JP7131803B2 - Preventive or therapeutic agent for diseases caused by Gb3 accumulation - Google Patents

Description

TECHNICAL FIELD The present invention relates to preventive or therapeutic agents for diseases caused by Gb3 accumulation.

Accumulation of globotriaosylceramide (Gb3) in the body is known to cause various diseases. For example, Fabry disease is caused by the accumulation of Gb3 in the body due to genetic deficiency or reduced activity of the enzyme that degrades Gb3 (α-galactosidase (GLA)), resulting in various abnormalities such as cardiac and renal dysfunction. is a disease with In addition, it has been reported that blood Gb3 concentration may be elevated even without GLA gene mutation, and that the elevation is associated with heart disease.

As therapeutic agents for Fabry disease, GLA recombinant proteins such as Fabrizyme are known. However, because it is a recombinant protein, it is 1) expensive, 2) anti-GLA antibodies appear with continuous administration, reducing the therapeutic effect, and 3) the therapeutic effect is limited in patients with advanced disease. Problems such as a low point have been 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 they have a therapeutic effect on Gb3 accumulation-induced diseases such as Fabry disease.

Japanese Patent Publication No. 2009-504743

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

As a result of intensive research in view of the above problems, the present inventors have found that chloroquine and chloroquine derivatives are effective in preventing or treating 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 aspects:
Section 1. A prophylactic or therapeutic agent for diseases caused by Gb3 accumulation, containing at least one selected from the group consisting of chloroquine and chloroquine derivatives.
Section 2. Item 2. The preventive or therapeutic agent according to Item 1, wherein the chloroquine derivative is hydroxychloroquine.
Item 3. Item 3. The preventive 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.
Section 4. Item 4. The preventive or therapeutic agent according to any one of Items 1 to 3, wherein the Gb3 accumulation-induced 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 cardiac rhythm.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a prophylactic or therapeutic agent for diseases caused by Gb3 accumulation, containing a low-molecular-weight compound as an active ingredient.

(a) shows the results of immunostaining of Fabry disease model cardiomyocytes (Example 1). LAMP1 shows the staining image of the lysosomal marker (LAMP1), mTOR shows the staining image of mTOR, and Merge shows the superimposed image of both images. -DOX/+DOX used cardiomyocytes obtained by culturing Fabry disease model iPSCs (Reference Example 2) in the presence (+DOX) or in the absence (-DOX) of DOX, followed by cardiomyocyte differentiation (Reference Example 3). HCQ indicates the case where hydroxychloroquine was added to the medium. (b) Shows the results of pulsation evaluation of Fabry disease model cardiomyocytes (Reference Example 4, Example 1). The vertical axis indicates the relaxation duration. In the horizontal axis, -D/+D are myocardium obtained by culturing Fabry disease model iPSCs (Reference Example 2) in the presence (+D) or in the absence of DOX (-D), followed by myocardial differentiation (Reference Example 3). HCQ indicates the case where hydroxychloroquine was added to the medium, Fz indicates the case when Fabrizyme was added to the medium, and Mock indicates the case when no drug was added. (c) shows the results of cell area measurement of Fabry disease model cardiomyocytes (Reference Example 4, Example 1). The vertical axis indicates cell area. In the horizontal axis, -D/+D are myocardium obtained by culturing Fabry disease model iPSCs (Reference Example 2) in the presence (+D) or in the absence of DOX (-D), followed by myocardial differentiation (Reference Example 3). Cells are used, HCQ indicates the case where hydroxychloroquine was added to the medium, and Mock indicates the case where no drug was added. (d) shows the results of pulsation evaluation of Fabry disease patient-derived cardiomyocytes (Example 1). The vertical axis indicates the relaxation duration. In the horizontal axis, HCQ indicates the case when hydroxychloroquine was added to the medium (values are concentrations in the medium (unit: μM)), Fz indicates the case when Fabrizyme was added to the medium, and Mock indicates the case when no drug was added. indicates

As used herein, the expressions "contain" and "include" include the concepts "contain", "include", "consist essentially of" and "consist only of".

In one aspect of the present invention, a Gb3 accumulation-causing The present invention relates to a prophylactic or therapeutic agent for sexually transmitted diseases (in this specification, it may be referred to as “the agent of the present invention”). This will be explained below.

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

The chloroquine derivative is not particularly limited, and known derivatives can be employed. Among chloroquine derivatives, preferably a derivative (derivative 1) 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, one of the dialkylamino groups of chloroquine and derivative 1 or derivatives in which two alkyl groups are substituted with hydroxy groups (derivative 2), more preferably derivative 2, and still more preferably hydroxychloroquine:

is mentioned.

The active ingredient of the present invention preferably includes chloroquine, hydroxychloroquine and the like, and more preferably includes hydroxychloroquine.

Salt forms are also included in the active ingredients of the present invention. Salts are not particularly limited as long as they are pharmaceutically acceptable salts, and both acid salts and basic salts can be employed. Examples of acid salts include mineral salts such as hydrochloride, hydrobromide, sulfate, nitrate, phosphate; acetate, propionate, tartrate, fumarate, maleate, apple organic acid salts such as acid salts, citrates, methanesulfonates, and paratoluenesulfonates; and amino acid salts such as aspartates and glutamates. 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, acid salts are preferred, inorganic acid salts are more preferred, and sulfates, phosphates, hydrochlorides and the like are more preferred.

The active ingredients of the present invention also include solvate forms. 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 one class of poisons or a combination of two or more.

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

Diseases caused by Gb3 accumulation include, for example, Fabry disease (preferably Fabry disease associated with heart disease) caused by accumulation of Gb3 due to GLA gene mutation. In addition, it has been reported that blood Gb3 concentration may increase even without GLA gene mutation, and that the increase is associated with heart disease. is also included. The Gb3 accumulation-induced disease is preferably a disease associated with cardiac rhythm abnormalities.

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 of only the active ingredient of the present invention, or may further contain other ingredients as necessary. may Other ingredients are not particularly limited as long as they are pharmaceutically acceptable ingredients. Other components include additives as well as components having pharmacological action. Examples of additives include bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, humectants, colorants, perfumes, A chelating agent and the like are included.

Subjects to which the drug of the present invention is applied are not particularly limited, but examples of mammals include humans, monkeys, mice, rats, dogs, cats, rabbits, pigs, horses, cows, sheep, goats, and deer.

The agent of the present invention can be in any dosage form, such as tablets (including orally disintegrating tablets, chewable tablets, effervescent tablets, lozenges, jelly drops, etc.), pills, granules, fine granules, powders, Oral dosage forms such as hard capsules, soft capsules, dry syrups, liquids (including drinks, suspensions, and syrups), jelly, and injection preparations (e.g., drip injections (e.g., intravenous drip preparations, etc.), intravenous injections, intramuscular injections, subcutaneous injections, intradermal injections), external preparations (e.g., ointments, poultices, lotions), suppository inhalants, eye preparations, eye ointments, drops Parenteral formulations such as nose drops, ear drops, and liposomes can be used.

The route of administration of the agent of the present invention is not particularly limited as long as the desired effect is obtained, and includes oral administration, enteral administration such as tube feeding and 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 mode of use, the target of application, the condition of the target of application, etc., and is not limited, but is for example 0.0001 to 100% by weight, preferably 0.001 to 50% by weight. %.

When the drug of the present invention is administered to animals, the dosage is not particularly limited as long as it is an effective amount that exhibits efficacy. 1000 mg/kg body weight, preferably 0.5-500 mg/kg body weight per day, and 0.01-100 mg/kg body weight per day, preferably 0.05-50 mg/kg body weight for parenteral administration . The above dosage can be adjusted appropriately according to age, condition and symptoms.

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

Reference example 1. Generation of iPSCs from AFD patients Hematopoietic cytokines (10 ng/ml IL-3 (R&D System), 100 ng/ml IL-6 (R&D Systems), 300 ng/ml SCF (R&D Systems), 300 ng/ml TPO (R&D Systems), 300 ng/ml Flt3L (308-FK, R&D)) in medium StemSpan ACF (Stem Cell Technologies) Selected and propagated by culturing for a week. The cultured cells were then reprogrammed into iPS cells (iPSCs) using episomal vectors under feeder-free conditions. Specifically, four pCXLE episomal non-integrating plasmids encoding Epstein-Barr nuclear antigen 1 (EBNA1) and shRNAs against human OCT4, SOX2, KLF4, LIN28, L-MYC and p53 were transfected into 500,000 cells. 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 of 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 of StemFit AK02N (Ajinomoto) was added twice to the culture every 2 days. All media were then supplemented with fresh StemFit AK02N every other day until iPSC colonies appeared. To establish stable cell lines, 10 colonies were picked and cultured for over 10 passages before analysis. GLA mutations in patient-specific iPSCs were confirmed by PCR amplification of the genomic region containing the mutation, followed by Sanger sequencing.

Reference example 2. Generation of Fabry disease model iPSCs A genome editing system called CRISPR interference (CRISPRi) was used for human iPSCs. In this system, a fusion protein of dead Cas9 (dCas9) with deficient nuclease activity and KRAB, a transcriptional repression domain, is injected onto the target gene using a single guided RNA (gRNA) that guides it to the transcription initiation region of the target gene. It is a system that localizes to the target gene and specifically suppresses the transcriptional expression of the target gene. Using a human iPSC strain (CRIPSRi iPSC strain) that can suppress the expression of dCas9-KRAB in a doxycycline (DOX)-dependent manner, gRNA targeting the GLA gene was introduced into the strain, and the expression of the GLA gene was suppressed in a DOX-dependent manner. Cells were generated that could be suppressed. Specifically, it was carried out as follows.

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

<Reference example 2-2. Design of gRNA and cloning into gRNA expression vector>
The sgRNA was designed around 200 bp upstream of the transcription start site (TSS) of the GLA gene. TSS locations were determined using the UCSC genome browser (https://genome.ucsc.edu). gRNA oligos were 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. gRNA Nucleofection and Selection of Stable CRISPRi Clones>
sgRNA expression vectors were transfected into well-characterized CRIPSRi iPSC lines that have a normal karyotype and differentiate into three germ layers in vitro and in teratomas, followed by treatment of transfected iPSCs with DOX. . Five to six days after initiation of DOX treatment, RNA was harvested from cells and examined for GLA expression by RT-qPCR. Based on the obtained expression level, we established a cell line (GLA CRISPRi iPSC line) that can efficiently suppress the expression of GLA in a DOX-dependent manner.

Reference example 3. Myocardial differentiation from iPSCs and myocardial purification purification
1 ml of StemFit AK02N containing Y-27632 and iMatrix-511 silk was seeded in a 12-well plate, and iPSCs were seeded there at 40,000 to 120,000 cells. After that, it was cultured for 3-4 days until the cell density reached 70-80%. Subsequently, cells were treated with 6-12 μM CHIR99021 for 24 hours to initiate myocardial differentiation. Treatment with CHIR99021 was followed 48 hours by treatment with 5 μM IWP2 for 2 days. As the basal medium, RPMI1640 medium without insulin was used until day 6 of differentiation, and RPMI1640 medium containing insulin was used until day 15 thereafter. On day 15 of differentiation, the cells that had differentiated into myocardium were detached with 0.25% trypsin. After cells were suspended 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 iPSCs was purified using lactic acid medium. A lactic acid medium was prepared by adding 4 mM lactic acid, Glutamax, and non-essential amino acids to a glucose-free DMEM medium. The medium was exchanged every other day for 5 days to purify the myocardium.

Reference example 4. Evaluation of Fabry disease model cardiomyocytes Fabry disease model iPSCs (Reference example 2) were cultured in the presence of DOX and myocardial differentiation (Reference example 3) was performed to obtain Fabry disease model cardiomyocytes. Quantification of movement was performed. Specifically, it was carried out as follows.

Myocardial beating was measured using a Sony SI8000 video microscope. Video images of human iPSC-derived myocardial beats were captured at a frame rate of 150 fps, with a resolution of 2048x2048 pixels and a 10x lens for 10 seconds. The Sony SI8000 analysis software analyzes the vectors obtained from all myocardial contraction movements captured in 10 seconds, and then converts the average vector length per unit time into a time-motion velocity plot to obtain functional parameters. (Contraction speed, relaxation speed, contraction duration, relaxation duration, contraction distance, relaxation distance, contraction-relaxation duration) were analyzed.

As a result, it was confirmed that cardiomyocytes with accumulated Gb3 (Fabry disease model cardiomyocytes) due to DOX-induced suppression of GLA expression exhibited abnormalities in cardiomyocyte contraction and relaxation (Fig. 1b). .

Furthermore, the obtained cells were fluorescently stained using an antibody against actinin that is specifically expressed in myocardium, and then observed using a fluorescence microscope. On the plate, a plurality of myocardial cells were aggregated to form clumps, and some myocardial cells were dissociated from the clumps and existed alone. The cell area of singly present cardiomyocytes was analyzed using image analysis software Image J. As a result, it was revealed that the myocardial area was expanded by suppressing GLA expression (Fig. 1c).

Example 1. Evaluation of therapeutic effect on Gb3 accumulation-induced abnormalities The effect of hydroxychloroquine on Gb3 accumulation-induced abnormalities in cardiomyocytes was investigated. Specifically, it was carried out as follows.

<Example 1-1. Immunostaining>
Fabry disease model iPSCs (Reference Example 2) were cultured in the presence or absence of DOX, and cardiomyocytes were differentiated (Reference Example 3). Hydroxychloroquine was added to the culture medium (concentration in the medium: 0.3 μM). After 4 to 5 days, the lysosomal marker (LAMP1) and mTOR were immunostained. Specifically, it was carried out as follows. Cardiomyocytes were 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% skimmed milk in PBS for 30 minutes at room temperature. did. Cells were reacted with anti-LAMP1 and anti-mTOR antibodies. After washing twice with PBS, cells were briefly incubated with 2.5% skimmed milk in PBS. Cells were then stained with a secondary antibody.

<Example 1-2. Beating evaluation of Fabry disease model cardiomyocytes>
Cardiomyocyte pulsation obtained by culturing Fabry disease model iPSCs (Reference Example 2) in the absence of DOX and causing cardiomyocyte differentiation (Reference Example 3), and Fabry disease model iPSCs (Reference Example 2) in the presence of DOX Add hydroxychloroquine or Fabrizyme (concentration in medium: hydroxychloroquine 0.3 μM, Fabrizyme 5 μg/ml) to cardiomyocytes obtained by culture and myocardial differentiation. It was measured in the same manner as in Reference Example 4.

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

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

<Example 1-5. Results>
The results of immunostaining are shown in FIG. 1a, the results of cell area measurements are shown in FIG. 1c, and the results of pulsatile assessment are shown in FIGS. 1b and 1d. Cardiomyocytes with DOX-induced repression of GLA showed decreased localization of mTOR in lysosomes as a result of immunostaining, whereas hydroxychloroquine treatment increased their colocalization (Fig. 1a). In both cases of myocardial differentiation of Fabry disease model iPSCs (Reference Example 2) and myocardial differentiation of iPSCs derived from Fabry disease patients (Reference Example 1), abnormal beating (increased Relaxation Duration) was observed. , improved by hydroxychloroquine (Figs. 1b and 1d). The degree of improvement was similar to that of Fabrizyme, an existing therapeutic agent. Furthermore, myocardial hypertrophy was dependent on GLA expression suppression. This hypertrophy was suppressed by hydroxychloroquine treatment (Fig. 1c).

Claims (4)

A prophylactic or therapeutic agent for diseases caused by Gb3 accumulation, containing hydroxychloroquine. 2. The prophylactic or therapeutic agent according to claim 1, wherein the Gb3 accumulation-induced disease is at least one disease selected from the group consisting of Fabry disease and Gb3 accumulation-induced heart disease. 3. The preventive or therapeutic agent according to claim 1 or 2, wherein the Gb3 accumulation-induced disease is Fabry disease. The prophylactic or therapeutic agent according to any one of claims 1 to 3, wherein the Gb3 accumulation-induced disease is a disease accompanied by Gb3 accumulation-induced cardiac rhythm abnormality.

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