of mTOR in lysosomes decreased as a result of immunostaining, but these colocalizations were increased by hydroxychloroquine treatment (FIG. 1a). In both cases of myocardial differentiation of Fabry disease model iPSC (Reference Example 2) and iPSCs derived from Fabry disease patients (Reference Example 1), abnormal pulsation (increase in relaxation duration) This was improved by hydroxychloroquine (FIGS. 1b and 1d). The degree of improvement was similar to that of the existing therapeutic agent Fabryzyme. Furthermore, myocardial hypertrophy was dependent on GLA expression suppression. This enlargement was suppressed by hydroxychloroquine treatment (FIG. 1c).

Claims (5)

A prophylactic or therapeutic agent for Gb3 accumulation-induced diseases, comprising at least one selected from the group consisting of chloroquine and chloroquine derivatives. The preventive or therapeutic agent according to claim 1, wherein the chloroquine derivative is hydroxychloroquine. The preventive or therapeutic agent according to claim 1 or 2, wherein the Gb3 accumulation-induced disease is at least one disease selected from the group consisting of Fabry disease and heart disease. The preventive or therapeutic agent according to any one of claims 1 to 3, wherein the Gb3 accumulation-causing disease is Fabry disease. The preventive or therapeutic agent according to any one of claims 1 to 4, wherein the Gb3 accumulation-induced disease is a disease accompanied by abnormal heart beat.