Novel lipid accumulation inhibitors and lipid storage disease treatments containing the same

Abstract

The present invention aims to provide a novel pharmaceutical composition that can be used for the treatment or prevention of lysosomal storage diseases, particularly Niemann-Pick disease. [Solution] The present invention provides a pharmaceutical composition for the treatment or prevention of lysosomal storage diseases, characterized by containing a modified γ-cyclodextrin as an active ingredient, wherein the modified γ-cyclodextrin has a monovalent group derived from galacturonic acid that is bonded to a sugar residue of the γ-cyclodextrin via an amide bond.

Description

[Technical Field]

[0001] The present invention relates to a novel inhibitor of lipid, particularly cholesterol, accumulation. The present invention also relates to a therapeutic agent for lipid, particularly cholesterol accumulation-related diseases, comprising the said lipid accumulation inhibitor. [Background technology]

[0002] When enzymes related to lysosomes, which are organelles within cells, are genetically deficient or mutated, substances that should be broken down or transported accumulate as foreign substances within the cell. Congenital metabolic disorders caused by this phenomenon are known as lysosomal storage diseases. Lysosomal storage diseases vary in name and symptoms depending on the deficient enzyme, and currently about 50 types of diseases are known. Examples of lysosomal storage diseases include cholesterol storage disorders and sphingolipidosis, in which lipids called sphingolipids accumulate, as well as Niemann-Pick disease, Gaucher disease, GM1 gangliosidosis, GM2 gangliosidosis, and Fabry disease.

[0003] Niemann-Pick disease type C (NPC) is a congenital lysosomal storage disorder caused by abnormalities in the membrane protein NPC1, which is responsible for the transport of lipids, primarily cholesterol, within cells, or the secreted protein NPC2, which coexists with NPC1 in endosomes. In Niemann-Pick disease type C (NPC), the deficiency of cholesterol transport proteins (NPC1 and NPC2) leads to the accumulation of free cholesterol and other substances in the central nervous system and organs. Neurological disorders (ataxia, seizures, intellectual disability) and liver damage develop in infancy, and death occurs by around age 20. The approved drug miglustat has limited efficacy and is ineffective against cholesterol accumulation. There are no other effective treatments.

[0004] Clinical trials of 2-hydroxypropyl-β-cyclodextrin (HPBCD) are underway in Europe and the United States. However, HPBCD is known to be ototoxic, which is hindering its application to patients (Non-Patent Literature 1). Furthermore, in Japan, HPBCD has caused severe lung damage, and systemic administration has been discontinued. Therefore, the development of a drug with superior efficacy and safety is urgently needed.

[0005] To date, the present inventors have found and reported that 2-hydroxypropyl-γ-cyclodextrin (HPGCD) has a cholesterol accumulation reduction effect equivalent to that of HPBCD, but with fewer side effects than HPBCD (Non-Patent Document 2, Patent Document 1). However, HPGCD still exhibits ototoxicity, albeit much less than that of HPBCD (Non-Patent Document 3).

[0006] Furthermore, cyclodextrin derivatives have been reported in which the hydroxyl group at position 6 of the sugar residue of γ-cyclodextrin is glucosidicated to the anomeric hydroxyl group of glucose or maltose (the carbon at position 6 of the sugar residue of cyclodextrin is bonded to the carbon at position 1 of the glucosyl or maltosyl group via an -O- link) (Non-Patent Literature 4). It has been reported that γ-cyclodextrin derivatives with attached glucosyl or maltosyl groups exhibit significantly reduced ototoxicity when administered subcutaneously to mice compared to HPBCD and HPGCD, but retain equivalent ototoxicity when administered intracerebroventricularly (Non-Patent Literature 4).

[0007] GM1 gangliosidosis is caused by a mutation in lysosomal β-galactosidase, a glucose hydrolase. Due to the deficiency of β-galactosidase, glycolipids such as GM1-ganglioside and asialoGM1-ganglioside, which are its substrates, accumulate in the brain and internal organs (liver, spleen), and mucopolysaccharides such as keratan sulfate accumulate in the bones. There are three types: an infantile type (Type 1) that develops in early infancy and is accompanied by widespread central nervous system disorders including spastic paraplegia, cherry-red spots in the fundus, hepatosplenomegaly, and bone abnormalities; a juvenile type (Type 2) that develops in early childhood and is characterized by progressive central nervous system disorders; and an adult type (Type 3) that appears from school age onward and is characterized by extrapyramidal symptoms, such as dysarthria. GM2 gangliosidosis is a disease caused by the accumulation of a lipid called GM2 ganglioside in nerve cells due to a deficiency in hexosaminidase A, a glucose hydrolase. Tay-Sachs disease and Sandhoff disease fall into this category.

[0008] While enzyme replacement therapy has been the primary treatment for these diseases, several problems exist. Enzyme preparations have difficulty reaching the central nervous system, resulting in little therapeutic effect on the nervous system, including the brain. Furthermore, expensive intravenous treatment with enzyme preparations must be continued for life. Therefore, new treatments for these lysosomal storage diseases are highly desired.

[0009] Various cyclodextrin derivatives have been developed for purposes such as improving the solubility of cyclodextrins in water and organic solvents, making them insoluble in water, modifying polymer surfaces, and adding properties. One such derivative is branched cyclodextrin, in which sugars are attached to cyclodextrin in a branched manner. Specifically, branched cyclodextrins with sugars such as glucose, maltose, maltooligosaccharides, galactose, and mannose as branched structures are known (Non-Patent Documents 5-7, Patent Documents 2-5). These branched cyclodextrins are synthesized by enzymatic reactions, and a hydroxyl group attached to the carbon atom at position 1 of the sugar undergoes dehydration condensation to form a glycosidic bond, so none of them have reducing properties.

[0010] In addition, a novel reducing cyclodextrin derivative having a branched structure derived from uronic acid in cyclodextrin, which has a structure bonded by an amide bond between an amino group of any of aminoated α-cyclodextrin, β-cyclodextrin or γ-cyclodextrin and a carboxyl group of uronic acid, and a method for producing the same have been reported (Patent Document 6).

Prior Art Documents

Patent Documents

[0011]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

Patent Document 6

Non-Patent Documents

[0012]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Summary of the Invention

Problems to be Solved by the Invention

[0013] The object of the present invention is to provide a novel lipid accumulation inhibitor and / or lipid accumulation disorder treatment agent that has an effect equivalent to or greater than that of 2-hydroxypropyl-γ-cyclodextrin (HPGCD) reported by the present inventors, particularly in reducing lipid accumulation, especially cholesterol accumulation, and has fewer side effects than HPGCD. [Means for solving the problem]

[0014] The present inventors, after diligently studying cyclodextrin derivatives, discovered a cyclodextrin derivative with a galacturonic acid-derived group (represented as "RGalγ-CD" or "GalGCD") (RGal6γCD and RGal3γCD, which have galacturonic acid-derived groups at the 6th and 3rd positions of the sugar, respectively), which has a cholesterol accumulation reduction effect equivalent to HPGCD but with fewer side effects, and thus completed the present invention. This invention includes the following: [1] A pharmaceutical composition for the treatment or prevention of lysosomal storage disease, characterized by containing a modified γ-cyclodextrin as an active ingredient, wherein the modified γ-cyclodextrin has a monovalent group derived from galacturonic acid that is bonded to a sugar residue of the γ-cyclodextrin via an amide bond. [2] The pharmaceutical composition according to [1] above, wherein the modified γ-cyclodextrin has a monovalent group derived from galacturonic acid bonded to the 6th and / or 3rd carbon of the sugar residue of the γ-cyclodextrin via an amide bond. [3] The modified γ-cyclodextrin is given by the following formula (1):

[0015] [ka] (In the above formula, m is 0 to 7, n is 0 to 8, o is 0 to 8, and m + n + o = 8, and n R 1 and R 2 Each of these is independently a hydroxyl group or a group represented by the following formula (a); -NH-Z ···(a) [In the formula, Z is a monovalent group formed by removing the hydroxyl group (-OH) from the carboxyl group (-COOH) of galacturonic acid.] and o R 3 , R 4 and R 5 Each of these independently represents either a hydroxyl group or a group represented by formula (a) above. Here, in equation (1), if n+o is 2 or greater, the arrangement of structures (A), (B), and (C) below is arbitrary, and the n (B)s may be the same or different, and the o (C)s may be the same or different.

[0016] [ka] The pharmaceutical composition described above [1], which is a compound represented by [1]. [4] The modified γ-cyclodextrin is given by the following formula (2):

[0017] [ka] (In the above formula, m is 0 to 7, n is 1 to 8, and m + n = 8, and n R 1 or R 2 Each of these is independently a hydroxyl group or a group represented by the following formula (a); -NH-Z ···(a) [In the formula, Z is a monovalent group formed by removing the hydroxyl group (-OH) from the carboxyl group (-COOH) of galacturonic acid.] Here, in equation (2), if n is 2 or greater, the arrangement of structure (A) and structure (B) below is arbitrary, and the n B's may be the same or different.

[0018] [ka] Or the following equation (3):

[0019] [ka] (In the above formula, m is 0 to 7, o is 1 to 8, and m + o = 8. o R 3 's, R 4 and R 5 are each independently a hydroxyl group or a group represented by the following formula (a): -NH-Z ···(a) [In the formula, Z is a monovalent group formed by removing a hydroxyl group (-OH) from the carboxyl group (-COOH) of galacturonic acid.] is shown. Here, in formula (3), when o is 2 or more, the arrangements of the following structures (A) and structure (C) are arbitrary, and the o (C)'s may be the same or different.)

[0020]

Chemical formula

[10] The pharmaceutical composition according to any one of [1] to [8] above, characterized in that the pharmaceutical composition is administered as an injectable preparation.

[0021]

[11] A method for treating or preventing a lysosomal storage disease, comprising administering to a subject in need of such treatment a therapeutically effective amount of a modified γ-cyclodextrin in which a monovalent group derived from galacturonic acid is bonded to a sugar residue of γ-cyclodextrin via an amide bond.

[12] The modified γ-cyclodextrin is the method according to

[11] , wherein a monovalent group derived from galacturonic acid is bonded to the 6th and / or 3rd carbon of the sugar residue of the γ-cyclodextrin via an amide bond.

[13] The modified γ-cyclodextrin is represented by formula (1) above (the definitions of which are the same as above), according to the method described in

[11] above.

[14] The modified γ-cyclodextrin is represented by formula (2) (the definition of which is the same as above) or formula (3) (the definition of which is the same as above), as described in

[13] above.

[15] The modified γ-cyclodextrin is represented by formula (2), where n is 1, R 1 This is the above equation (a), R 2 The method described in

[13] above, wherein is OH.

[16] The modified γ-cyclodextrin is represented by formula (3), where o is 1, R 3 and R 4 OH, R 5 The method described in

[13] above, wherein is the above formula (a).

[17] The method according to any one of the above

[11] to

[16] , wherein the lysosomal storage disease is a lipid storage disorder (e.g., cholesterol storage disorder, sphingolipidosis or lysosomal acid lipase deficiency, in particular Niemann-Pick disease, Gaucher disease, GM1 gangliosidosis, GM2 gangliosidosis, or Fabry disease).

[18] The method according to any one of the above

[11] to

[16] , wherein the lysosomal storage disease is selected from the group consisting of Niemann-Pick disease type C, GM1 gangliosidosis, and GM2 gangliosidosis. [Effects of the Invention]

[0022] The pharmaceutical composition of the present invention, which contains γ-cyclodextrin (GalGCD) as an active ingredient, in which a monovalent group derived from galacturonic acid is amide-bonded to a sugar residue of γ-cyclodextrin, is effective in the treatment or prevention of lysosomal storage diseases, particularly lipid storage diseases (e.g., Niemann-Pick disease, GM1 gangliosidosis, and GM2 gangliosidosis). The GalGCD contained in the pharmaceutical composition of the present invention has lower ototoxicity and is useful compared to conventional cyclodextrin derivatives such as HPBCD and HPGCD. [Brief explanation of the drawing]

[0023] [Figure 1] Figure 1 shows the total protein mass measured when γ-cyclodextrin derivatives of various concentrations were added. From left to right, the results are for Gal6GCD, Gal3GCD, Glc6GCD, and Glc3GCD. Figures 2 through 4 are similar. [Figure 2] Figure 2 is a graph showing the results of the response to GalGCD addition concentration on the improvement of intracellular cholesterol (total cholesterol) accumulation in Npc1-deficient CHO cells. The data represent Mean ± SEM (N=3) values, where * indicates p<0.05 (compared to wild-type (WT)) and # indicates p<0.05 (compared to Npc1 null). The total cholesterol accumulation rate is shown in nmol / mg protein in the upper panel and as a percentage with WT set to 100% in the lower panel. [Figure 3]Figure 3 is a graph showing the results of the response of GalGCD addition concentration to the reduction of intracellular cholesterol (free cholesterol) accumulation in Npc1-deficient CHO cells. The data represent Mean ± SEM (N=3) values, where * indicates p<0.05 (compared to WT) and # indicates p<0.05 (compared to Npc1 null). The free cholesterol accumulation rate is shown in nmol / mg protein in the upper panel and as a percentage with WT set to 100% in the lower panel. [Figure 4] Figure 4 is a graph showing the results of the response of GalGCD addition concentration to the effect of improving intracellular cholesterol accumulation (ratio of total cholesterol to esterified cholesterol) in Npc1-deficient CHO cells. The data represent Mean ± SEM (N=3) values, where * indicates p<0.05 (compared to WT) and # indicates p<0.05 (compared to Npc1 null). The upper panel shows the ratio of total cholesterol to esterified cholesterol, and the lower panel shows the percentage when WT is set to 100%. [Figure 5] Figure 5 shows the results of the response of GalGCD concentration to the improvement of intracellular free cholesterol accumulation in neural stem cells differentiated from NPC-derived iPS cells. The data represent Mean ± SEM (N=3) values, where * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.001 (compared to NPCs without compound treatment). A visualizes the effect of GalGCD on cholesterol accumulation reduction by Filipin staining, showing free cholesterol. B graphs the degree of reduction in A. [Figure 6] Figure 6 shows the results of evaluating the effect of GalGCD on reducing LC3B-II protein expression (A) and insoluble p62 (B) using neural stem cells derived from NPC-iPS cell lines. A and B are the results of Western blotting, and C is the result graphed after correcting protein expression levels with tubulin expression. Normal represents neural stem cells derived from iPS cell lines from normal humans, and NPC represents neural stem cells derived from iPS cell lines from NPC patients. - indicates no addition of GalGCD or HPGCD. [Figure 7]Figure 7 shows the results of repeated intravenous administration of GalGCD (A) and HPGCD (B) into normal adult mice, followed by measurement of auditory brainstem response (ABR). Each dot represents the results for an individual mouse. [Modes for carrying out the invention]

[0024] The present invention will be described below, with illustrative embodiments as examples, along with preferred methods and materials that may be used in carrying out the invention, but the present invention is not limited to the embodiments described below. Unless otherwise specified herein, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present invention pertains. Any materials and methods equivalent to or similar to those described herein may be used in carrying out the present invention. Furthermore, all publications and patents cited herein in connection with the present invention are cited herein and constitute part of this specification, for example, as indicating methods, materials, and other matters that may be used in the present invention.

[0025] In this specification, the notation "A to B" indicating a numerical range means a numerical range that includes the endpoints A and B. The same applies to "A to B". In this specification, "approximately" means that a tolerance of ±10% is allowed.

[0026] 1. Modified γ-cyclodextrin The pharmaceutical composition of the present invention contains γ-cyclodextrin (GalGCD), in which a monovalent group derived from galacturonic acid is amide-bonded to a sugar residue of γ-cyclodextrin, as an active ingredient. The GalGCD used in this invention is a compound represented by the following formula (1).

[0027] [ka]

[0028] In equation (1) above, m is between 0 and 7, n is between 0 and 8, o is between 0 and 8, and m + n + o = 8. That is, at least one of n or o is 1 or greater. n R 1 and R 2 Each of these independently represents a hydroxyl group or a group represented by the following formula (a). -NH-Z ···(a) In formula (a), Z represents a monovalent group formed by removing a hydroxyl group (-OH) from a carboxyl group (-COOH) of galacturonic acid. o R 3 , R 4 and R 5 Each of these independently represents either a hydroxyl group or a group represented by formula (a) above. Furthermore, in equation (1), if n+o is 2 or greater, the arrangement of structures (A), (B), and (C) below is arbitrary, and the n (B)s may be the same or different, and the o (C)s may be the same or different.

[0029] [ka]

[0030] In one embodiment, the GalGCD of the present invention is a compound represented by the following formula (2).

[0031] [ka] In equation (2) above, m is between 0 and 7, n is between 1 and 8, and m + n = 8. n R 1 and R 2 Each of these independently represents a hydroxyl group or a group represented by the following formula (a). -NH-Z ···(a) In formula (a), Z represents a monovalent group formed by removing a hydroxyl group (-OH) from a carboxyl group (-COOH) of galacturonic acid. Here, in equation (2), if n is 2 or greater, the arrangement of structures (A) and (B) below is arbitrary, and the n (B)s may be the same or different.

[0032] [ka]

[0033] In one preferred embodiment, the GalGCD of the present invention is represented by formula (2) above, where n is 1, R 1 This is the above equation (a), R 2 It is OH.

[0034] In one embodiment, the GalGCD of the present invention is a compound represented by the following formula (3).

[0035] [ka] In equation (3) above, m is between 0 and 7, o is between 1 and 8, and m + o = 8. o R 3 , R 4 and R 4 Each of these independently represents a hydroxyl group or a group represented by the following formula (a). -NH-Z ···(a) In formula (a), Z represents a monovalent group formed by removing a hydroxyl group (-OH) from a carboxyl group (-COOH) of galacturonic acid. Here, in equation (3), if o is 2 or more, the arrangement of structures (A) and (C) below is arbitrary, and the o (C)s may be the same or different.

[0036] [ka]

[0037] In one preferred embodiment, the GalGCD of the present invention is represented by the above formula (3), where o is 1, R 3 and R 4 OH, R5 This is given by equation (a) above.

[0038] In the GalGCD used in the present invention, the group represented by formula (a) is bound to the 6th and / or 3rd and / or 2nd positions of the sugar residue of γ-cyclodextrin, and one, two, or three groups represented by formula (a) may be bound to the same sugar residue. In the GalGCD of the present invention, the group represented by formula (a) may be present on one sugar residue of cyclodextrin, on multiple sugar residues, or even on all eight sugar residues. When two or more groups represented by formula (a) are present in the GalGCD, the binding position of the group represented by formula (a) to the sugar is selected from the 6th, 3rd, or 2nd position, and these may be the same or different. For example, when three groups represented by formula (a) are present in the GalGCD, their binding positions to the sugar residues may all be the same, two may be the same, or all may be different. Furthermore, if there are two or more sugar residues to which the group represented by formula (a) is attached within a GalGCD, the positions of those sugar residues within a GCD containing eight sugar residues are arbitrary.

[0039] The GalGCD used in the present invention preferably has a group represented by formula (a) at the 6th or 3rd position, more preferably at the 6th position.

[0040] In the present invention, the GalGCD used in formula (1) above has n+o as 1 to 8, preferably 1 to 3, more preferably 1 to 2, and even more preferably 1. For example, a GalGCD in which n is 1, o is 0, and the above formula (a) is bonded to the 6th carbon can be represented by the following formula (4).

[0041] [ka]

[0042] When n is 0 and o is 1, the GalGCD in which the above formula (a) is bonded to the 3rd carbon is represented by the following formula (5).

[0043] [ka]

[0044] The GalGCD of the present invention can be produced by condensing the carboxyl group of galacturonic acid with the amino group of aminated γ-cyclodextrin (aminated GCD), which is obtained by substituting the hydroxyl group of a sugar residue of γ-cyclodextrin (GCD) with an amino group, in the presence of a condensing agent. That is, the GalGCD of the present invention can be produced by condensing aminated cyclodextrin, which is obtained by aminating one or more hydroxyl groups at the 6th, 3rd, or 2nd positions of any one or more sugar residues constituting cyclodextrin, with galacturonic acid in the presence of a condensing agent, thereby producing the GalGCD represented by formula (1) above. For example, by the above production method, a GalGCD having a structure in which the amino group of aminated GCD and the carboxyl group of the 6th carbon of galacturonic acid are linked by an amide bond can be obtained. Amination of GCD is a GCD in which an amino group is attached to one or more carbon atoms at position 6, 3, or 2 of a sugar residue. GCDs with amino groups attached to different sugar residues can be used, and GCDs with amino groups attached to multiple carbon atoms within the same sugar residue can also be used. There are no particular restrictions on the number of amino groups, and GCDs with multiple amino groups attached to a single sugar residue or with amino groups attached to multiple sugar residues can be used. An amination of GCD in which the hydroxyl group at position 6 of one sugar residue of GCD is replaced with an amino group, and a GalGCD in which the carboxyl group at position 6 of galacturonic acid is bonded by an amide bond, are represented by formula (4) above. An amination of GCD in which the hydroxyl group at position 3 of one sugar residue of GCD is replaced with an amino group, and a GalGCD in which the carboxyl group at position 6 of galacturonic acid is bonded by an amide bond, are represented by formula (5) above.

[0045] The GalGCD of the present invention can be manufactured using methods known in the art, for example, by referring to Patent Document 7 (Japanese Patent Application Publication No. 2020-37673). Specifically, it can be manufactured as follows: Amination of GCD can be prepared by tosyling GCD, azidating the resulting tosylated GCD, and aminating the resulting azidated GCD. For example, the conversion of the hydroxyl group at position 6 of GCD to an amino group can be achieved by tosyling the hydroxyl group with, for example, p-toluenesulfonyl chloride (tosyl chloride). Then, the tosyled hydroxyl group is converted to an azide group with sodium amide, and finally, the azide group is reduced with triphenylphosphine to obtain the amination of GCD. Alternatively, the tosyled hydroxyl group can be converted to an amino group more simply by reacting it with aqueous ammonia to obtain the amination of GCD, although it may be synthesized by other methods. Furthermore, amination of GCD can also be prepared by chlorinating GCD, azidating the resulting chlorinated GCD, and aminating the resulting azidated GCD. In addition, amination of GCD with a specific degree of substitution can be synthesized by separating tosyled GCD or chlorinated GCD with a specific degree of substitution by liquid chromatography after tosyling or chlorination, and then azidating and aminating them. Furthermore, various types of aminated GCDs sold as reagents can be purchased and used. In addition, aminated GCD salts, which are obtained by dissolving aminated GCDs in an acid such as hydrochloric acid, can also be used.

[0046] The condensing agent used in the production of GalGCD of the present invention can be any agent capable of forming the above-mentioned amide bond, and any commonly used condensing agent can be used. Specifically, as condensing agents, 1H-benzotriazole-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP reagent), 1-hydroxybenzotriazole (HOBt reagent), 1H-benzotriazole-1-yloxytripyrrolidinophosphonium hexafluorophosphate (PyBOP reagent), N,N-dicyclohexylcarbodiimide (DCC reagent), 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide (WSC reagent), N,N'-diisopropylcarbodiimide (DIC reagent), 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride (DMT-MM reagent), O-(benzotriazole-1-yl)-N,N,N',N')-tetramethyluronium hexafluorophosphate (HBTU reagent), etc., can be used.

[0047] The condensation reaction using the condensing agent in the production of GalGCD of the present invention can be appropriately adjusted depending on the properties of the condensing agent used. For example, in the case of BOP reagent, the reaction can be carried out at room temperature in N,N-dimethylformamide (DMF) for 3 hours.

[0048] 2. Pharmaceutical Compositions The pharmaceutical composition of the present invention contains GalGCD as an active ingredient and can be used as a therapeutic agent for lysosomal storage diseases, which are lipid storage disorders. Examples of lysosomal storage diseases, which are lipid storage disorders, include cholesterol storage disorders, sphingolipidosis, and lysosomal acid lipase deficiency. Cholesterol storage disorders are diseases characterized by the accumulation of cholesterol, and examples include Niemann-Pick disease type C, Wolman disease, Cholesterol Ester Storage Disease (CESD), Tangier disease, and Familial Hypercholesterolemia. The pharmaceutical composition of the present invention is particularly preferred for Niemann-Pick disease type C. Examples of sphingolipidosis include GM1 gangliosidosis, GM2 gangliosidosis, Gauche disease, and Fabry disease. The pharmaceutical composition of the present invention is preferably used for GM1 gangliosidosis and GM2 gangliosidosis.

[0049] The modified γ-cyclodextrin, which is the active ingredient of the pharmaceutical composition of the present invention, is GalGCD as described above, and the pharmaceutical composition of the present invention reduces the accumulation of lipids, particularly cholesterol, and / or inhibits the disruption of the autophagy pathway.

[0050] "Reducing cholesterol accumulation" or "a reduction in cholesterol accumulation" means, for example, that when administered to NPC model cells (e.g., neural stem cells derived from iPS cells from NPC patients) at a concentration of, for example, 1 mM, the accumulation of free cholesterol in the cells decreases compared to when not administered, preferably by 10% or more, 20% or more, 30% or more, 40% or more, 45% or more, or about 50%. Alternatively, "reducing cholesterol accumulation" means, for example, that when administered to NPC model cells at a concentration of, for example, 1 mM, the amount of esterified cholesterol in the cells (the ratio of esterified cholesterol to total cholesterol) increases compared to when not administered, preferably by 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 times or more, or about 2.5 times. In one embodiment, the pharmaceutical composition of the present invention exhibits an effect of "reducing cholesterol accumulation" to the same extent as a pharmaceutical composition containing 2-hydroxypropyl-β-cyclodextrin (HPBCD) or 2-hydroxypropyl-γ-cyclodextrin (HPGCD) at the same concentration.

[0051] "Suppressing the disruption of the autophagy pathway" means that when administered to NPC model cells (for example, neural stem cells derived from iPS cells from NPC patients) at a concentration of, for example, 1 mM, the autophagosome volume decreases, preferably by 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, or approximately 30% compared to when not administered. In one embodiment, the pharmaceutical composition of the present invention exhibits the function of "suppressing the disruption of the autophagy pathway" to the same extent as a pharmaceutical composition containing 2-hydroxypropyl-β-cyclodextrin (HPBCD) or 2-hydroxypropyl-γ-cyclodextrin (HPGCD) at the same concentration.

[0052] GalGCD is less toxic than HPBCD, HPGCD, glucosyl-GCD, and maltosyl-GCD, which are particularly problematic in terms of ototoxicity. Non-limitingly, "low ototoxicity" means, for example, that the effect of administration on the auditory threshold is lower than that of HPBCD, HPGCD, glucosyl-GCD, and maltosyl-GCD, and preferably shows a significantly lower sound pressure threshold over a wide frequency range (low to high frequencies).

[0053] The target population for the pharmaceutical composition of the present invention may include, for example, humans and non-human mammals (e.g., guinea pigs, rats, mice or other rodents, rabbits, cats, dogs, pigs, sheep, goats, cattle, horses, donkeys, and non-human primates), and is preferably humans.

[0054] The pharmaceutical composition of the present invention is not limited to the present invention, but can take the form of parenteral administration, preferably in the form of an injectable formulation. The parenteral agent of the present invention (for example, an injectable formulation) can be administered intravenously, intramuscularly, subcutaneously, or directly into the cerebral ventricle. The pharmaceutical composition of the present invention can take the form of either a water-soluble formulation or a lyophilized formulation, preferably an aqueous injectable formulation or a lyophilized, ready-to-use injectable formulation.

[0055] The compositions of the present invention may contain sugars, preservatives, stabilizers, and antistatic agents commonly used in injectable preparations. The compositions of the present invention may also contain pharmacologically acceptable pH adjusters. The pH adjusters used in the present invention are not particularly limited as long as they are pharmacologically acceptable substances that can be used in pharmaceutical applications, but are preferably sodium hydroxide, carbonate buffer, phosphate buffer, citrate buffer, acetate buffer, and hydrochloric acid. These pH adjusters may be used individually or in combination of two or more. The compositions of the present invention may also contain osmotic pressure adjusters or isotonic agents, and may include at least one of the following, for example, sodium chloride or dextrose.

[0056] The effective dose of the pharmaceutical composition of the present invention can be appropriately selected depending on the type of disease, the severity of the disease, the treatment plan, the route of administration, body weight, age, sex, and the patient's (genetic) racial background. However, the pharmaceutically effective dose is generally determined based on factors such as clinically observed symptoms and the degree of disease progression. The route of administration is not particularly limited, but examples include intravenous infusion, intracerebroventricular infusion, intrathecal infusion, subarachnoid infusion, lumbar puncture infusion, and nasal administration. Depending on the route of administration, the daily dose is, for example, about 1 mg / kg to about 2 g / kg, preferably about 5 mg / kg to about 500 mg / kg. The dose is lower for intracerebroventricular infusion, intrathecal infusion, subarachnoid infusion, and lumbar puncture infusion compared to intravenous infusion. The drug may be administered in a single dose, in multiple doses, or continuously over time via intravenous infusion. Preferably, it should be administered via intravenous infusion over several hours or longer, for example, several hours to approximately 10 hours. The drug may be administered daily or intermittently, and the appropriate method can be selected depending on the condition of the patient, but intermittent administration is preferred. For example, in the case of intracerebroventricular infusion, approximately 1 mg / kg to approximately 50 mg / kg, preferably approximately 10 mg / kg to approximately 30 mg / kg, can be administered weekly or bi-weekly for several months to several years. The dosage may be adjusted as appropriate, taking into consideration the condition of the patient (e.g., the appearance of ototoxicity).

[0057] Furthermore, the pharmaceutical composition of the present invention has excellent safety and can be administered over a long period of time. In other words, lysosomal storage diseases, which are targeted by the pharmaceutical composition of the present invention, are genetic diseases, and administration is often necessary as long as the patient is alive. The pharmaceutical composition of the present invention is particularly suitable for such use due to its excellent safety. The period over which the pharmaceutical of the present invention can be administered is not particularly limited, but the pharmaceutical of the present invention can be administered over a long period of time, for example, at least several weeks, preferably several months, and more preferably several years.

[0058] In this specification, “therapeutic dose” means the amount of GalGCD of the present invention, which is the amount effective in producing the desired therapeutic effect at a reasonable benefit / risk ratio, e.g., a reduction in cholesterol accumulation and / or inhibition of autophagy pathway dysfunction. The therapeutic dose may vary depending on the route of administration used, as is known to those skilled in the art. Furthermore, the therapeutic dose may be appropriately determined by the subject being treated, the severity of the symptoms, the route of administration, the frequency of administration, the judgment of the prescribing physician, and other relevant factors.

[0059] As detailed below, neural stem cells derived from iPS cells of NPC patients accumulate free cholesterol intracellularly, similar to what is observed in patients. When the effect of GalGCD, the active ingredient of the pharmaceutical composition of the present invention, on this accumulation was examined, it showed a reduction effect comparable to that of HPGCD, which was used as a control. Furthermore, GalGCD exhibited extremely low levels of auditory toxicity even with repeated intraventricular administration. In addition, while abnormalities in autophagy function were observed in neural stem cells derived from NPC patients, this abnormal phenotype improved with GalGCD treatment, confirming its effect on neuronal dysfunction. [Examples]

[0060] The present invention will be specifically described below with reference to examples, but the present invention is not limited to the following examples. (Example 1) Synthesis of mono-6-Op-toluenesulfonyl-γ-cyclodextrin 50 g of γ-cyclodextrin was dissolved in 500 mL of 4-methylpyridine and cooled on ice. 11 g of p-toluenesulfonyl chloride was added to the solution and reacted at room temperature for 15 hours. The reaction mixture was added to 3.5 L of acetone, the resulting precipitate was collected, and washed with acetone. The collected precipitate was dissolved in 200 mL of boiling water, cooled to 4°C, and recrystallized. After a total of three recrystallization treatments, 11.3 g of white powder was obtained by vacuum drying. FT-IR and NMR confirmed that the white powder was mono-6-Op-toluenesulfonyl-γ-cyclodextrin.

[0061] (Example 2) Synthesis of 6-amino-6-deoxy-γ-cyclodextrin 9 g of mono-6-Op-toluenesulfonyl-γ-cyclodextrin prepared in Example 1 was dissolved in 200 mL of 28% aqueous ammonia solution and reacted at 60°C for 16 hours. The reaction solution was added to 2.5 L of acetone, and the resulting precipitate was collected and washed with acetone. The collected precipitate was dried under reduced pressure to obtain 7.1 g of yellowish-white powder. FT-IR and NMR confirmed that the yellowish-white powder was 6-amino-6-deoxy-γ-cyclodextrin.

[0062] (Example 3) Synthesis of Gal6GCD (γ-cyclodextrin having a monovalent group derived from galacturonic acid at the 6th carbon) 3 g of 6-amino-6-deoxy-γ-cyclodextrin prepared in Example 2 was dissolved in 30 mL of dimethylformamide (DMF), and 1.3 g of 1H-benzotriazole-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP reagent) and 1.5 mL of N,N-diisopropylethylamine (DIEA) were added. 0.64 g of galacturonic acid monohydrate was added to the above solution, and the mixture was sealed with Ar gas and reacted at room temperature for 3 hours. The reaction mixture was added to 500 mL of acetone, and the resulting precipitate was collected and washed with acetone and methanol. The collected precipitate was dried under reduced pressure and then separated by preparative high-performance liquid chromatography using an ODS column. The separated solution was freeze-dried to recover 0.97 g of white powder. NMR confirmed that the above white powder was Gal6GCD (the compound of formula (4) below).

[0063] [ka]

[0064] (Example 4) Synthesis of 3A-amino-3A-deoxy-(2AS,3AS)-γ-cyclodextrin 7.6 g of mono-2-Op-toluenesulfonyl-γ-cyclodextrin was dissolved in 300 mL of 10% (w / w) ammonium bicarbonate aqueous solution and reacted at 60°C for 5 hours. The reaction solution was added to 4 L of acetone, the precipitate was collected, and washed with acetone. The collected precipitate was dried under reduced pressure to obtain a white powder. 6.6 g of the obtained white powder was dissolved in 100 mL of 28% ammonia aqueous solution and reacted at 60°C for 5 hours. The reaction solution was added to 1.5 L of acetone, the precipitate was collected, and washed with acetone. The collected precipitate was dried under reduced pressure to obtain 7.1 g of white powder. Dissolve in 300 mL of 10% (w / w) ammonium bicarbonate aqueous solution and react at 60°C for 5 hours. The reaction solution was added to 4 L of acetone, the precipitate was collected, and washed with acetone. The collected precipitate was dried under reduced pressure to obtain 5.4 g of yellowish-white powder. FT-IR and NMR confirmed that the above white powder is 3A-amino-3A-deoxy-(2AS,3AS)-γ-cyclodextrin.

[0065] (Example 5) Synthesis of Gal3GCD (γ-cyclodextrin having a monovalent group derived from galacturonic acid at the 3rd carbon) 2 g of 3A-amino-3A-deoxy-(2AS,3AS)-γ-cyclodextrin prepared in Example 4 was dissolved in 25 mL of DMF, and 0.82 g of BOP reagent and 0.97 mL of DIEA were added. 0.39 g of galacturonic acid monohydrate was added to the above solution, and the mixture was sealed with Ar gas and reacted at room temperature for 3 hours. The reaction mixture was added to 500 mL of acetone, and the resulting precipitate was collected and washed with acetone and methanol. The collected precipitate was dried under reduced pressure and then separated by preparative high-performance liquid chromatography using an ODS column. The separated solution was freeze-dried to collect 1.8 g of white powder. NMR confirmed that the above white powder was Gal3GCD (the compound of formula (5) below).

[0066] [ka]

[0067] (Comparative Example 1) Synthesis of Glc6GCD (γ-cyclodextrin having a monovalent group derived from glucuronic acid at the 6th carbon) 3.6 g of 6-amino-6-deoxy-γ-cyclodextrin prepared in Example 2 was dissolved in 30 mL of DMF, and 1.8 g of BOP reagent and 2.2 mL of DIEA were added. 0.81 g of glucuronic acid was added to the above solution, and the mixture was sealed with Ar gas and reacted at room temperature for 3 hours. The reaction mixture was added to 500 mL of acetone, and the resulting precipitate was collected and washed with acetone and methanol. The collected precipitate was dried under reduced pressure and then separated by preparative high-performance liquid chromatography using an ODS column. The separated solution was freeze-dried to recover 0.78 g of white powder. NMR confirmed that the above white powder was Glc6GCD (the compound of formula (6) below).

[0068] [ka]

[0069] (Comparative Example 2) Synthesis of Glc3GCD (γ-cyclodextrin having a monovalent group derived from glucuronic acid at the 3rd carbon) 3.0 g of 3A-amino-3A-deoxy-(2AS,3AS)-γ-cyclodextrin prepared in Example 4 was dissolved in 25 mL of DMF, and 1.2 g of BOP reagent and 1.5 mL of DIEA were added. 0.54 g of glucuronic acid was added to the above solution, and the mixture was sealed with Ar gas and reacted at room temperature for 3 hours. The reaction mixture was added to 500 mL of acetone, and the resulting precipitate was collected and washed with acetone and methanol. The collected precipitate was dried under reduced pressure and then separated by preparative high-performance liquid chromatography using an ODS column. The separated solution was freeze-dried to collect 1.1 g of white powder. NMR confirmed that the above white powder was Glc3GCD (the compound of formula (7) below).

[0070] [ka]

[0071] (Example 6) Effects of GalGCD on NPC model cells Npc1-deficient CHO cells (Npc1 null CHO cells: Higaki, K et al., J. Biochem. 129, 875-880. (2001)) were used as NPC model cells. After pre-culturing NPC model cells for 24 hours, Gal6GCD and Gal3GCD were added to the culture medium at concentrations ranging from 0.001 to 10 mM. After 24 hours, the amount of total cholesterol (TC; nmol) and free cholesterol (FC; nmol) per 1 mg of protein in the cells were measured. Wild-type (WT) CHO cells were used as a control (without the addition of CD). Glc6GCD and Glc3GCD, as well as hydroxypropyl-β-cyclodextrin (HP-β-CD) and hydroxypropyl-γ-cyclodextrin (HP-γ-CD), were used as reference compounds. Specifically, NPC model cells were treated for 24 hours in or without each γ-cyclodextrin derivative, and then lysed. The protein concentration in the lysate was measured using a protein assay kit. The results of the protein concentration measurements are shown in Figure 1. No significant decrease in total protein content was observed with the addition of γ-cyclodextrin derivatives at concentrations up to 10 mM. Next, cholesterol was extracted from the lysate according to a standard method, and the cholesterol content was measured. A portion of the extract was incubated with esterase to measure total cholesterol, and the other portion was incubated without esterase to measure free cholesterol. The cholesterol content was calculated as nmol / mg protein. Furthermore, the cholesterol content in Npc1-deficient CHO cells treated with γ-cyclodextrin derivatives is shown as the cholesterol accumulation rate, with the intracellular cholesterol content of wild-type cells set to 100. The results of the total cholesterol measurement are shown in Figure 2, and the results for free cholesterol are shown in Figure 3. The ratio of esterified cholesterol to total cholesterol is shown in Figure 4. Note that there was no effect on the amount of cellular protein due to the addition of GCD.

[0072] In NPC model cells, both GlcGCD and GalGCD showed a significant concentration-dependent decrease in total cholesterol, free cholesterol, and esterified cholesterol ratio, demonstrating a remarkable improvement in intracellular cholesterol accumulation. Furthermore, both CDs with Glc or Gal bonded to the 6th carbon and CDs with Glc or Gal bonded to the 3rd carbon also showed significant improvement. Specifically, when GlcGCD and GalGCD (1 mM) were administered, intracellular free cholesterol accumulation decreased by approximately 50% compared to control groups, and intracellular esterified cholesterol more than doubled compared to control groups. The improvement in intracellular cholesterol accumulation for both GlcGCD and GalGCD was comparable to that of HP-β-CD and HP-γ-CD. Furthermore, since no significant decrease in total protein content was observed even when using GalGCD concentrations 20-100 times higher (10 mM) compared to the effective concentration (0.1-0.5 mM) at which GalGCD shows improvement, it can be estimated that the cytotoxicity is low.

[0073] (Example 7) Effect of GalGCD on neural stem cells induced from NPC-iPS cell lines An NPC-iPS cell line was used, derived from patients with the NPC1 mutation. Table 2015 / 083736, which discloses the creation of the NPC-iPS cell line, is incorporated herein by reference. Neural stem cells derived from iPS cells from NPC patients accumulate free cholesterol intracellularly, similar to what is observed in patients. Normal cell lines (iPS cell lines derived from normal fibroblasts) and NPC-iPS cell lines were differentiated into neural stem cells using known methods. After pre-culturing each differentiated cell, Gal6GCD and Gal3GCD were added to the culture medium at concentrations of 100 μM, 300 μM, and 1 mM. After 96 hours, Filipin staining was performed, followed by analysis using an IN CELL ANALYZER (GE Healthcare). Filipin staining can detect free cholesterol in cells and confirm cholesterol accumulation. HPGCD was used as the reference compound. In addition, to confirm the presence of cells, the nuclei were stained with propidium iodide and Hoechst 33258. The results of nuclear staining and Filipin staining observed with a fluorescence microscope are shown in Figure 5A, and the degree of decrease in fluorescence from Filipin staining is graphed in Figure 5B.

[0074] Compared to normal cell lines, NPC-iPS cell lines showed significant intracellular cholesterol accumulation. However, the addition of Gal3GCD, Gal6GCD, and HPGCD resulted in a concentration-dependent decrease in intracellular cholesterol accumulation.

[0075] (Example 8) Confirmation of the effect of GalGCD on restoring autophagy function In the disease, the protein expression of LC3B-II, an autophagy initiation marker, is elevated, indicating activated autophagy. However, the accumulation of insoluble p62 protein suggests that autophagy is not progressing and is stagnant. Similarly, neural stem cells differentiated from NPC-iPS cell lines created from patients with NPC1 mutations show abnormalities in the autophagy function of the cells. Using neural stem cells induced from NPC-iPS cell lines, we confirmed the effect of GalGCD on reducing LC3B-II protein expression (A) and insoluble p62 (B), and evaluated the effect of GalGCD on restoring autophagy function. Gal3GCD, Gal6GCD, and HPGCD were added at a concentration of 1 mM, and LC3B-II protein and insoluble p62 protein were measured after 72 hours. The results are shown in Figure 6. Based on the above, abnormalities in autophagy function were observed in neural stem cells derived from NPCs, but this abnormal phenotype was improved by GalGCD treatment.

[0076] (Example 9) Confirmation of ototoxicity of GalGCD To evaluate auditory toxicity, Gal3GCD and Gal6GCD were repeatedly administered into the ventricles of normal adult mice at 9 weeks of age, and auditory brainstem response (ABR) was measured. Administration was performed every two weeks at a dose of 0.029 nmol / kg / dose, for a total of five doses. ABR was measured three days after the final dose. ABR was measured in each animal at five frequencies (4, 8, 12, and 20 kHz). HPGCD was used as the control compound and administered in the same manner. The results are shown in Figure 7.

[0077] Non-patent document 4 reports that γ-cyclodextrin derivatives with a glucosyl group attached exhibit octotoxicity equivalent to that of HPGCD when administered intracerebroventricularly. On the other hand, both Gal3GCD and Gal6GCD of the present invention were found to have significantly lower octotoxicity compared to HPGCD. Based on the above findings, it was found that the two compounds, Gal3GCD and Gal6GCD, have cholesterol accumulation reduction and nerve function improvement effects equivalent to those of conventional HPGCD, and also have weaker ototoxic side effects compared to HPGCD.

[0078] The above detailed description merely illustrates the object and subject matter of the present invention and does not limit the scope of the appended claims. Various modifications and substitutions to the embodiments described without departing from the scope of the appended claims will be apparent to those skilled in the art from the teachings described herein. [Industrial applicability]

[0079] The present invention provides a pharmaceutical composition that can be used for the treatment or prevention of lysosomal storage diseases, particularly Niemann-Pick disease. The cyclodextrin derivative, which is the active ingredient of the pharmaceutical composition of the present invention, has comparable cholesterol accumulation reduction and nerve function improvement effects to conventional cyclodextrins, and is useful because it has low ototoxicity.

Claims

1. A pharmaceutical composition for the treatment or prevention of lysosomal storage diseases, characterized by containing a modified γ-cyclodextrin as an active ingredient, wherein the modified γ-cyclodextrin has a monovalent group derived from galacturonic acid bonded to a sugar residue of the γ-cyclodextrin via an amide bond.

2. The pharmaceutical composition according to claim 1, wherein the modified γ-cyclodextrin has a monovalent group derived from galacturonic acid bonded to the 6th and / or 3rd carbon of the sugar residue of the γ-cyclodextrin via an amide bond.

3. The modified γ-cyclodextrin is given by the following formula (1): 【Chemistry 1】 (In the above formula, m is 0 to 7, n is 0 to 8, o is 0 to 8, and m + n + o = 8, and n R １ and R ２ Each of these is independently a hydroxyl group or a group represented by the following formula (a); -NH-Z...(a) [In the formula, Z is a monovalent group formed by removing a hydroxyl group (-OH) from the carboxyl group (-COOH) of galacturonic acid.] and o R ３ , R 4 and R 5 Each of these independently represents either a hydroxyl group or a group represented by formula (a) above. Here, in equation (1), if n + o is 2 or greater, the arrangement of structures (A), (B), and (C) below is arbitrary, and the n (B)s may be the same or different, and the o (C)s may be the same or different. 【Chemistry 2】 The pharmaceutical composition according to claim 1, wherein the compound is represented by the compound.

4. The modified γ-cyclodextrin is given by the following formula (2): 【Transformation 3】 (In the above formula, m is 0 to 7, n is 1 to 8, and m + n = 8, and n R 1 or R ２ Each of these is independently a hydroxyl group or a group represented by the following formula (a); -NH-Z...(a) [In the formula, Z is a monovalent group formed by removing a hydroxyl group (-OH) from a carboxyl group (-COOH) of galacturonic acid.] Here, in equation (2), if n is 2 or greater, the arrangement of structure (A) and structure (B) below is arbitrary, and the n B's may be the same or different. 【Chemistry 4】 Or the following formula (3): 【Transformation 5】 (In the above formula, m is 0 to 7, o is 1 to 8, and m + o = 8, and the o Rs ３ , R 4 and R 5 are each independently a hydroxyl group or a group represented by the following formula (a): -NH-Z...(a) [In the formula, Z is a monovalent group formed by removing a hydroxyl group (-OH) from a carboxyl group (-COOH) of galacturonic acid.] Here, in equation (3), if o is 2 or more, the arrangement of structure (A) and structure (C) below is arbitrary, and the o (C)s may be the same or different. 【Transformation 6】 The pharmaceutical composition according to claim 1, wherein the compound is represented by [the compound].

5. The modified γ-cyclodextrin is represented by formula (2), where n is 1, R １ R is the above formula (a), ２ The pharmaceutical composition according to claim 4, wherein is OH.

6. The modified γ-cyclodextrin is represented by formula (3), where o is 1, R ３ and R 4 OH, R 5 The pharmaceutical composition according to claim 4, wherein is the above formula (a).

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein the lysosomal storage disease is a lipid storage disorder.

8. The pharmaceutical composition according to claim 7, wherein the lipid storage disorder is selected from the group consisting of Niemann-Pick disease type C, GM1 gangliosidosis, and GM2 gangliosidosis.

9. The pharmaceutical composition according to any one of claims 1 to 6, characterized in that the pharmaceutical composition is administered parenterally.

10. The pharmaceutical composition according to any one of claims 1 to 6, characterized in that the pharmaceutical composition is administered as an injectable preparation.

Images