Pharmaceutical composition for treating non-alcoholic steatohepatitis

Abstract

One problem addressed by the present invention is to provide a composition and treatment method for use in treating non-alcoholic steatohepatitis. As one means for solving this problem, the present invention provides a composition for use in treating non-alcoholic steatohepatitis, the composition containing an acid-decomposable polyrotaxane compound that includes a plurality of cyclic molecules and a straight-chain molecule having a terminal group. The polyrotaxane compound used in the present invention may be a compound that decomposes in an acidic environment within cells or in an acidic environment having a pH of 4.0-6.0. The cyclic molecules in the polyrotaxane can be β-cyclodextrins, and the hydroxyl groups of the β-cyclodextrins may be modified by water-soluble functional groups.

Description

[Technical Field]

[0001] The present invention relates to a composition for use in the treatment of non-alcoholic steatohepatitis. More specifically, the present invention relates to a composition for use in the treatment of non-alcoholic steatohepatitis, comprising an acid-degradable polyrotaxane. The present invention also relates to the use of the acid-degradable polyrotaxane in the treatment of non-alcoholic steatohepatitis. [Background technology]

[0002] Nonalcoholic steatohepatitis (NASH) is one of the lifestyle-related diseases with the greatest unmet medical needs. While previously considered to have a favorable prognosis, clinical evidence has revealed that a subset of patients with fatty liver disease can progress to NASH and further develop into cirrhosis and hepatocellular carcinoma. NASH is thought to develop when liver metabolic abnormalities, such as fat accumulation and insulin resistance, are combined with various inflammatory stimuli, including inflammatory cytokines, endotoxin, and oxidative stress. However, much of its pathogenesis remains unknown. Furthermore, a highly invasive liver biopsy is required for definitive diagnosis and assessment of treatment efficacy, and there are currently no specific therapeutic agents for NASH.

[0003] Meanwhile, Suganami, Ito, and others found that mice lacking the melanocortin 4 receptor (MC4R), a gene known to cause human hereditary obesity, fed a high-fat diet developed fatty liver, NASH, and hepatocellular carcinoma over time, against the backdrop of obesity and insulin resistance (Non-Patent Document 1, Patent Document 1, Patent Document 2). This model closely reflects the characteristics of human NASH and hepatocellular carcinoma in terms of liver histopathology and gene expression profile, and can be considered an animal model of NASH and hepatocellular carcinoma that develops against the backdrop of obesity and insulin resistance. Furthermore, Suganami, Ito, and others found histological features of Kupffer cells accumulating around hepatocyte death due to excessive lipid accumulation (CLS: crown-like structures) in this NASH mouse model and in human NASH, demonstrating that CLS is the starting point for liver fibrosis (Non-Patent Document 2).

[0004] Polyrotaxanes are compounds with a structure in which a linear molecule (also called the main axis) serves as an axis and passes through multiple macrocyclic molecules (rings). Bulky moieties are attached to both ends of the axis, preventing the macrocyclic molecules from slipping off the axis due to steric hindrance. The bulky moieties at both ends are called stoppers, caps, or terminal groups. If there are no stoppers, or if the stoppers are present but are not bulky enough, the ring and axis may separate, resulting in a compound called pseudopolyrotaxane.

[0005] Tamura et al. have reported an acid-degradable polyrotaxane containing β-cyclodextrin as a cyclic molecule (Non-Patent Document 3, Patent Document 3). This acid-degradable polyrotaxane can decompose in the intracellular environment and release the β-cyclodextrin that had been embedded within the cell. Because conventional free β-cyclodextrin primarily interacts with the cell membrane, acid-degradable polyrotaxanes are being investigated for their potential application in disease treatment as compounds capable of altering the site of action of β-cyclodextrin. The above documents (Non-Patent Document 3 and Patent Document 3) tested the therapeutic effect on Niemann-Pick disease type C, in which cholesterol accumulates in the lysosomes of cells, and reported that acid-degradable polyrotaxanes improved cholesterol accumulation at lower concentrations than conventional β-cyclodextrins. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Patent No. 6670556 [Patent Document 2] Patent Publication No. 2020-065454 [Patent Document 3] Patent No. 6464087 [Non-patent literature]

[0007] [Non-Patent Document 1] Am. J. Pathol. 2018, 188, 1213-1224, Am. J. Pathol. 2011, 179, 2454-2463 [Non-patent document 2] JCI Insight 2017, 2, e92902, PLoS ONE 2013, 8, e82163 [Non-patent document 3] J. Control. Release 2018, 269, 148-158 Summary of the Invention [Problem to be solved by the invention]

[0008] An object of the present invention is to provide a method for treating non-alcoholic steatohepatitis. Another object of the present invention is to provide a composition for use in treating non-alcoholic steatohepatitis. A further object of the present invention is to provide a method for suppressing liver fibrosis and a composition for use therein.

[0009] The present inventors have demonstrated that Kupffer cells, upon interaction with dead cells, transform into CD11c-positive cells and acquire pro-fibrotic properties, and that the progression from fatty liver to NASH can be suppressed by eliminating CD11c-positive Kupffer cells. Furthermore, while accumulating knowledge about the molecular pathogenesis of CLS, they have also identified hepatic metabolic alterations that are key to the formation and activation of CLS. Specifically, while in simple fatty liver, non-cytotoxic triglycerides accumulate within hepatocytes, in NASH, free cholesterol accumulates and crystallizes. Interestingly, the present inventors have demonstrated the presence of cholesterol crystals within CLS, indicating that the CD11c-positive Kupffer cells that comprise CLS are in a state of cholesterol overload.

[0010] Based on these findings, we hypothesized that reducing excess cholesterol in CD11c-positive Kupffer cells, which constitute the CLS, would lead to the suppression of fibrosis. Based on this hypothesis, we investigated the use of supramolecular polyrotaxanes to reduce cholesterol accumulation. The polyrotaxanes we used consist of a cyclic oligosaccharide, β-cyclodextrin (β-CD), with a polymer perforated into the cavity. These polyrotaxanes are designed to decompose in the acidic intracellular environment and release β-CD. Such polyrotaxanes have previously been shown to suppress intracellular cholesterol accumulation and promote its excretion, and have been shown to exhibit superior cholesterol accumulation suppression and excretion promotion effects compared to β-CD alone in Niemann-Pick disease type C (NPC) model mice (J. Control. Release 2018, 269, 148-158).

[0011] In the NASH model described above, continuous subcutaneous administration of low-dose polyrotaxane (30 mg / kg / day) using an osmotic minipump for 4–6 weeks resulted in a significant decrease in cholesterol content in CD11c-positive Kupffer cells, which in turn led to a marked decrease in the number of CLS in the liver and suppression of liver fibrosis. However, administration of this low-dose polyrotaxane did not significantly affect cholesterol levels in the entire liver. Thus, low-dose polyrotaxane suppresses liver fibrosis by targeting intracellular cholesterol in CD11c-positive Kupffer cells, which constitute the CLS, and may be useful as a novel therapeutic agent for NASH. [Means for solving the problem]

[0012] The present invention is based on these findings, and its aspects relate to the following matters. [1] A pharmaceutical composition for use in treating non-alcoholic steatohepatitis, comprising an acid-degradable polyrotaxane compound. [2] A pharmaceutical composition for use in inhibiting liver fibrosis, comprising an acid-degradable polyrotaxane compound. [3] A pharmaceutical composition for use in inhibiting cholesterol accumulation in macrophages, comprising an acid-degradable polyrotaxane compound. [4] The pharmaceutical composition according to any one of aspects [1] to [3], which is degraded in an acidic environment within a cell or an acidic environment of pH 4.0 to 6.0. [5] The pharmaceutical composition according to any one of aspects [1] to [4], wherein the cyclic molecule of the polyrotaxane compound is β-cyclodextrin. [6] The pharmaceutical composition according to embodiment [5], wherein the hydroxyl group of the β-cyclodextrin is modified with a water-soluble functional group. [7] The water-soluble functional group is a 2-(2-hydroxyethoxy)ethyl (HEE) group, a 2-[2-[2-hydroxyethoxy]ethoxy]ethyl (HEEE) group, a 2-[2-[2-(2-hydroxyethoxy]ethoxy]ethoxy]ethyl (HEEEE) group, a 2-(2-methoxyethoxy)ethyl (MEE) group, a 2-[2-[2-methoxyethoxy]ethoxy]ethyl (MEEE) group, a 2-[2-[2-(2-methoxyethoxy]ethoxy]ethoxy]ethyl (MEEEE) group, a 2-(2-ethoxyethoxy)ethyl (EEE) group, a 2-[2-[2-ethoxyethoxy]ethoxy]ethyl (EEEE) group, a 2-[2-[2-(2-ethoxyethoxy]ethoxy]ethoxy]ethyl (EEEEE) group, a hydroxyethyl group, a hydroxypropyl group, a hydroxybutyl group, a carboxymethyl group, carboxyethyl group , a carboxypropyl group, a methyl group, a sugar chain, polyethylene glycol, transferrin, an antibody, and an oligopeptide. [8] The pharmaceutical composition according to embodiment [6] or [7], wherein the β-cyclodextrin has 1 to 21 water-soluble functional groups per molecule. [9] The pharmaceutical composition according to any one of aspects [1] to [8], wherein the linear molecule of the polyrotaxane compound comprises polyethylene glycol (PEG) and / or polypropylene glycol (PPG).

[10] The pharmaceutical composition according to any one of aspects [1] to [9], wherein the linear molecule of the polyrotaxane compound has an acid-decomposable bond selected from an acetal bond, a ketal bond, a disulfide bond, an ester bond, an orthoester bond, a vinyl ether bond, a hydrazide bond, and an amide bond.

[11] The pharmaceutical composition according to any one of aspects [1] to

[10] , wherein the linear molecule of the polyrotaxane compound comprises a poloxamer.

[12] The pharmaceutical composition according to any one of aspects [1] to

[11] , wherein the molecular weight of the linear molecule of the polyrotaxane compound is 4,000 to 12,000.

[13] The pharmaceutical composition according to any one of aspects [1] to

[12] , wherein the ratio of the number of linear molecules to the number of cyclic molecules of the polyrotaxane compound is 1:10 to 1:20.

[14] The pharmaceutical composition according to any one of aspects [1] to

[13] , wherein the terminal group of the polyrotaxane compound is selected from the group consisting of an O-triphenylmethyl group with or without a substituent, an S-triphenylmethyl group with or without a substituent, and an N-triphenylmethyl group with or without a substituent.

[15] The pharmaceutical composition according to any one of aspects [1] to

[14] , wherein an end group of the polyrotaxane compound is linked to a linear molecule of the polyrotaxane compound via a peptide bond, a carbamate bond, an ester bond, or an ether bond.

[16] An acid-decomposable polyrotaxane compound containing β-cyclodextrin and poloxamer, having the following structural formula: [ka] where: m is an integer of 0 to 200 that represents the number of repeating polypropylene glycol units in the poloxamer; n is an integer of 0 to 200 that represents the number of polyethylene glycol repeating units in the poloxamer; The average molecular weight of the poloxamer is 4,000 to 12,000, x is an integer of 10 to 20 indicating the number of β-cyclodextrins; β-cyclodextrin has 1 to 21 water-soluble functional groups R per molecule, The water-soluble functional group may be a 2-(2-hydroxyethoxy)ethyl (HEE) group, a 2-[2-[2-hydroxyethoxy]ethoxy]ethyl (HEEE) group, a 2-[2-[2-(2-hydroxyethoxy]ethoxy]ethoxy]ethyl (HEEEE) group, a 2-(2-methoxyethoxy)ethyl (MEE) group, a 2-[2-[2-methoxyethoxy]ethoxy]ethyl (MEEE) group, a 2-[2-[2-(2-methoxyethoxy]ethoxy]ethoxy]ethyl (MEEEE) group, a 2-(2-ethoxyethoxy)ethyl (EEE) group, a 2-[2-[2-ethoxyethoxy]ethoxy]ethyl (EEEE) group, a 2-[2-[2-(2-ethoxyethoxy]ethoxy]ethoxy]ethyl (EEEEE) group, a hydroxyethyl group, a hydroxypropyl group, a hydroxybutyl group, a carboxymethyl group, carboxyethyl group , a carboxypropyl group, and a methyl group; L is a single bond, a carbamate bond (-O-CO-NH-), an ester bond (-O-CO-), a carbonate bond (-O-CO-O-), or an ether bond (-O-), The terminal group Z is an N-triphenylmethyl group, Y is a single bond or a peptide bond, a carbamate bond, an ester bond, or an ether bond; The pharmaceutical composition according to any one of aspects [1] to

[15] , comprising a polyrotaxane compound having an overall average molecular weight of 20,000 to 50,000.

[17] The pharmaceutical composition according to any one of aspects [1] to

[16] , which is a composition for subcutaneous, intraperitoneal, or intravenous administration.

[18] The pharmaceutical composition according to any one of aspects [1] to

[17] , which is a composition administered at a dose of 3 to 35 mg / kg / day.

[19] A method for treating non-alcoholic steatohepatitis, comprising administering an acid-degradable polyrotaxane compound to a subject.

[20] A method for inhibiting liver fibrosis, comprising administering an acid-degradable polyrotaxane compound to a subject.

[21] A method for inhibiting cholesterol accumulation in macrophages, comprising administering an acid-degradable polyrotaxane compound to a subject.

[22] The method according to any one of embodiments

[19] to

[21] , wherein the administration is subcutaneous, intraperitoneal, or intravenous.

[23] The method according to any one of embodiments

[19] to

[22] , wherein the dosage is 3 to 35 mg / kg / day.

[24] Use of an acid-degradable polyrotaxane compound in the manufacture of a medicament for use in the treatment of non-alcoholic steatohepatitis.

[25] Use of an acid-degradable polyrotaxane compound in the manufacture of a medicament for use in inhibiting liver fibrosis.

[26] Use of an acid-degradable polyrotaxane compound in the manufacture of a medicament for use in inhibiting cholesterol accumulation in macrophages.

[27] The method according to any one of embodiments

[24] to

[26] , wherein the agent is for subcutaneous, intraperitoneal, or intravenous administration.

[28] The pharmaceutical composition according to any one of aspects

[24] to

[27] , wherein the drug is administered at a dose of 3 to 35 mg / kg / day. [Brief explanation of the drawings]

[0013] [Figure 1] Figure 1 shows the results of continuous subcutaneous administration of HEE-PRX for 6 weeks by subcutaneously implanting it into an osmotic minipump at 30 mg / kg / day into a mouse. After the administration period, blood was collected from the mouse and neutral fat and total cholesterol were measured using a biochemical colorimetric method. [Figure 2] FIG. 2 is a graph showing the results of measuring blood alanine aminotransferase (ALT) by biochemical colorimetry, similar to FIG. [Figure 3] FIG. 3 is a graph showing the results of extracting livers from MC4R-deficient mice that had been given continuous subcutaneous administration of HEE-PRX for 6 weeks, extracting lipids, and measuring them by a biochemical colorimetric method. [Figure 4]Figure 4 shows the results of histological analysis of livers taken from MC4R-deficient mice that had received continuous subcutaneous administration of HEE-PRX for 6 weeks, which were fixed in formalin and immunohistochemically analyzed for F4 / 80, a macrophage marker, to evaluate crown-like structures (CLS) in the tissue. [Figure 5] FIG. 5 shows images of fibrotic tissue stained with Sirius Red, and graphs showing the results of quantifying the fibrotic area from the stained images. [Figure 6] FIG. 6 is a graph showing that administration of HEE-PRX suppresses gene expression (mRNA level) of profibrotic factors (Pdgfb, Spp1) and extracellular matrix factors (Col1a1, Col4a1) in the liver. [Figure 7] FIG. 7 is a graph showing that cholesterol crystals increase the mRNA levels of Pdgfb and Spp1 in cultured macrophages, and that the addition of HEE-PRX suppresses the effect of cholesterol crystals. DETAILED DESCRIPTION OF THE INVENTION

[0014] The present invention is described in detail below. The present inventors have discovered that the use of polyrotaxane, which decomposes at an acidic pH to release β-cyclodextrin, reduces cholesterol accumulation in macrophages and inhibits liver fibrosis.

[0015] Polyrotaxane (PRX) compounds Rotaxanes are macrocyclic molecules threaded by linear molecules, with bulky moieties attached to both ends of the linear molecules, preventing the ring from slipping off the axis due to steric hindrance. In polyrotaxanes, one linear molecule threads through the rings of multiple macrocyclic molecules.

[0016] The linear molecules and cyclic molecules used in the present invention are not particularly limited, but the linear molecules are preferably one or more selected from the group consisting of polyethylene glycol, polypropylene glycol, copolymers of polyethylene glycol and polypropylene glycol (poloxamer), polyethyleneimine, polyamino acids, and polymethyl vinyl ether. The average molecular weight of the linear molecules is preferably 1,000 to 20,000, particularly 2,000 to 15,000 or 4,000 to 12,000. For example, a poloxamer with a molecular weight of approximately 5,000 can be used.

[0017] The cyclic molecule is preferably α-, β-, or γ-cyclodextrin, but may also have a similar cyclic structure, such as cyclic polyether, cyclic polyester, cyclic polyetheramine, cyclic polyamine, etc. From the viewpoint of cholesterol encapsulation ability, the preferred cyclic molecule is β- or γ-cyclodextrin, with β-cyclodextrin being particularly preferred.

[0018] The cyclic molecules contained in the polyrotaxane according to the present invention may be chemically modified at their functional groups to impart water solubility. The cyclic molecules are preferably cyclodextrins, and the hydroxyl groups of the cyclodextrins may be modified with water-soluble functional groups. The cyclodextrin is preferably β-cyclodextrin. The β-cyclodextrin may be modified to have, for example, 1 to 21, preferably 2 to 7, water-soluble functional groups per molecule. Examples of the functional group include a 2-(2-hydroxyethoxy)ethyl (HEE) group, a 2-[2-[2-hydroxyethoxy]ethoxy]ethyl (HEEE) group, a 2-[2-[2-(2-hydroxyethoxy]ethoxy]ethoxy]ethyl (HEEEE) group, a 2-(2-methoxyethoxy)ethyl (MEE) group, a 2-[2-[2-methoxyethoxy]ethoxy]ethyl (MEEE) group, a 2-[2-[2-(2-methoxyethoxy]ethoxy]ethoxy]ethyl (MEEEE) group, a 2-(2-ethoxyethoxy)ethyl (EEE) group, a 2-[2-[2-ethoxyethoxy]ethoxy]ethyl (EEEE) group, a 2-[2-[2-(2-ethoxyethoxy]ethoxy]ethoxy]ethyl (EEEEE) group, a hydroxyethyl group, a hydroxypropyl group, a hydroxybutyl group, a carboxymethyl group, carboxyethyl group Examples of suitable linkers include carboxypropyl groups, methyl groups, sugar chains such as mannose and galactose, water-soluble polymers such as polyethylene glycol, protein molecules such as transferrin and antibodies, and oligopeptide molecules such as oligoarginine. These groups may be directly bonded to the cyclic molecule or may be bonded via a linker. The linker is not particularly limited and can be appropriately selected depending on the purpose. Examples include a carbamate ester bond (-O-CO-NH-), an ester bond (-O-CO-), a carbonate bond (-O-CO-O-), and an ether bond (-O-). In one embodiment, a carbamate ester bond (-O-CO-NH-) is preferably used as the linker. Therefore, the carboxy group may be in the form of a carboxymethyl carbamate ester group, a carboxyethyl carbamate ester group, or a carboxypropyl carbamate ester group.

[0019] A preferred combination of linear molecules and cyclic molecules is a combination of β-cyclodextrin and poloxamer. The synthesis of polyrotaxanes using a combination of β-cyclodextrin and poloxamer is also disclosed in the aforementioned Japanese Patent No. 6464087, the contents of which are incorporated herein by reference. The ratio of the number of linear molecules to the number of cyclic molecules is preferably 1:4 to 1:50, more preferably 1:8 to 1:20, for example, 1:10 to 1:20. That is, preferably, one linear molecule contains 4 to 50 cyclic molecules, more preferably 8 to 30 cyclic molecules, for example, 10 to 20 cyclic molecules.

[0020] Examples of terminal groups (also referred to as bulky substituents) used in the present invention include, but are not limited to, O-triphenylmethyl (O-Trt) groups, S-triphenylmethyl (S-Trt) groups, and N-triphenylmethyl (N-Trt) groups. The terminal groups prevent the cyclic molecules of polyrotaxane from escaping from the linear molecule due to a steric hindrance effect. The terminal groups may be substituted O-triphenylmethyl groups, S-triphenylmethyl groups, N-triphenylmethyl groups, etc. N-triphenylmethyl groups are preferred. The N-Trt groups decompose in a weakly acidic environment, causing the polyrotaxane skeleton to collapse and releasing cyclic molecules such as β-CD. The terminal groups can be linked to the linear molecule via peptide bonds, carbamate bonds, ester bonds, ether bonds, etc., but peptide bonds are preferred. Therefore, one aspect of the present invention relates to an acid-decomposable polyrotaxane compound comprising a β-cyclodextrin having a substituent as a cyclic molecule and a poloxamer having an N-triphenylmethyl group at its terminal as a linear molecule. The substituent of the β-cyclodextrin is preferably, but not limited to, an HEE group, for example, a 2-[2-[2-hydroxyethoxy]ethoxy]ethyl (HEEE) group, a 2-[2-[2-(2-hydroxyethoxy]ethoxy]ethoxy]ethyl (HEEEE) group, a 2-(2-methoxyethoxy)ethyl (MEE) group, a 2-[2-[2-methoxyethoxy]ethoxy]ethyl (MEEE) group, 2-[2-[2-(2-methoxyethoxy]ethoxy]ethoxy]ethyl (MEEEE) group, 2-(2-ethoxyethoxy)ethyl (EEE) group, 2-[2-[2-ethoxy]ethoxy]ethyl (EEEE) group, 2-[2-[2-(2-ethoxy]ethoxy]ethoxy]ethyl (EEEEE) group, hydroxyethyl group, hydroxypropyl group, hydroxybutyl group, carboxymethyl group, carboxyethyl groupThe cyclic molecule may be a carboxypropyl group, a methyl group, a sugar chain such as mannose or galactose, a water-soluble polymer such as polyethylene glycol, a protein molecule such as transferrin or an antibody, or an oligopeptide molecule such as oligoarginine. These groups may be directly bonded to the cyclic molecule or may be bonded via a linker. The linker is not particularly limited and can be appropriately selected depending on the purpose. Examples of the linker include a carbamate ester bond (-O-CO-NH-), an ester bond (-O-CO-), a carbonate bond (-O-CO-O-), and an ether bond (-O-).

[0021] The polyrotaxane compound according to the present invention may have a structure containing an acid-decomposable bond within a linear molecule, or a structure in which a terminal group is connected to a linear molecule via an acid-decomposable bond. Examples of acid-decomposable bonds include, but are not limited to, acetal bonds, ketal bonds, disulfide bonds, ester bonds, orthoester bonds, vinyl ether bonds, hydrazide bonds, and amide bonds. These may be used alone or in combination. Examples of terminal groups that may be used include, but are not limited to, N-tritylglycine, groups having one or more benzene rings, and groups having one or more tertiary butyl groups. Examples of groups having one or more benzene rings include, but are not limited to, benzyloxycarbonyl (Z) groups, 9-fluorenylmethyloxycarbonyl (Fmoc) groups, and benzyl ester (OBz) groups. Examples of groups having one or more tertiary butyl groups include, but are not limited to, tertiary butylcarbonyl (Boc) groups and amino acid tertiary butyl ester (OBu) groups. The acid-decomposable bond and the terminal group do not necessarily have to be directly linked, but may be linked via a linker moiety known to those skilled in the art.

[0022] The number average molecular weight of the polyrotaxane according to the present invention is not particularly limited and can be appropriately selected depending on the purpose, but is preferably about 10,000 to 100,000.

[0023] The polyrotaxane compound according to the present invention is particularly preferably a polyrotaxane having a linear molecule that is a poloxamer containing polyethylene glycol (PEG) and polypropylene glycol (PPG), a cyclic molecule that is β-cyclodextrin, and an N-triphenylmethyl terminal group that is linked to the linear molecule via a peptide bond. In this case, the molecular weight of the poloxamer may be 4,000 to 12,000 (e.g., about 5,000), and the number of β-cyclodextrins per linear molecule may be 10 to 20 (e.g., about 16). Therefore, the polyrotaxane compound according to the present invention includes a polyrotaxane having the following chemical structure: [ka] Here, m is an integer representing the number of repeating polypropylene glycol units in the poloxamer (here, three repeating polypropylene glycol units are listed in the parentheses, so "m / 3" is written, but m does not need to be a multiple of 3). n is an integer representing the number of repeating polyethylene glycol units. m and n are each, for example, integers from 0 to 200, from 10 to 100, or from 30 to 70, and can be appropriately determined depending on the poloxamer used. x is an integer representing the number of β-cyclodextrins. R represents a substituent and is bonded to the β-cyclodextrin via a chemical bond L. The chemical bond L can be, for example, a carbamate ester bond (-O-CO-NH-), an ester bond (-O-CO-), a carbonate bond (-O-CO-O-), or an ether bond (-O-) formed by modifying a hydroxyl group of the cyclodextrin. In the present disclosure, the substituent R is a water-soluble functional group, such as a 2-(2-hydroxyethoxy)ethyl (HEE) group, a 2-[2-[2-hydroxyethoxy]ethoxy]ethyl (HEEE) group, a 2-[2-[2-(2-hydroxyethoxy]ethoxy]ethoxy]ethyl (HEEEE) group, a 2-(2-methoxyethoxy)ethyl (MEE) group, a 2-[2-[2-methoxyethoxy]ethoxy]ethyl (MEE) group, a 2-[2-[2-methoxyethoxy]ethoxy]ethyl (MEE) group, a 2-[2-[2-methoxyethoxy]ethoxy]ethyl (MEE) group, a 2-[2-[2-hydroxy ... EE) group, 2-[2-[2-(2-methoxyethoxy]ethoxy]ethoxy]ethyl (MEEEE) group, 2-(2-ethoxyethoxy)ethyl (EEE) group, 2-[2-[2-ethoxyethoxy]ethoxy]ethyl (EEEE) group, 2-[2-[2-(2-ethoxyethoxy]ethoxy]ethoxy]ethyl (EEEEE) group, hydroxyethyl group, hydroxypropyl group, hydroxybutyl group, carboxymethyl group, carboxyethyl groupThe substituent may be a carboxypropyl group, a methyl group, a sugar chain such as mannose or galactose, a water-soluble polymer such as polyethylene glycol, a protein molecule such as transferrin or an antibody, or an oligopeptide molecule such as oligoarginine. For convenience of illustration, only one substituent is shown on the β-cyclodextrin, but the number of substituents may be multiple. In some cases, the chemical bond L may not be interposed. Z represents a capping molecule (e.g., a terminal group such as an N-triphenylmethyl group) and is bonded to both ends of the main polymer axis via chemical bonds Y (e.g., peptide bonds). Chemical bonds Y may be acid-decomposable bonds, but in some cases, the chemical bond Y may not be interposed.

[0024] intracellular environment The polyrotaxane compound according to the present invention is an acid-decomposable polyrotaxane compound that decomposes in an acidic environment, for example, in an acidic environment of pH 4.0 to 6.0. As described above, when an N-Trt group is used as the terminal group, the N-Trt group is decomposed in a weakly acidic environment, causing the polyrotaxane skeleton to collapse and releasing cyclic molecules such as β-CD.

[0025] Vesicles such as lysosomes and late endosomes exist within the cells of eukaryotes, including humans, and the lumen of these vesicles is known to be acidic. For example, the pH of the lumen of a lysosome is approximately 5. Therefore, the polyrotaxane compound of the present invention can be degraded by being incorporated into these vesicles. Upon degradation, the polyrotaxane compound of the present invention releases cyclic molecules such as β-CD.

[0026] As described above, β-cyclodextrin is known to have the ability to encapsulate cholesterol. Those skilled in the art will understand that encapsulation of intracellular cholesterol by β-cyclodextrin enables the treatment of diseases caused by excessive intracellular cholesterol. Therefore, one aspect of the present invention relates to a pharmaceutical composition for treating diseases caused by intracellular cholesterol accumulation, comprising an acid-degradable polyrotaxane compound comprising a plurality of cyclodextrin molecules modified with water-soluble functional groups. From another perspective, one aspect of the present invention relates to a method for treating or preventing NASH, comprising administering to a subject, preferably a human, in need of such treatment or prevention an acid-degradable polyrotaxane compound comprising a plurality of cyclodextrin molecules modified with water-soluble functional groups. Furthermore, one aspect of the present invention relates to the use of an acid-degradable polyrotaxane compound comprising a plurality of cyclodextrin molecules modified with water-soluble functional groups in the manufacture of a medicament for treating or preventing NASH.

[0027] Furthermore, one aspect of the present invention relates to a pharmaceutical composition for treating or preventing NASH, which contains an acid-degradable polyrotaxane compound comprising a plurality of chemically modified cyclodextrin molecules. From another perspective, one aspect of the present invention relates to a method for treating or preventing NASH, comprising administering to a subject, preferably a human, an acid-degradable polyrotaxane compound comprising a plurality of chemically modified cyclodextrin molecules. Another aspect of the present invention relates to the use of an acid-degradable polyrotaxane compound comprising a plurality of chemically modified cyclodextrin molecules in the manufacture of a medicament for treating or preventing NASH. Another aspect of the present invention also relates to an acid-degradable polyrotaxane compound, as described above, comprising a β-cyclodextrin having a substituent as a cyclic molecule and a poloxamer having an N-triphenylmethyl group at its terminal as a linear molecule. Furthermore, one aspect of the present invention relates to a pharmaceutical composition for treating or preventing NASH, which contains an acid-degradable polyrotaxane compound comprising, as a cyclic molecule, a β-cyclodextrin having a substituent and, as a linear molecule, a poloxamer having an N-triphenylmethyl group at its terminal.

[0028] Nonalcoholic steatohepatitis (NASH) refers to a liver disease accompanied by fat accumulation in hepatocytes, hepatocyte ballooning, increased apoptosis, infiltration of inflammatory cells into the central vein region of the liver, a phenomenon in which macrophages surround and phagocytose / process fatty liver cells (CLS), and deposition of excessive extracellular matrix in the liver. NASH of the present invention also includes cirrhosis or hepatocellular carcinoma that develops after the progression of these symptoms. Furthermore, NASH of the present invention also includes not only such histological changes but also high serum concentrations of at least one marker selected from alanine aminotransferase (ALT), aspartate aminotransferase (AST), free fatty acids, and triglycerides.

[0029] Pharmaceutical Composition The polyrotaxane compound according to the present invention can be used as an active ingredient in a pharmaceutical composition used for treating or preventing the above-mentioned diseases. Therefore, one aspect of the present invention relates to a pharmaceutical composition used for treating or preventing diseases. Other ingredients in the pharmaceutical composition according to the present invention are not particularly limited and can be selected appropriately depending on the purpose, and examples include pharmaceutically acceptable carriers. The carrier is also not particularly limited and can be selected appropriately depending on, for example, the dosage form. The content of the compound in the pharmaceutical composition according to the present invention is also not particularly limited and can be selected appropriately depending on the purpose. Preferably, the pharmaceutical composition according to the present invention is water-soluble near body temperature, for example, at 34°C to 42°C, more preferably at 35°C to 38°C or 37°C.

[0030] The dosage form of the pharmaceutical composition of the present invention is not particularly limited and can be appropriately selected depending on the desired administration method. Examples include injections (solutions, suspensions, solid preparations to be dissolved when needed, etc.) and inhalable powders. For example, injections for subcutaneous, intraperitoneal, intramuscular, intravenous, etc. can be prepared by adding a pH adjuster, buffer, stabilizer, isotonicity agent, local anesthetic, etc. to the polyrotaxane compound of the present invention in accordance with a conventional method. Examples of pH adjusters and buffers include sodium citrate, sodium acetate, and sodium phosphate. Examples of stabilizers include sodium pyrosulfite, EDTA, thioglycolic acid, and thiolactic acid. Examples of isotonicity agents include sodium chloride and glucose. Examples of local anesthetics include procaine hydrochloride and lidocaine hydrochloride.

[0031] The method of administration of the pharmaceutical composition of the present invention is not particularly limited, and either local administration or systemic administration can be selected depending on, for example, the dosage form of the pharmaceutical composition, the patient's condition, etc. Examples include intermittent subcutaneous administration using a pen-type syringe, as with insulin preparations, or continuous subcutaneous injection using a portable syringe pump. The duration of continuous subcutaneous administration is approximately one week, as with insulin preparations, with the device replaced. The upper limit of the duration can be determined based on the needs of the treatment, but treatment may be interrupted after, for example, one month, two months, three months, six months, or twelve months. After administration is interrupted, administration can be resumed after a period of, for example, two weeks, four weeks, six weeks, or two months.

[0032] The dosage can be, for example, 1 mg / kg / day, 3 mg / kg / day, 5 mg / kg / day, 10 mg / kg / day, 20 mg / kg / day, 30 mg / kg / day, 40 mg / kg / day, 50 mg / kg / day, 75 mg / kg / day, 100 mg / kg / day, or any range between 1 and 100 mg / kg / day, for example, 1 to 40 mg / kg / day or 3 to 35 mg / kg / day.

[0033] The subjects to which the pharmaceutical composition of the present invention is administered are not particularly limited and can be appropriately selected depending on the purpose. Examples include humans and non-human mammals such as mice, rats, cows, pigs, monkeys, dogs, and cats, with humans being preferred.

[0034] The dosage of the pharmaceutical composition of the present invention is not particularly limited and can be appropriately selected depending on the dosage form, the age and body weight of the subject, the degree of the desired effect, and the like.

[0035] The timing of administration of the pharmaceutical composition of the present invention is not particularly limited and can be appropriately selected depending on the purpose, and for example, it may be administered prophylactically to patients susceptible to the above-mentioned diseases, or may be administered therapeutically to patients exhibiting symptoms. The number of administrations is also not particularly limited and can be appropriately selected depending on the age, body weight, desired degree of effect, etc. of the subject to be administered.

[0036] The present invention will be specifically explained below by showing examples, but the present invention is not limited by these examples. [Example]

[0037] <Example 1: Preparation of HEE group-modified polyrotaxane> Polyrotaxanes were synthesized using Pluronic® P123 (Sigma-Aldrich Corporation, a copolymer of polyethylene glycol (PEG) and polypropylene glycol (PPG) polymerized in the order PEG-PPG-PEG; the number-average molecular weight of the PPG portion was 3,850, and the number-average molecular weight of the PEG portion was 860 × 2) as the linear polymer and β-CD (Nihon Shokuhin Kako Co., Ltd., trade name: Celdex B-100) as the cyclic molecule, according to the method described in Japanese Patent No. 6,464,087. Next, polyrotaxanes were obtained in which the β-CD in the polyrotaxane was modified with an HEE group via a carbamate bond (hereinafter referred to as "HEE-PRX") according to the method described in Japanese Patent No. 6,464,087. Proton nuclear magnetic resonance spectroscopy (Bruker) at 400 MHz measured in dimethyl sulfoxide-d6 (Kanto Chemical) showed that the average number of β-CD threadings per polyrotaxane molecule was 16.6, the number of HEE group modifications was 97.8, and the number-average molecular weight of HEE-PRX was 38,000. The general chemical structure of HEE-PRX is shown below: [ka] Here, m is an integer representing the number of repeating polypropylene glycol units in the poloxamer; n is an integer representing the number of repeating polyethylene glycol units; and l is an integer representing the number of β-cyclodextrins. β-cyclodextrins are shown here as ring structures in parentheses. For convenience of illustration, only one substituent on the β-cyclodextrin is shown, although multiple substituents are possible.

[0038] <Example 2: Creation of NASH model mice and administration of HEE-PRX> Mice lacking the melanocortin type 4 receptor (hereafter referred to as "MC4R-deficient mice"; genetic background: C57BL / 6J) were kindly provided by Dr. Joel K. Elmquist of the University of Texas Medical Center (see Balthasar N. et al., Cell, November 4, 2005, Vol. 123, No. 3, pp. 493-505). Wild-type C57BL / 6J mice were purchased from CLEA Japan, Inc. as a control.

[0039] Unless otherwise specified, these mice were maintained in a temperature-, humidity-, and lighting-period-controlled environment (12-hour light / dark cycle). After weaning (4 weeks after birth), they were maintained with ad libitum access to water and a standard diet (CLEA Japan, rodent breeding diet: CE-2, 343.1 kcal / 100 g, containing 12.6% fat).

[0040] Eight-week-old male MC4R-deficient mice were fed a high-fat diet (Research Diet, D12079B) for 18 weeks to induce NASH. Subsequently, HEE-PRX prepared in Example 1 was loaded into an osmotic minipump (ALZET) at 30 mg / kg / day and implanted subcutaneously into the mice for continuous subcutaneous administration for 6 weeks. During the administration period, the mice's body weights were measured over time. After the administration period, blood was collected from the mice, and blood glucose, triglycerides, and total cholesterol were measured using biochemical colorimetry. The results are shown in Figure 1. Similarly, blood alanine aminotransferase (ALT) was measured using biochemical colorimetry. The results are shown in Figure 2.

[0041] No change in the body weight of MC4R-deficient mice was observed during the HEE-PRX administration period. Furthermore, as shown in Figure 1, continuous subcutaneous administration of HEE-PRX did not significantly affect blood glucose levels or serum lipids (triglycerides and total cholesterol) in MC4R-deficient mice. However, as shown in Figure 2, ALT was reduced by HEE-PRX administration. These results suggest that continuous administration of HEE-PRX to MC4R-deficient mice improved liver damage without affecting whole-body glycolipid metabolism.

[0042] <Example 3: Cholesterol levels in liver macrophages in NASH model mice> Livers were collected from MC4R-deficient mice that had received continuous subcutaneous administration of HEE-PRX for 6 weeks, and lipids were extracted and measured by biochemical colorimetry. The results are shown in Figure 3. The livers were also dispersed with collagenase, and resident macrophages in the liver were separated according to their CD11c expression level using a cell sorter (FACSAria II, BD Biosciences). The free cholesterol content in the cells separated by the cell sorter was quantified using a gas chromatography mass spectrometer GCMS-QP2020 (Shimadzu Corporation). The amount of intracellular free cholesterol was expressed as cholesterol weight / cell number. The average values ​​are shown in Figure 3.

[0043] The results in Figure 3 show that there was no significant decrease in total cholesterol or triglyceride content in the liver. CD11c-positive macrophages play an important role in the development of fatty liver and NASH. The results in Figure 3 demonstrate that the cholesterol content of CD11c-positive macrophages was significantly reduced by HEE-PRX administration. These results suggest that continuous subcutaneous administration of HEE-PRX to MC4R-deficient mice resulted in HEE-PRX being taken up by CD11c-positive macrophages and acting on intracellular cholesterol to promote its excretion, or that HEE-PRX suppressed the accumulation of cholesterol in CD11c-positive macrophages.

[0044] <Example 4: Histological evaluation of NASH model mice> Livers taken from MC4R-deficient mice that had received continuous subcutaneous administration of the above-mentioned HEE-PRX for 6 weeks were fixed in formalin and subjected to histochemical analysis. Immunostaining for the macrophage marker F4 / 80 was performed to evaluate crown-like structures (CLS) in the tissue. The results are shown in Figure 4. Fibrotic tissue was also stained with Sirius Red, and the fibrotic area was quantified from the stained images. The results are shown in Figure 5.

[0045] In livers with NASH, crown-like structures (CLSs) are observed, where resident macrophages surround apoptotic hepatocytes, and these structures are the starting point for the pathogenesis of the disease. Figure 4 shows that the number of CLSs decreased with HEE-PRX administration. Figure 5 shows that HEE-PRX administration reduced the area of ​​fibrosis. Figure 6 shows that HEE-PRX administration suppressed the gene expression of pro-fibrotic factors (Pdgfb, Spp1) and extracellular matrix proteins (Col1a1, Col4a1) in the liver. Pdgfb and Spp1 are known to be primarily derived from macrophages. Figure 7 shows that cholesterol crystals increased Pdgfb and Spp1 mRNA levels in cultured macrophages, and the addition of HEE-PRX suppressed the effects of cholesterol crystals. These findings suggest that HEE-PRX inhibits liver fibrosis by affecting the activation state of macrophages associated with cholesterol accumulation. [Industrial Applicability]

[0046] As described above, the present inventors focused on cholesterol accumulation in CD11c-positive Kupffer cells that constitute the CLS and demonstrated that the intracellular cholesterol-lowering effect of polyrotaxane leads to the suppression of liver fibrosis in NASH. Because the polyrotaxane of the present invention targets the CLS, it is expected to be highly effective against liver fibrosis, which determines prognosis. Thus, administration of low-dose polyrotaxane has the effect of suppressing liver fibrosis by targeting intracellular cholesterol in CD11c-positive Kupffer cells that constitute the CLS, and can be used in the treatment and prevention of NASH.

Claims

1. A pharmaceutical composition for use in treating non-alcoholic steatohepatitis, comprising an acid-degradable polyrotaxane compound.

2. A pharmaceutical composition for use in inhibiting liver fibrosis, comprising an acid-decomposable polyrotaxane compound.

3. A pharmaceutical composition for use in inhibiting cholesterol accumulation in macrophages, comprising an acid-degradable polyrotaxane compound.

4. The pharmaceutical composition according to any one of claims 1 to 3, which is degraded in an acidic environment within a cell or an acidic environment at a pH of 4.0 to 6.

0.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the cyclic molecule of the polyrotaxane compound is β-cyclodextrin.

6. 6. The pharmaceutical composition according to claim 5, wherein the hydroxyl group of the β-cyclodextrin is modified with a water-soluble functional group.

7. The water-soluble functional group is a 2-(2-hydroxyethoxy)ethyl (HEE) group, a 2-[2-[2-hydroxyethoxy]ethoxy]ethyl (HEEE) group, a 2-[2-[2-(2-hydroxyethoxy]ethoxy]ethoxy]ethyl (HEEEE) group, a 2-(2-methoxyethoxy)ethyl (MEE) group, a 2-[2-[2-methoxyethoxy]ethoxy]ethyl (MEEE) group, a 2-[2-[2-(2-methoxyethoxy]ethoxy]ethoxy]ethyl (MEEEE) group, a 2-(2-methoxyethoxy) ...

7. The pharmaceutical composition of claim 6, wherein the hydroxyl group is selected from the group consisting of a 2-[2-[2-(2-ethoxy)ethoxy]ethyl (EEE) group, a 2-[2-[2-(2-ethoxy)ethoxy]ethoxy]ethyl (EEEE) group, a 2-[2-[2-(2-ethoxy)ethoxy]ethoxy]ethyl (EEEEE) group, a hydroxyethyl group, a hydroxypropyl group, a hydroxybutyl group, a carboxymethyl group, a carboxyethyl group, a carboxypropyl group, a methyl group, a sugar chain, polyethylene glycol, transferrin, an antibody, and an oligopeptide.

8. 8. The pharmaceutical composition according to claim 6, wherein the β-cyclodextrin has 1 to 21 water-soluble functional groups per molecule.

9. The pharmaceutical composition according to any one of claims 1 to 8, wherein the linear molecule of the polyrotaxane compound comprises polyethylene glycol (PEG) and / or polypropylene glycol (PPG).

10. The pharmaceutical composition according to any one of claims 1 to 9, wherein the linear molecule of the polyrotaxane compound has an acid-decomposable bond selected from an acetal bond, a ketal bond, a disulfide bond, an ester bond, an orthoester bond, a vinyl ether bond, a hydrazide bond, and an amide bond.

11. The pharmaceutical composition according to any one of claims 1 to 10, wherein the linear molecule of the polyrotaxane compound comprises a poloxamer.

12. The pharmaceutical composition according to any one of claims 1 to 11, wherein the molecular weight of the linear molecule of the polyrotaxane compound is 4,000 to 12,000.

13. The pharmaceutical composition according to any one of claims 1 to 12, wherein the ratio of the number of linear molecules to the number of cyclic molecules of the polyrotaxane compound is 1:10 to 1:

20.

14. The pharmaceutical composition according to any one of claims 1 to 13, wherein the terminal group of the polyrotaxane compound is selected from the group consisting of an O-triphenylmethyl group with or without a substituent, an S-triphenylmethyl group with or without a substituent, and an N-triphenylmethyl group with or without a substituent.

15. The pharmaceutical composition according to any one of claims 1 to 14, wherein an end group of the polyrotaxane compound is linked to a linear molecule of the polyrotaxane compound via a peptide bond, a carbamate bond, an ester bond, or an ether bond.

16. An acid-decomposable polyrotaxane compound containing β-cyclodextrin and poloxamer, having the following structural formula: 【Chemistry 1】 where: m is an integer from 0 to 200 that represents the number of repeating polypropylene glycol units in the poloxamer; n is an integer from 0 to 200 representing the number of polyethylene glycol repeating units in the poloxamer; the average molecular weight of the poloxamer is 4,000 to 12,000; x is an integer of 10 to 20 indicating the number of β-cyclodextrins; β-cyclodextrin has 1 to 21 water-soluble functional groups R per molecule, The water-soluble functional group is a 2-(2-hydroxyethoxy)ethyl (HEE) group, a 2-[2-[2-hydroxyethoxy]ethoxy]ethyl (HEEE) group, a 2-[2-[2-(2-hydroxyethoxy]ethoxy]ethoxy]ethyl (HEEEE) group, a 2-(2-methoxyethoxy)ethyl (MEE) group, a 2-[2-[2-methoxyethoxy]ethoxy]ethyl (MEEE) group, a 2-[2-[2-(2-methoxyethoxy]ethoxy]ethyl (MEEE) group, a 2-[2-[2-(2-methoxyethoxy]ethoxy]ethyl a 2-[2-[2-(2-ethoxy]ethoxy]ethoxy]ethyl (MEEEE) group, a 2-(2-ethoxy)ethyl (EEE) group, a 2-[2-[2-ethoxy]ethoxy]ethyl (EEEE) group, a 2-[2-[2-(2-ethoxy]ethoxy]ethoxy]ethyl (EEEEE) group, a hydroxyethyl group, a hydroxypropyl group, a hydroxybutyl group, a carboxymethyl group, a carboxyethyl group, a carboxypropyl group, and a methyl group; L is a single bond, a carbamate bond (—O—CO—NH—), an ester bond (—O—CO—), a carbonate bond (—O—CO—O—), or an ether bond (—O—), the terminal group Z is selected from the group consisting of an O-triphenylmethyl group with or without substitution, an S-triphenylmethyl group with or without substitution, and an N-triphenylmethyl group with or without substitution; Y is a single bond, a peptide bond, a carbamate bond, an ester bond, or an ether bond; The pharmaceutical composition according to any one of claims 1 to 15, comprising a polyrotaxane compound having an overall average molecular weight of 20,000 to 50,000.

17. The pharmaceutical composition according to any one of claims 1 to 16, which is a composition for subcutaneous administration, intraperitoneal administration, or intravenous administration.

18. The pharmaceutical composition according to any one of claims 1 to 17, which is administered at a dose of 3 to 35 mg / kg / day.

Images