JP7264452B2 - Pharmaceutical composition for the treatment or prevention of lysosomal storage diseases - Google Patents

Description

The present invention relates to pharmaceutical compositions for treating or preventing lysosomal storage diseases.

Lysosomal storage disease Lysosomes decompose carbohydrates and glycolipids in cells, and it is believed that about 60 types of hydrolases are involved in this decomposition. Lysosomal storage disease is a disease in which the degradative function of lysosomes is not exerted due to deficiency or abnormality of hydrolase, and substances that should be degraded accumulate in cells as foreign substances. About 30 types of lysosomal storage diseases are currently known, with different disease names and symptoms depending on the deficient enzyme. Examples of lysosomal storage diseases include sphingolipidosis, a type in which sphingolipids accumulate, and examples of sphingolipidosis include Niemann-Pick disease, GM1 gangliodosis, GM2 gangliodosis, and the like.

Niemann-Pick disease is a congenital metabolic disorder in which insoluble metabolites that are supposed to be decomposed accumulate in cells due to enzyme abnormalities caused by congenital gene mutations. It is roughly classified into two types, A type, B type, and C type, depending on the gene that mutates. Niemann-Pick disease types A and B are caused by mutations in a gene called SMPD1. In types A and B, sphingomyelin, a type of sphingolipid, mainly accumulates due to an abnormality in acid sphingomyelinase.

Niemann-Pick disease type C (NPC) is a recessive genetic disease caused by mutations in the NPC1 or NPC2 genes. Normally, free cholesterol in lysosomes is transported to the endoplasmic reticulum by the action of NPC1 and NPC2, where it is converted into ester-type cholesterol. In NPC cells, NPC protein dysfunction results in accumulation of free cholesterol and depletion of cholesterol esters (ie, cholesterol imbalance). As a result, lysosomes containing free cholesterol become stagnated and deposited, and cellular functions such as autophagy, which are important for maintaining vital activities, are significantly reduced. NPC lesions are particularly conspicuous in the central nervous system, exhibiting impaired motor function and poor prognosis.

Treatment of Niemann-Pick Disease Type C Until recently, no therapeutic drug has been found that is highly effective against NPC. Since 2009, 2-hydroxypropyl-β-cyclodextrin (HPBCD), a cyclic oligosaccharide, has been administered to NPC patients for humane use, has proven to have some therapeutic effect, and is currently on the III A phased clinical trial is underway. JP 2018-123153 (Patent Document 1) describes compositions based on methylcyclodextrin for treating and/or preventing diseases by increasing HDL cholesterol levels. The invention described in the document includes lysosomal storage diseases (sometimes referred to as “lysosomal storage diseases”) as target diseases. US Pat. No. 6,200,401 describes that a composition based on methylcyclodextrin may contain cyclodextrin substituted with hydroxypropyl groups, in particular β-cyclodextrin, ie HPBCD (patent Paragraphs 0084 and 0086 of claim 10, claim 12, etc. of Document 1).

However, it has been reported that administration of 2-hydroxypropyl-β-cyclodextrin (HPBCD) causes serious adverse events such as lung injury due to HPBCD administration. Furthermore, hearing impairment is seen in multiple patients in clinical trials and is regarded as a problem. At present, HPBCD is one of the most promising therapeutic drug candidates for NPC, but there is a demand for the development of a therapeutic drug that has equivalent therapeutic effects and is safer.

Retable 2015/083736 describes pharmaceutical compositions for the treatment or prevention of lysosomal storage diseases, characterized in that they contain hydroxypropyl-γ-cyclodextrin (HPGCD) as an active ingredient. However, for example, Annals of Clinical and Translational Neurology 2016, 3(5), p. 366-380 reported that HPGCD, like HPBCD, can also cause ototoxicity. In addition, HPGCD has the problem that it has a high viscosity in a high-concentration solution and requires a highly difficult and advanced synthesis technique in order to achieve a uniform degree of substitution with hydroxypropyl.

"Grants-in-Aid for Scientific Research Research Results Report Institution No. 34517 Substrate Research (C) (General) Project No. 26505012" describes the research results report on the development of maltosyl-β-cyclodextrin to treat Niemann-Pick type C disease. there is In the report of the research results, it was reported that treatment of NPC1-deficient cells with maltosyl-β-cyclodextrin (G2BCD) reduced intracellular cholesterol in a concentration-dependent manner with G2BCD, and also reduced lysosomal cholesterol in a time-dependent manner. It is However, the effect and safety of applying G2BCD to the living body have not been reported and are unknown. Development of a highly safe and practical therapeutic agent for lysosomal storage diseases containing NPC is desired.

JP 2018-123153 Re-table 2015/083736 Aanals of Clinical and Translational Neurology 2016, 3(5), p. 366-380 Shiraishi et al., Agric. Biol. Chem. , 1989, 53(5), p. 2181-2188 Abe et al., Crabohydrate Research. , 1988, 176, p. 87-95 Okada et al., Chem. Pharm. Bull. 1988, 36(6), p. 2176-2185 Comprehensive Supramolecular Chemistry, Volume 3 CYCLODEXTRINS, Pergamon Press, 1996, p. 127-138 KAKENHI Grants-in-Aid for Scientific Research Report on Research Results Organization No. 34517 Substrate Research (C) (General) Project No. 26505012

An object of the present invention is to provide novel pharmaceutical compositions for the treatment or prevention of lysosomal storage diseases.

The present inventors evaluated the biocompatibility of various cyclodextrin derivatives and their therapeutic effects on lysosomal storage diseases (especially NPC) in order to solve the above problems. As a result, it was clarified that 6-O-maltosyl-γ-cyclodextrin (G2GCD) ameliorated impaired intracellular cholesterol transport (accumulation of free cholesterol and depletion of ester cholesterol) in NPC model cells. In addition, it was found that when G2GCD was intracerebroventricularly administered to NPC model mice, it exhibited a remarkable life-extending effect similar to HPBCD. The inventors further found that G2GCD is less cytotoxic and less ototoxic compared to HPBCD and G2BCD. As a result of intensive research, the inventors have conceived the present invention based on the above findings.

The invention includes, but is not limited to, the following aspects.
[Aspect 1]
A pharmaceutical composition for treating or preventing lysosomal storage diseases, comprising 6-O-maltosyl-γ-cyclodextrin.
[Aspect 2]
The pharmaceutical composition according to aspect 1, wherein the lysosomal storage disease is Niemann-Pick disease.
[Aspect 3]
The pharmaceutical composition according to aspect 1 or aspect 2, wherein the lysosomal storage disease is Niemann-Pick disease type C.
[Aspect 4]
A pharmaceutical composition comprising 6-O-maltosyl-γ-cyclodextrin that inhibits lysosomal accumulation and/or impairment of the autophagy pathway.
[Aspect 5]
The pharmaceutical composition according to any one of aspects 1-4, which is administered parenterally.
[Aspect 6]
The pharmaceutical composition according to any one of aspects 1-5, which is administered as an injection.

The composition of the present invention containing 6-O-maltosyl-γ-cyclodextrin (G2GCD) is effective in treating or preventing lysosomal storage diseases, particularly Niemann-Pick disease type C. G2GCD is less cytotoxic and less ototoxic than conventional cyclodextrin derivatives such as HPBCD and G2BCD, and is useful.

FIG. 1 shows the results of investigating the effects of various cyclodextrin derivatives on abnormal intracellular cholesterol transport in NPC model cells. FIG. 1A shows the amount of free cholesterol per 1 mg protein of cells, and FIG. 1B shows the amount of ester-type cholesterol per 1 mg protein of cells. FIG. 2 shows the results of examining the effects of various cyclodextrin derivatives on lysosomal volume and autophagosome volume abnormalities in NPC model cells. The vertical axis is % when the lysosome volume (FIG. 2A) and autophagosome volume (FIG. 2B) of wild-type cells (CHO cells) are set to 100. FIG. 3 shows the results of examining the effects of various cyclodextrin derivatives on cell viability of wild-type cells and NPC model cells. The vertical axis indicates the % when each cyclodextrin derivative was added at the concentration indicated on the horizontal axis, with respect to the cell survival rate when no cyclodextrin derivative was added as 100%. FIG. 3A shows the results for wild-type cells, and FIG. 3B shows the results for NPC1 model cells. ▲: G2GCD, ■: G2BCD, ●: HPBCD FIG. 4 shows the results of examining the life prognosis improving effect of various cyclodextrin derivatives in NPC model mice. △: G2GCD, ■: HPBCD FIG. 5 shows the results of in vitro evaluation of the ototoxicity of various cyclodextrin derivatives by investigating the effect of adding various cyclodextrin derivatives on cell viability of mouse inner ear hair cell model cell HEI-OC1. The vertical axis represents the % of the concentration of each cyclodextrin derivative added on the horizontal axis, relative to the cell viability of 100% when no cyclodextrin derivative was added. :HP BCD FIG. 6 shows the results of examining the effects of G2GCD and HPBCD administration on the hearing threshold of wild-type mice. The horizontal axis of FIG. 6 indicates frequency (kHz), and the vertical axis indicates sound pressure threshold (db) at each frequency. ▲: G2GCD, ●: HPBCD

1. Pharmaceutical Compositions The present invention relates to pharmaceutical compositions for the treatment or prevention of lysosomal storage diseases.

The pharmaceutical composition comprises 6-O-maltosyl-γ-cyclodextrin (G2GCD).

6-O-maltosyl-γ-cyclodextrin (G2GCD) has the structure:

Ｇ２ＧＣＤは分岐されたシクロデキストリン類であり、例えば、マルトースおよびγ－シクロデキストリンから、Ｋｌｅｂｓｉｅｌｌａ ｐｎｅｕｍｏｎｉａｅ由来のプルラナーゼまたはＰｓｅｕｄｏｍｏｎａｓのイソアミラーゼの逆反応（ｒｅｖｅｒｓｅ ｒｅａｃｔｉｏｎ）によって、合成することができる（例えば、Ｓｈｉｒａｉｓｈｉら、Ａｇｒｉｃ．Ｂｉｏｌ．Ｃｈｅｍ．，１９８９、５３（５）、ｐ．２１８１－２１８８；Ａｂｅら、Ｃｒａｂｏｈｙｄｒａｔｅ Ｒｅｓｅａｒｃｈ．，１９８８，１７６、ｐ．８７－９５等を参照）。Ｇ２ＧＣＤはマルトシル基の置換数を容易に制御することができ、単離も容易である。一方、ＨＰＢＣＤ及びＨＰＧＣＤは、ヒドロキシプロピル基の置換数を制御すること及び単離精製を行うことは困難であり、ヒドロキシプロピル基の置換数が異なる混合物として用いざるを得ない。

G2GCD is a class of branched cyclodextrins, which can be synthesized, for example, from maltose and γ-cyclodextrin by the reverse reaction of pullulanase from Klebsiella pneumoniae or isoamylase of Pseudomonas (e.g., Shiraishi et al. Chem., 1989, 53(5), pp. 2181-2188; Abe et al., Crabohydrate Research., 1988, 176, pp. 87-95, etc.). G2GCD can easily control the number of substitutions of maltosyl groups, and can be easily isolated. On the other hand, HPBCD and HPGCD are difficult to control the number of hydroxypropyl group substitutions and difficult to isolate and purify, and must be used as a mixture with different numbers of hydroxypropyl group substitutions.

The chemistry of G2GCD is described, for example, in Okada et al., Chem. Pharm. Bull. 1988, 36(6), p. 2176-2185; Comprehensive Supramolecular Chemistry, Volume 3 CYCLODEXTRINS, Pergamon Press, 1996, p. 127-138, etc. Aqueous solutions of G2GCD have significantly lower surface activity and viscosity compared to HPBCD.

"Lysosomal storage disease" is a general term for diseases in which substances to be decomposed accumulate in cells due to deficiency of hydrolase associated with lysosomes. About 30 kinds of lysosomal storage diseases have different disease names and symptoms depending on the defective enzymes, and about 30 kinds of diseases are currently known, and they are designated as intractable diseases. Examples of lysosomal storage diseases include sphingolipidosis, a type in which sphingolipids accumulate, glycogen storage disease type II, a type in which glycogen accumulates, mucopolysaccharidosis, a type in which mucopolysaccharides accumulate, and abnormal glycoprotein metabolism. including type of disease. Other lysosomal storage diseases whose precise classification is under investigation include sialidosis and galactosialidosis.

Examples of sphingolipidosis include Niemann-Pick disease, GM1 gangliodosis, GM2 gangliodosis. Metachromatic leukodystrophy, Fabry disease, Farber disease, Gaucher disease, etc.

Glycogen storage disease type II includes Pompe disease. Dysglycoprotein metabolism types include mucolipidosis.

Niemann-Pick disease, included in sphingolipidosis, is a congenital metabolic disorder in which insoluble metabolites that are supposed to be decomposed accumulate in cells due to enzyme abnormalities caused by congenital gene mutations. It is roughly classified into two types, A type, B type, and C type, depending on the gene that mutates. Niemann-Pick disease types A and B are caused by mutations in a gene called SMPD1. In types A and B, sphingomyelin, a type of sphingolipid, mainly accumulates due to an abnormality in the acid sphingomyelinase enzyme. Type A (acute neuronal form) is a severe form with a sphingomyelinase enzymatic activity of 5% or less. Type B is characterized by a sphingomyelinase enzyme activity of 5% or more and no neurological symptoms.

Niemann-Pick disease type C (NPC) is a recessive genetic disease caused by mutations in the NPC1 or NPC2 genes. NPC1 type caused by NPC1 mutation and NPC2 type caused by NPC2 mutation are known. Normally, free cholesterol in lysosomes is transported to the endoplasmic reticulum by the action of NPC1 and NPC2, where it is converted into ester-type cholesterol. In NPC cells, NPC protein dysfunction results in accumulation of free cholesterol and depletion of cholesterol esters (ie, cholesterol imbalance).

In one non-limiting aspect, the lysosomal storage disease targeted by the pharmaceutical composition is Niemann-Pick disease. In one aspect, the lysosomal storage disease is Niemann-Pick disease type C.

The present invention also relates to pharmaceutical compositions comprising 6-O-maltosyl-γ-cyclodextrin (G2GCD) that inhibit lysosomal accumulation and/or impairment of the autophagy pathway.

"Suppress lysosomal accumulation" means, for example, when administered to NPC model cells (for example, at a concentration of 1 mM), the accumulation of free cholesterol in the cells decreases, preferably, compared to when not administered. It means a decrease of 10% or more, a decrease of 20% or more, a decrease of 30% or more, a decrease of 40% or more, a decrease of 45% or more, and a decrease of about 50%. Alternatively, "suppressing lysosomal accumulation" means, for example, when administered to NPC model cells (for example, at a concentration of 1 mM), compared to when not administered, intracellular ester cholesterol increases, preferably , 1.1 times or more, 1.3 times or more, 1.5 times or more, 1.8 times or more, 2 times or more, 2.2 times or more, 2.4 An increase of more than a factor of 2 means an increase of about 2.5 times. Alternatively, "suppressing lysosomal accumulation" means, for example, when administered to NPC model cells (for example, at a concentration of 1 mM), the lysosomal volume is reduced, preferably 5% or more, compared to when not administered. It means to decrease, decrease by 10% or more, decrease by 15% or more, decrease by 20% or more, decrease by 25% or more, decrease by 30% or more, decrease by about 33%. In one aspect, the pharmaceutical composition functions to "inhibit lysosomal accumulation" to the same extent as a pharmaceutical composition comprising the same concentration of 2-hydroxypropyl-β-cyclodextrin (HPBCD) or maltosyl-β-cyclodextrin (G2BCD). play.

"Suppressing the impairment of the autophagy pathway" means that when administered to NPC model cells (for example, at a concentration of 1 mM), the autophagosome volume is reduced, preferably by 5% or more, compared to when it is not administered. decrease by 10% or more, decrease by 15% or more, decrease by 20% or more, decrease by 25% or more, decrease by about 30%. In one aspect, the pharmaceutical composition "suppresses impairment of the autophagy pathway" to the same extent as a pharmaceutical composition comprising the same concentration of 2-hydroxypropyl-β-cyclodextrin (HPBCD) or maltosyl-β-cyclodextrin (G2BCD). "Do" function.

G2GCD has been shown to have low cytotoxicity and good prognosis in NPC model mice. Non-limitingly, "low cytotoxicity" means, for example, HPBCD and G2BCD in vitro at a concentration such that the cell viability is 0%, even if administered to wild-type cells or NPC model cells, 30 % or higher, 40% or higher, 50% or higher, or 60% or higher survival rate.

G2GCD is clearly less toxic than HPBCD, HPGCD, and G2BCD, which are particularly problematic in terms of ototoxicity. Non-limitingly, "low ototoxicity" means that, for example, in HPBCD and G2BCD in vitro, a concentration such that the cell viability is 0%, even if administered to wild-type ear model cells, 40 % or higher, 50% or higher, 60% or higher, or 70% or higher survival rate. Alternatively, "low ototoxicity" means, for example, that the effect of administration on hearing threshold is lower than that of HPBCD, HPGCD, and G2BCD, preferably in a wide range of frequencies (low to high range), and significantly lower sound pressure thresholds. means to indicate

2. Mode of administration The mode of administration of the pharmaceutical composition of the present invention is not particularly limited.

6-O-maltosyl-γ-cyclodextrin (G2GCD) can be administered in the form of a pharmaceutical composition, optionally in combination with a pharmaceutically or pharmacologically acceptable carrier. One or more types of pharmaceutically or pharmacologically acceptable carriers may be used. "Pharmaceutically" or "Pharmaceutically Acceptable Carrier" means any carrier, diluent or excipient that is compatible with the other ingredients of the formulation and not deleterious to the subject. Based on the disclosure herein, it is within the skill of the art to formulate pharmaceutical compositions comprising G2GCD.

For example, pharmaceutical compositions can be formulated according to standard techniques in the pharmaceutical arts. See, eg, Alfonso Gennaro, ed. , Remington's Pharmaceutical Sciences, 18th Ed. , (1990) Mack Publishing Co.; , Easton, Pa.

The administration target of the pharmaceutical composition is not particularly limited. Non-limitingly, in one aspect, the subject is a human. Alternatively, the subject is a non-human mammal (e.g., rabbit, guinea pig, rat, mouse or other rodent (including any animal within the order Rodentia), cat, dog, pig, sheep, goat, bovine (cattle, for example, dairy cows or any animal within the order Bovida), horses (including any animal within the order Equidae), donkeys, and non-human primates).

The administration route is not particularly limited. Parenteral administration such as intravenous administration such as intravenous infusion or injection, transdermal administration such as a patch, and intraperitoneal administration may be used. In addition, administration by intramuscular administration such as intramuscular injection, enteral administration, topical administration and the like is also possible. In one aspect, the pharmaceutical composition is administered as an injection.

For injections, for example, pharmaceutical compositions containing G2GCD are dissolved in aqueous injections together with dispersants, preservatives, tonicity agents, etc., or in vegetable oils such as olive oil, sesame oil, cottonseed oil, corn oil, propylene glycol, etc. , by suspending or emulsifying and molding as an oily injection.

External preparations are produced, for example, by making a pharmaceutical composition containing G2GCD into a solid, semi-solid or liquid composition. For example, the above-mentioned solid composition is produced by powdering the pharmaceutical composition as it is or by adding and mixing an excipient, a thickening agent, and the like. The above-mentioned liquid composition is produced by making an oily or aqueous suspension in almost the same manner as for injections. Semi-solid compositions are preferably in the form of aqueous or oily gels or ointments. Any of these compositions may also contain buffering agents, preservatives and the like. External preparations include, for example, creams, lotions, gels, ointments and the like for topical administration.

A pharmaceutical composition comprising G2GCD may optionally further comprise one or more other active ingredients. The “other active ingredient” may be an ingredient that has the function of treating or preventing lysosomal storage diseases, or suppressing lysosomal accumulation and/or disorders of the autophagy pathway, similar to G2GCD. Alternatively, it may be a component having functions other than these.

Pharmaceutical compositions comprising G2GCD may further contain stabilizers, antioxidants and preservatives, if desired. Suitable antioxidants include, for example, sulfites, ascorbic acid, citric acid and its salts, and sodium EDTA. Suitable preservatives include, for example, benzalkonium chloride, methyl- or propyl-paraben, and chlorbutanol.

The amount and duration of administration of the pharmaceutical composition will depend on the individual patient, including the size, mass, age and sex of the subject being administered, the nature and stage of the disease being treated, the aggressiveness of the disease, the route of administration, and the specific toxicity of radiation. determined by the particular circumstances of Dosages and durations may also be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data. Concentration ranges set forth herein are for exemplary purposes only and are not intended to limit the scope or practice of the claimed compositions.

For example, the dosage in the pharmaceutical composition is about 1 to about 100 mg/kg/day, more preferably about 5 to about 80 mg/kg/day of the active ingredient G2GCD. A dose of about 15 to about 50 mg/kg/day, such as about 35 mg/kg/day (eg, 2.1 g for an adult weighing 60 kg) is a particularly suitable dose. The pharmaceutical composition may be administered once daily, or may be divided into a number of lower doses administered simultaneously or at intervals of time. The dose may be given in multiple doses, eg two doses of 20 mg/kg. Higher or lower doses are possible.

The pharmaceutical composition of the present invention, especially the pharmaceutical composition containing G2GCD, is excellent in safety and can be administered for a long period of time. The lysosomal storage diseases targeted by the pharmaceutical composition of the present invention are hereditary diseases, and administration is often required as long as the patient lives.

The pharmaceutical compositions of the present invention are particularly well suited for such uses due to their superior safety. The period during which the medicament of the present invention can be administered is not particularly limited. can be administered as

3. Treatment or prevention method, use for manufacturing pharmaceutical composition concerning methods of prevention.

The invention further relates to the use of 6-O-maltosyl-γ-cyclodextrin (G2GCD) for the manufacture of a pharmaceutical composition for the treatment or prevention of lysosomal storage diseases.

The definitions of "6-O-maltosyl-γ-cyclodextrin (G2GCD)", "lysosomal storage disease" and the like are as described above.

EXAMPLES The present invention will be described in detail below based on examples, but the present invention is not limited to these examples. A person skilled in the art can easily make modifications and changes to the present invention based on the description of this specification, and they are included in the technical scope of the present invention.

Example 1 Effects of Various Cyclodextrin Derivatives on Abnormal Intracellular Cholesterol Transport in NPC Model Cells In this example, the effects of various cyclodextrin derivatives on abnormal intracellular cholesterol transport in NPC model cells were examined.

Cyclodextrin derivatives 6-O-maltosyl-γ-cyclodextrin (G2GCD) and 6-O-maltosyl-β-cyclodextrin (G2BCD) are described by Shiraishi et al., Agric. Biol. Chem. , 1989, 53(5), p. 2181-2188, using Klebsiella pneumoniae-derived pullulanase (manufactured by Amano Enzyme Co., Ltd.). The same applies to the following examples.

HPBCD was synthesized by reacting propylene oxide and β-cyclodextrin under alkaline conditions and adding a 2-hydroxypropyl group to the hydroxyl group of β-cyclodextrin.

Retable 2015/083736 discloses NPC-iPS cell lines generated from patients with NPC1 mutations. NPC-derived iPS cells exhibit an NPC phenotype and provide an in vitro system for screening drug candidates for the treatment of NPC. The extreme cholesterol accumulation observed in NPC iPS cell-derived HLCs allows it to be used to examine the effects of various drug treatments on this process. NPC-derived iPS cells were used as NPC model cells (Npc1-deficient CHO cells).

For NPC model cells, 1 mM each of G2GCD, G2BCD and HPBCD was added, and after 24 hours, the amounts of free cholesterol and ester cholesterol nmol per 1 mg protein in the cells were measured. Specifically, cells were treated with or without G2BCD for 24 hours and then lysed. Protein concentrations in the lysates were determined by the Pierce BCA protein assay kit (Thermo Fisher Scientific Inc.). Cholesterol was then extracted from the lysates using a mixture of chloroform, 2-propanol and NP-40 (7:11:0.1). Samples were centrifuged (15,000 xg for 10 minutes at 4°C) and the chloroform layer was collected. The chloroform layer was evaporated and the residue dissolved in a 2-propanol, polyoxyalkylene alkyl ether, and polyoxylauryl ether mixture (87:10:3). A portion of the solution was incubated with esterase to measure total cholesterol and another portion was incubated without esterase to measure free cholesterol. Total cholesterol levels were measured by a Determiner L FC (Kyowa Hakko Kirin Co., Ltd., Tokyo, Japan) using a microplate reader (Tecan Co., Ltd., Mennedorf, Switzerland). Cholesterol esters were calculated by subtracting free cholesterol levels from total cholesterol levels. Intracellular cholesterol levels were expressed as nmol/mg protein.

The results are shown in FIG. In the NPC model cells, a significant increase in free cholesterol and a decrease in ester cholesterol were observed as compared with wild-type cells (CHO cells), indicating that intracellular cholesterol disorder occurred. On the other hand, HPBCD, G2BCD and G2GCD (1 mM) showed remarkable improvement effects. Specifically, administration of HPBCD, G2BCD and G2GCD (1 mM) reduced the intracellular accumulation of free cholesterol by about 50% compared to the case of no administration. In addition, intracellular ester cholesterol increased by about 2.4 to 2.5 times compared to the case without administration.

Mean±SEM (N=3)
^** p<0.01 compared to Npc1 null group
Example 2 Effect of Various Cyclodextrin Derivatives on Lysosome and Autophagosome Volume Abnormalities in NPC Model Cells
In this example, the effects of various cyclodextrin derivatives on lysosomal and autophagosome volume abnormalities in NPC model cells were investigated.

For NPC model cells, 1 mM each of G2GCD, G2BCD and HPBCD was added, and the lysosome volume and autophagosome volume were measured 24 hours later. Specifically, cells were stained with LysoTracker Green® DND-26 for 15 minutes at 37° C. (for lysosomal volume measurements) or with Cyto-ID® for 20 minutes at 37° C. (for aurophagosome for volumetric measurements). Analysis was performed on a flow cytometer (BD Biosciences Accuri™ C6, Becton Dickinson Biosciences, Franklin Lakes, USA) using a 488 nm laser. The resulting fluorescence was detected in the FL1 channel using a 530±30 nm filter. Data from 10,000 cells were collected and analyzed using the attached software.

The results are shown in FIG. The vertical axis is % when the lysosome volume and autophagosome volume of wild-type cells (CHO cells) are taken as 100. In NPC model cells, significant increases in lysosome and autophagosome volumes were observed compared to wild-type cells, indicating intracellular lysosome accumulation and impaired autophagy pathway (impaired autolysosome formation). On the other hand, G2GCD, G2BCD and HPBCD (1 mM) showed remarkable improvement effects. Specifically, administration of G2GCD, G2BCD and HPBCD (1 mM) all decreased lysosomal volume by about 33% compared to no administration. Also, autophagosome volume decreased by about 30% compared to the case without administration.

Mean±SEM (N=3)
^** p<0.01 compared to Npc1 null group
Example 3 Effect of addition of various cyclodextrins on cell viability of wild-type cells and NPC model cells
In this example, the effect of adding various cyclodextrins on the cell viability of wild-type cells and NPC model cells was investigated.

0 mM-100 mM G2GCD, G2BCD and HPBCD were administered to wild-type CHO cells and NPC model cells, and cell viability after 12 hours was assessed by the WST-8 assay, which is an indicator of live cytotoxic cells.

The reduction reaction of the tetrazolium salt occurs when the reductase is not inactivated. Therefore, this reduction reaction is often used to measure living cells. An example of a tetrazolium salt used in this reaction is WST (Water soluble Tetrazolium salts), particularly WST-8(2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5- (2,4-disulfophenyl)-2H-tetrazolium) is known. WST-8 is reduced by intracellular dehydrogenases to produce water-soluble formazan. Since the number of cells and the amount of formazan produced are in a linear proportional relationship, it is possible to easily count the number of living cells by directly measuring the absorbance of formazan at 450 nM to 460 nM. In this example, a kit called Cell Counting Kit-8 from Dojindo Laboratories was used.

The results are shown in FIG. G2GCD was shown to be less cytotoxic compared to HPBCD and G2BCD in both wild-type CHO cells and NPC model cells at all concentrations tested up to 100 mM.

Mean±SEM (N=3)
^** p<0.01 compared to Npc1 null group
Example 4 Life prognosis improving effect of HPBCD and G2GCD in NPC model mice
In this example, the life prognosis improving effect of HPBCD and G2GCD in NPC model mice was examined.

Model mice (NPC mice) are deficient in transport of cholesterol from lysosomes to the ER due to a spontaneous mutation in the Npc1 gene. This mouse model also exhibits symptoms similar to those of human disease with cholesterol accumulation in the liver and brain. This model mouse exhibits liver damage and neurological dysfunction and dies at 12 weeks of age without treatment.

4-week-old NPC mice (N = 6) (Director of Sanin Rosai Hospital (former Professor of Department of Neuropediatrics, Faculty of Medicine, Tottori University) Dr. Kosaku Ohno, Dr. Katsumi Higaki, Associate Professor of Research Platform Center, Organization for Research Promotion, Tottori University A single intracerebroventricular injection of HPBCD and G2GCD at 30 and 35 mg/kg (both 21.4 μmol/kg in terms of moles), respectively, was administered to the animals bred at the Kumamoto University Life Resources Research and Support Center). were performed and the survival times of the mice were compared.

The results are shown in FIG. It was revealed that G2GCD has the same prognostic effect as HPBCD.

Example 5 Effect of Addition of Various Cyclodextrin Derivatives on Cell Viability of Mouse Inner Ear Hair Cell Model Cells HEI-OC1 The effect of addition of dextrin derivatives was investigated and the ototoxicity of various cyclodextrin derivatives was evaluated in vitro.

Mouse inner ear hair cell model cell HEI-OC1 (cells established by Professor Federico Kalinec of David Geffen School of Medicine, University of California, Los Angeles) was co-applicant Dr. Toru Miwa, Department of Otolaryngology, Head and Neck Surgery, Kumamoto University School of Medicine ) was cultured at 33° C. and 10% CO ₂ conditions. G2GCD, G2BCD and HPBCD were added to HEI-OC1 cells at respective concentrations shown in FIG. 5, and cell viability was evaluated 12 hours later by WST-8 assay, which is an indicator of viable cytotoxic cells.

The results are shown in FIG. At all concentrations tested up to 100 mM, G2GCD was shown to be less cytotoxic to ear model cells compared to HPBCD and G2BCD.

Mean±SEM (N=3)
Example 6 Effects of G2GCD and HPBCD Administration on Hearing Thresholds in Wild-Type Mice
In this example, the effects of G2GCD and HPBCD administration on hearing thresholds in wild-type mice were investigated. G2GCD and HPBCD were given a single subcutaneous dose of 5.7 mmol/kg to 8-week-old male wild-type mice. Seven days after administration, auditory brain-stem response (ABR) was measured.

The results are shown in FIG. G2GCD exhibited significantly lower sound pressure thresholds at all frequencies examined compared to HPBCD. That is, it was suggested that G2GCD is less ototoxic than HPBCD.

Mean±SEM (N=7)
^* p<0.051 compared to HPBCD group
^** p<0.01 compared to HPBCD group

The compositions of the invention, including G2GCD, are useful for treating or preventing lysosomal storage diseases, particularly Niemann-Pick disease type C. Aqueous solutions of G2GCD have remarkably low surface activity and viscosity compared to HPBCD, and have excellent chemical properties assuming injections for clinical use. G2GCD is a chemically pure compound with a single maltosyl group substitution. G2GCD is less cytotoxic, especially less ototoxic, than HPBCD and G2BCD and is useful.

Claims (4)

A pharmaceutical composition for the treatment or prevention of Niemann-Pick disease type C lysosomal storage disease, comprising 6-O-maltosyl-γ-cyclodextrin. A pharmaceutical composition comprising 6-O-maltosyl-γ-cyclodextrin for suppressing lysosomal accumulation and/or impairment of the autophagy pathway associated with cholesterol accumulation,
Here, the suppression of the lysosomal accumulation means that when the pharmaceutical composition is administered, the accumulation of free cholesterol in cells is reduced and the amount of esterified cholesterol in cells is increased, compared to when the pharmaceutical composition is not administered. , or lysosomal volume is decreased,
Said pharmaceutical composition . 3. The pharmaceutical composition according to any one of claims 1 or 2 , which is administered parenterally. The pharmaceutical composition according to any one of claims 1-3 , which is administered as an injection.

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