Pharmaceutical Compositions for Treating Lipid Storage Diseases - Patent application

Abstract

The purpose of the present invention is to provide a pharmaceutical composition for treating lipid storage disorder. More specifically, the purpose of the present invention is to provide a pharmaceutical composition for treating lipid storage disorder, the pharmaceutical composition exhibiting a therapeutic effect by improving lipid storage at least in the brain. According to the present invention, there is provided a pharmaceutical composition for treating the storage of lipids in the brain, the pharmaceutical composition containing lactose-modified hydroxypropyl-β-cyclodextrin or lactose-modified hydroxypropyl-γ-cyclodextrin as an active ingredient, wherein the pharmaceutical composition is characterized in that the compound migrates at least into the brain when administered in vivo.

Description

[Technical field]

[0001] The present invention relates to a pharmaceutical composition for treating lipid storage diseases. [Background technology]

[0002] Niemann-Pick disease type C (NPC), a lipid storage disease, is characterized by the accumulation of lipids, including free cholesterol, in the late endosomal / lysosomal system (endolysosomes) due to abnormalities in the membrane protein NPC1 or the soluble protein NPC2. The incidence of NPC is approximately 1 in 150,000, and approximately 95% of patients have the NPC1 gene. Mutations in the NPC1 gene have been found in approximately 500 families worldwide. There are an estimated 150 patients in Japan. The main symptoms of NPC vary depending on the time of onset, but neurological symptoms and hepatosplenomegaly are seen in all age groups. In other words, the target tissues are the brain and liver.

[0003] Hydroxypropyl-β-cyclodextrin (HP-β-CyD) is widely used as a drug solubilizer, and because it can incorporate cholesterol into its cavity, basic and clinical research into the application of HP-β-CyD as a therapeutic agent for NPC has been actively conducted in recent years (Non-Patent Document 1: Liu B. et al., Proc Natl Acad Sci US A. 2009; 106(7):2377-82, Non-Patent Document 2: Ory DS et al., Lancet. 2017; 390(10104):1758-1768). Since 2009, humanitarian clinical application of HP-β-CyD has been started in the United States for twin NPC patients by intravenous administration, and since 2011, intrathecal administration has also been performed with the aim of further improving neurological symptoms. Currently, CTD Holdings, Inc. of the United States is conducting Phase I / II clinical trials of HP-β-CyD by intravenous administration, and Mallinckrodt, Inc. of the United States is conducting Phase IIb / III clinical trials of HP-β-CyD by intrathecal administration. In particular, intrathecal administration has been shown to be highly promising, as it has delayed the progression of neurological symptoms in 14 NPC patients and has only mild side effects such as hearing loss. However, considering that intrathecal administration places a great burden on patients and that most NPC patients are children, there is a strong need for treatment by subcutaneous or intravenous injection, which places a smaller burden on patients.

[0004] On the other hand, subcutaneous or intravenous injection of HP-β-CyD is likely to cause side effects such as lung damage due to large doses (Non-Patent Document 3: Yin-Hsiu Chien et al., Mol Genet Metab. 2013; 109(2):231-2) and hearing impairment (Non-Patent Document 2). In fact, in a humanitarian clinical application under the supervision of a doctor at Saga University in Japan, a high dose of 40 g was administered intravenously to a child, and abnormal shadows were observed in the lungs, so the administration was switched to intraventricular administration (Non-Patent Document 4: Matsuo M. et al., Mol. Genet. Metab. Rep. 2014; 1:391-400). In addition, intravenous administration of 5 g / kg HP-β-CyD three times a week also induced transient lung damage, and further, it has been confirmed that intrathecal administration of 200 mg / kg HP-β-CyD once every two weeks worsens hearing loss. Therefore, there is a strong need for a treatment that can be administered by subcutaneous or intravenous injection and has fewer side effects. In particular, since most patients are children and the symptoms progress quickly, the development of a new drug is an urgent task.

[0005] The method to improve the therapeutic efficiency of subcutaneous and intravenous injection of HP-β-CyD is clear: to improve neurological symptoms, deliver HP-β-CyD to the brain, and to improve hepatosplenomegaly, deliver HP-β-CyD to the liver. The present inventors have previously accidentally discovered that lactose-modified β-cyclodextrin migrates to the liver, and have already reported this (Non-Patent Document 5: Motoyama et al., Beilstein J. Org. Chem. 13, 10-18, 2017). There, lactose-modified β-cyclodextrin designed to treat hepatosplenomegaly in Niemann-Pick disease type C is described. Therefore, the present inventors further synthesized lactose-modified HP-β-CyD (Lac-HP-β-CyD) and evaluated its blood-brain barrier (BBB) ​​permeability in vitro and brain migration in vivo, finding that BBB permeability and brain migration were observed, and have filed a patent application (Patent Application 2018-245538).

[0006] In addition, Alzheimer's disease is known as a lipid storage disease, and it has been reported that increased cholesterol occurs in brain cells, inhibiting autophagosome-lysosome fusion and reducing the clearance of amyloid beta (Non-Patent Document 6: Nixon RA et al., J. Neuropathol. Exp. Neurol., 2005, 64, 113-122; Non-Patent Document 7: Barbero-Camps E. et al., Autopagy, 2018, 14, 1129-1154). [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Liu B. et al., Proc Natl Acad Sci US A. 2009; 106(7):2377-82 [Non-Patent Document 2] Ory DS et al., Lancet. 2017; 390(10104):1758-1768 [Non-Patent Document 3] Yin-Hsiu Chien et al. Mol Genet Metab. 2013; 109(2):231-2 [Non-Patent Document 4] Matsuo M. et al., Mol. Genet. Metab. Rep. 2014; 1:391-400 [Non-Patent Document 5] Motoyama et al., Beilstein J. Org. Chem. 13, 10-18, 2017 [Non-Patent Document 6] Nixon RA et al., J. Neuropathol. Exp. Neurol., 2005, 64, 113-122 [Non-Patent Document 7] Barbero-Camps E. et al., Autopagy, 2018, 14, 1129-1154 Summary of the Invention [Problem to be solved by the invention]

[0008] An object of the present invention is to provide a pharmaceutical composition for treating lipid storage diseases, more specifically, to provide a pharmaceutical composition for treating lipid storage diseases that exhibits a therapeutic effect by improving lipid accumulation at least in the brain. [Means for solving the problem]

[0009] Means for Solving the Problems The present inventors have conducted intensive research to solve the above problems and have found that hydroxypropyl-β-cyclodextrin or hydroxypropyl-γ-cyclodextrin, or derivatives thereof, can penetrate the blood-brain barrier and be transported into neurons in the brain, thereby improving lipid accumulation in neurons, thereby completing the present invention. The present invention includes the following:

[0010] [1] A pharmaceutical composition for treating lipid accumulation in the brain, comprising as an active ingredient a compound selected from the group consisting of lactose-modified hydroxypropyl-β-cyclodextrin and lactose-modified hydroxypropyl-γ-cyclodextrin, wherein the compound is transported at least into the brain when administered to a living body. [2] The pharmaceutical composition according to [1] above, wherein the transfer of the compound into the brain includes, in addition to penetration of the compound through the blood-brain barrier, transfer of the compound into neurons in the brain. [3] The pharmaceutical composition according to [1] or [2] above, which is administered intravenously or subcutaneously. [4] The pharmaceutical composition according to any one of the above [1] to [3], wherein the degree of substitution of lactose for cyclodextrin in the compound is 4 or more. [5] The pharmaceutical composition according to any one of the above [1] to [3], wherein the degree of substitution of lactose for cyclodextrin in the compound is 8 or more. [6] The pharmaceutical composition according to any one of the above-mentioned [1] to [5], wherein the accumulation of lipids is caused by a lipid storage disease that causes accumulation of lipids in the brain. [7] The pharmaceutical composition according to [6] above, wherein the lipid storage disease is a lysosomal disease. [8] The pharmaceutical composition described in [6] above, wherein the lipid storage disease is Niemann-Pick disease. [9] The pharmaceutical composition according to [6] above, wherein the lipid storage disease is Alzheimer's disease.

[10] The pharmaceutical composition according to any one of the above [1] to [9], further comprising another drug that exhibits a pharmacological effect in the brain.

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

[10] , which is administered continuously once or twice a week for several weeks or more.

[0011]

[12] A pharmaceutical composition for treating lipid accumulation in the brain, comprising as an active ingredient a compound selected from the group consisting of lactose-modified hydroxypropyl-β-cyclodextrin and lactose-modified hydroxypropyl-γ-cyclodextrin, wherein the compound is administered for the purpose of reducing the amount of lipids (preferably cholesterol) in at least neurons in the brain.

[13] The pharmaceutical composition according to

[12] above, which is administered intravenously or subcutaneously.

[14] The pharmaceutical composition according to

[12] or

[13] above, wherein the degree of substitution of lactose for cyclodextrin in the compound is 4 or more.

[15] The pharmaceutical composition according to

[12] or

[13] above, wherein the degree of substitution of lactose for cyclodextrin in the compound is 8 or more.

[16] The pharmaceutical composition according to any one of the above-mentioned

[12] to

[15] , wherein the accumulation of lipids is caused by a lipid storage disease that causes accumulation of lipids in the brain.

[17] The pharmaceutical composition according to

[16] above, wherein the lipid storage disease is a lysosomal disease.

[18] The pharmaceutical composition described in

[16] above, wherein the lipid storage disease is Niemann-Pick disease.

[19] The pharmaceutical composition according to

[16] above, wherein the lipid storage disease is Alzheimer's disease.

[20] The pharmaceutical composition according to any one of the above

[12] to

[19] , further comprising another drug that exhibits a pharmacological effect in the brain.

[21] The pharmaceutical composition according to any one of [12 to

[20] above, which is administered continuously once or twice a week for several weeks or more.

[0012]

[21] A method for treating a lipid storage disease, comprising administering to a patient suffering from a lipid storage disease at least intracerebrally an effective amount of lactose-modified hydroxypropyl-β-cyclodextrin or lactose-modified hydroxypropyl-γ-cyclodextrin.

[22] The method of treatment according to the above

[21] , wherein the compound is administered into the brain via passage of the compound through the blood-brain barrier.

[23] The method for treatment according to the above

[21] or

[22] , wherein the compound is administered intravenously or subcutaneously.

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

[21] to

[23] , wherein the substitution degree of lactose for cyclodextrin in the compound is 4 or more.

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

[21] to

[23] , wherein the degree of substitution of lactose for cyclodextrin in the compound is 8 or more.

[26] The method for treating the lipid storage disease according to any one of

[21] to

[25] above, wherein the lipid storage disease is a lysosomal disease.

[27] The method for treating the lipid storage disease according to any one of

[21] to

[25] above, wherein the lipid storage disease is Niemann-Pick disease.

[28] The method for treating the lipid storage disease according to any one of

[21] to

[25] above, wherein the lipid storage disease is Alzheimer's disease.

[29] The method for treatment according to any one of the above

[21] to

[28] , further comprising another drug that exhibits a pharmacological effect in the brain. Effect of the Invention

[0013] The present invention provides a pharmaceutical composition for treating lipid storage diseases. [Brief description of the drawings]

[0014] [Figure 1] Schematic diagram of some examples of lactose-modified hydroxypropyl-β-cyclodextrins, in which four hydroxypropyl-substituted β-cyclodextrins are modified with two, four, or eight lactoses, respectively, as indicated by DSL=2, 4, or 8, respectively. [Diagram 2] FIG. 1 is a diagram showing an outline of an example of a process for synthesizing Lac-HP-β-CyD from HP-β-CyD. [Diagram 3] The left graph shows the results of measuring the MALDI-TOF MS spectrum of Lac-HP-β-CyD synthesized in Example 1, and the right graph shows the results of measuring the 1H-NMR spectrum. [Figure 4] The permeation of Lac-HP-β-CyD fluorescently labeled with HiLyte through hCMEC / D3 cell monolayers was measured using an in vitro BBB model. The left panel shows the permeation over time, and the right panel (table) shows the permeation coefficient. Each value represents the mean ± SE of three experiments. * indicates p<0.05 compared to HP-β-CyD, + indicates p<0.05 compared to Lac-HP-β-CyD (DSL2), and ++ indicates p<0.05 compared to Lac-HP-β-CyD (DSL4). [Diagram 5] The results confirm the competitive inhibition of lactose against the permeation of Lac-HP-β-CyD in an in vitro BBB model. The results confirm the inhibitory effect of lactose, glucose, or phloretin. The left figure shows the amount of permeation over time, and the right figure (table) shows the permeation coefficient. Each value shows the average ± SE of three experiments. * indicates p<0.05 compared to HP-β-CyD. [Figure 6] Using an in vitro BBB model, the uptake of fluorescently labeled Lac-HP-β-CyD into SH-SY5Y cells after BBB penetration was confirmed and measured using a flow cytometer. [Figure 7]These are the results of confirming the cytotoxicity of Lac-HP-β-CyD and its effect on tight junctions in the blood-brain barrier. A: The results of confirming the cytotoxicity to hCMEC / D3 cells. Each value indicates the mean ± SE of three experiments. * indicates p<0.05 compared to HP-β-CyD. B: The results of confirming the effect on tight junctions of hCMEC / D3 cell monolayers. Each value indicates the mean ± SE of three experiments. [Figure 8] These are the results of confirming the transfer of Lac-HP-β-CyD to each organ after intravenous injection into mice. The fluorescence of each excised organ was observed using an in vivo imaging system (IVIS) (Ex: 535 nm, Em: DsRed). Representative images from three experiments are shown. [Figure 9] These are the results of confirming the transfer of Lac-HP-β-CyD to each organ after subcutaneous administration to AD model mice (AppNL-GF mice). The fluorescence of each excised organ was observed using IVIS. The left image shows a representative image from three experiments. The right image is a graph showing the results of the fluorescence intensity of each organ. * indicates p<0.05 when compared to HP-β-CyD. [Figure 10] This is the result of confirming the transfer of Lac-HP-β-CyD into the brain after intravenous injection into mice. The left image shows the results observed using an IVIS, and the right image shows the results of measuring the fluorescence intensity using a microreader. Representative images from four experiments are shown. * indicates p<0.05 compared to HP-β-CyD. [Figure 11] This shows the results of observing the localization of Lac-HP-β-CyD in various brain tissues (cerebral vascular endothelial cells, cerebral cortex, and hippocampus). Representative images from three experiments are shown. [Figure 12] These are the results of observing the localization of Lac-HP-β-CyD in various brain tissues (cerebral vascular endothelial cells, cerebral cortex, and hippocampus) of AD model mice. Representative images from three experiments are shown. The left image shows the results for the cerebral cortex, and the right image shows the results for the hippocampus. [Figure 13]This is the result of confirming the transfer of Lac-HP-β-CyD to the brain after intravenous administration of TPPS to mice. Each value shows the mean ± SE of three experiments. * indicates p<0.05 compared to TPPS, and + indicates p<0.05 compared to HP-β-CyD / TPPS. [Figure 14] The results of blood biochemistry tests after intravenous administration of Lac-HP-β-CyD. LDH, lactate dehydrogenase; AST, aspartate aminotransferase; ALT, alanine aminotransferase; BUN, blood urea nitrogen; CRE, creatinine. Each value shows the mean ± SE of three experiments. [Figure 15] Blood biochemistry results after subcutaneous administration of Lac-HP-β-CyD to NPC model mice. Each value shows the mean ± SE of 3-5 experiments. * indicates p<0.05 compared to saline-administered Npc1- / - mice, and + indicates p<0.05 compared to HP-β-CyD-administered Npc1- / - mice. [Figure 16] The results of blood biochemistry tests after subcutaneous administration of Lac-HP-β-CyD to AD model mice. Each value shows the mean ± SE of three experiments. [Figure 17] The results show that labeled Lac-HP-β-CyD was taken up into cells by using SH-SY5Y cells treated with U18666A. Each value shows the mean ± SE of three experiments. * indicates p<0.05 compared to HP-β-CyD, + indicates p<0.05 compared to Lac-HP-β-CyD (DSL2), and ++ indicates p<0.05 compared to Lac-HP-β-CyD (DSL4). [Figure 18]The results were obtained by confirming the competitive inhibition of lactose against the permeation of Lac-HP-β-CyD using SH-SY5Y cells treated with U18666A. Each value shows the mean ± SE of three experiments. * indicates p<0.05 compared to HP-β-CyD, + indicates p<0.05 compared to Lac-HP-β-CyD + lactose, and ++ indicates p<0.05 compared to Lac-β-CyD. [Figure 19] The results show that asialofetuin (AF) competitively inhibits the permeation of Lac-HP-β-CyD using HepG2 cells treated with U18666A. Each value shows the mean ± SE of three experiments. * indicates p<0.05 compared to control, + indicates p<0.05 compared to HP-β-CyD, and ++ indicates p<0.05 compared to Lac-β-CyD+AF. [Figure 20] The effect of Lac-HP-β-CyD on free cholesterol accumulated in endolysosomes was confirmed using SH-SY5Y cells treated with U18666A. Cholesterol and endolysosomes were stained with Filipin III and LysoTracker® Green DND-26, respectively, and observed with a confocal laser scanning microscope. [Figure 21] The effect of Lac-HP-β-CyD on free cholesterol accumulated in endolysosomes was confirmed using SH-SY5Y cells treated with U18666A. The figure shows the amount of cholesterol in endolysosomes calculated from the fluorescence intensity measured using a BZ-II analyzer. Each value shows the mean ± SE of three experiments. * indicates p<0.05 compared to the control (no CyD added), and + indicates p<0.05 compared to HP-β-CyD. [Figure 22]The results were obtained by confirming the competitive inhibition of lactose against the effect of Lac-HP-β-CyD in reducing the amount of free cholesterol accumulated in endolysosomes, using SH-SY5Y cells treated with U18666A. Cholesterol and endolysosomes were stained with Filipin III and LysoTracker® Green DND-26, respectively, and observed with a confocal laser scanning microscope. [Diagram 23] The results were obtained by confirming the competitive inhibition of lactose against the effect of Lac-HP-β-CyD on the reduction of free cholesterol accumulated in endolysosomes using SH-SY5Y cells treated with U18666A. The figure shows the amount of cholesterol in endolysosomes calculated from the fluorescence intensity measured using a BZ-II analyzer. Each value shows the mean ± SE of three experiments. * indicates p<0.05 compared to the control (no CyD added), + indicates p<0.05 compared to HP-β-CyD, and § indicates p<0.05 compared to Lac-HP-β-CyD + lactose. [Figure 24] The results show that Lac-HP-β-CyD is cytotoxic to NPC-like nerve cells. A shows the results of confirming the cytotoxicity to SH-SY5Y cells treated with U18666A. Each value shows the average ± SE of 6-8 experiments. * indicates p<0.05 compared to HP-β-CyD. B shows the results of confirming the effect on cholesterol leakage from the plasma membrane of SH-SY5Y cells treated with U18666A. Each value shows the average ± SE of 4-6 experiments. * indicates p<0.05 compared to the control. + indicates p<0.05 compared to HP-β-CyD. [Diagram 25] The results are the results of measuring the body weight of NPC model mice (Npc1- / -) subcutaneously administered Lac-HP-β-CyD or HP-β-CyD over time. Each value shows the mean ± SE of five mice. * indicates p<0.05 compared to HP-β-CyD. [Figure 26]The results of the Beam Walking Test of NPC model mice (Npc1- / -) subcutaneously administered Lac-HP-β-CyD or HP-β-CyD. A is the number of slips, and B is the moving speed. The results are of body weight measured over time. Each value shows the mean ± SE of 5-7 mice. * indicates p<0.05 compared to saline, and + indicates p<0.01 compared to HP-β-CyD. [Figure 27] The results show the survival rate of NPC model mice (Npc1- / -) subcutaneously administered Lac-HP-β-CyD or HP-β-CyD. Each value shows the mean ± SE of 5-7 mice. * indicates p<0.05 compared to saline, and + indicates p<0.01 compared to HP-β-CyD. [Figure 28] The effect of Lac-HP-β-CyD on intracellular accumulated Aβ was confirmed using SH-SY5Y cells treated with HiLyte-Aβ and U18666A. Cholesterol and endolysosomes were stained with Filipin III and LysoTracker (registered trademark) Red, respectively, and observed with a confocal laser scanning microscope. [Figure 29] The effect of Lac-HP-β-CyD on the intracellular accumulation of autophagosomes was confirmed using SH-SY5Y cells treated with U18666A. Cell nuclei and endolysosomes were stained with Hoechst 33342 and LysoTracker® Red, respectively, and autophagy vesicles were stained with CYTO-ID® Green. The results were then observed using a confocal laser scanning microscope. [Diagram 30] The effect of Lac-HP-β-CyD on autophagic flux was confirmed using SH-SY5Y cells treated with U18666A. The cells were stained with Hoechst 33342 and anti-p62 / SQSTM1 antibodies, and then observed with a confocal laser scanning microscope. [Diagram 31]Results of the Y-maze test using AD model mice. Each value shows the mean ± SE of 15-17 mice per group. * indicates p<0.05 compared to saline, and + indicates p<0.01 compared to HP-β-CyD. [Diagram 32] The results of the Barnes maze test using AD model mice. A shows the time spent on the target up to day 4, B shows the time spent on each area (TQ, LQ, OQ, RQ) on day 5, C shows the time spent on the target on day 5, D shows the time spent on each area (TQ, LQ, OQ, RQ) on day 9, and E shows the time spent on the target on day 9. Each value shows the mean ± SE of 15 mice per group. * indicates p<0.05 compared to saline, and + indicates p<0.01 compared to HP-β-CyD. [Diagram 33] The effect of Lac-HP-β-CyD on intracerebral cholesterol was confirmed using AD model mice. Each value represents the mean ± SE of three experiments. * indicates p<0.05 compared to saline. [Diagram 34] The effect of Lac-HP-β-CyD on free cholesterol in the brain was confirmed using AD model mice. Each value shows the mean ± SE of three experiments. * indicates p<0.05 compared to saline. [Diagram 35] These are the results of confirming Aβ accumulation in the brains of AD model mice that were subcutaneously administered Lac-HP-β-CyD. Images of half brains under each condition are shown. Representative images from three experiments are shown. [Diagram 36] The results confirm Aβ accumulation in the brain of AD model mice subcutaneously administered Lac-HP-β-CyD. The number of Aβ plaques in the hemibrain under each condition is shown. Each value shows the mean ± SE of three experiments. * indicates p<0.05 compared to saline. [Figure 37]This shows the accumulation of Aβ in the brain of AD model mice after subcutaneous administration of Lac-HP-β-CyD. Representative images from three experiments are shown. [Figure 38] The accumulation of Aβ in the brain of AD model mice subcutaneously administered Lac-HP-β-CyD was confirmed by ELISA. Each value shows the mean ± SE of three experiments. * indicates p<0.05 compared to saline. [Figure 39] The effect of Lac-HP-β-CyD on brain glial cells was confirmed. Representative images from three experiments are shown. [Diagram 40] These are the results of confirming the effect of Lac-HP-β-CyD on hearing. Each value shows the average ±SE of experiments on 4 to 5 mice. Saline: -■-, Lac-HP-β-CyD: --▲--, HP-β-CyD: -●-. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] The present invention will be described below with exemplary embodiments as examples, together with preferred methods and materials that can be used in the practice of the present invention. Unless otherwise specified in the text, all technical and scientific terms used in the present specification have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. In addition, any materials and methods equivalent or similar to those described in the present specification can be used in the practice of the present invention. In addition, all publications and patents cited in the present specification in relation to the invention described in the present specification are incorporated as part of the present specification, for example, as a description of methods, materials, etc. that can be used in the present invention. In the present specification, the expression "X to Y" is used to mean that X is the lower limit and Y is the upper limit, or that X is the upper limit and Y is the lower limit. In the present specification, "about" is used to mean that ±10% is allowed.

[0016] (1) Lactose-modified hydroxypropyl cyclodextrin β-Cyclodextrin (β-CyD) and γ-Cyclodextrin (γ-CyD) are oligosaccharides in which 7 or 8 glucose units are linked in a ring. Hydroxypropyl-β-cyclodextrin (HP-β-CyD) and hydroxypropyl-γ-cyclodextrin (HP-γ-CyD) (hereinafter, in this specification, collectively referred to as "hydroxypropyl-cyclodextrin (HP-CyD)" unless otherwise clearly indicated by the context) are β-cyclodextrin or γ-cyclodextrin (hereinafter, in this specification, collectively referred to as "cyclodextrin (CyD)" unless otherwise clearly indicated by the context) in which the hydroxyl groups at the 2-, 3-, and 6-positions, preferably the hydroxyl groups at the 2- or 6-positions, of the glucose skeleton are randomly substituted with hydroxypropyl groups, preferably 2-hydroxypropyl groups. The number of modifications of hydroxypropyl groups is expressed as the average substitution degree per molecule of HP-CyD. The average degree of substitution is not particularly limited, and may be, for example, 2, 3, 4, 5, 6, 7, 8, or 9. In general, the average degree of substitution is about 4 to 8. In this specification, CyD in which an average of 4 hydroxyl groups are substituted with hydroxypropyl groups is referred to as HP-CyD (DS 4). The modification of CyD with hydroxypropyl groups can be carried out by appropriately referring to known methods. In addition, HP-CyD is commercially available, so it can be purchased and used.

[0017] In the present invention, lactose-modified HP-β-CyD (Lac-HP-β-CyD) or lactose-modified HP-γ-CyD (Lac-HP-γ-CyD) (hereinafter, in this specification, collectively referred to as "lactose-modified hydroxypropyl cyclodextrin (Lac-HP-CyD)" unless otherwise clearly indicated by the context) is a compound in which the hydroxyl groups or hydroxypropyl groups at the 2-, 3-, and 6-positions of glucose in HP-β-CyD or HP-γ-CyD, preferably the hydroxyl group at the 2- or 6-position of glucose, more preferably the hydroxyl group at the 6-position of glucose, are randomly substituted with one, two, or more lactoses. The number of lactose modifications is expressed as the average substitution degree per molecule of Lac-HP-CyD. The average substitution degree of lactose is not particularly limited, but is at least 2 or more, preferably 4 or more, and more preferably 8 or more. Lac-HP-CyD is preferably Lac-HP-β-cyclodextrin. In this specification, HP-CyD substituted with an average of 8 lactoses is referred to as Lac-HP-CyD (DSL 8). Figure 1 shows a schematic diagram of an example of HP-CyD modified with lactose. As an example, Lac-HP-CyD having a hydroxypropyl group substitution degree of 4 and lactose substitution degrees of 2, 4, and 8 are shown.

[0018] Modification of HP-CyD with lactose can be performed with reference to known reports. For example, but not limited to, ethylenediamine is added to HP-β-CyD dissolved in DMSO, and reacted with CDI to form a urethane bond between the hydroxyl group of glucose or hydroxypropyl group and ethylenediamine to obtain NH2-HP-β-CyD (ethylenediamine). This is dissolved again in DMSO, and sodium cyanoborohydride and lactose are added to react, and reductive amination reaction is performed between the amino group of ethylenediamine and the hydroxyl group at the 1-position of lactose to obtain HP-β-CyD modified with lactose. Furthermore, lactose can be bound to HP-CyD using another arbitrary linker. In addition, lactose modification can be easily performed on highly reactive functional groups present in cyclodextrin, such as amino groups and carboxyl groups, and even when using these groups, lactose can be further linked to HP-CyD using any linker.

[0019] In this specification, the term "hydroxypropyl-β-cyclodextrin modified with lactose" or "hydroxypropyl-γ-cyclodextrin modified with lactose" refers to any of the following: a lactose molecule is directly bonded to a group (e.g., a hydroxyl group) of HP-β-CyD or HP-γ-CyD; a lactose molecule is bonded via an atomic group for bonding; and a lactose molecule is bonded via a linker. "Via an atomic group for bonding" can refer to, for example, a case where HP-CyD is modified with an arbitrary group in order to bond lactose to HP-CyD, and lactose is bonded via the group.

[0020] The lactose-modified HP-CyD can also be modified with PEG, etc., which is also included in the lactose-modified HP-CyD of the present invention. Modification with PEG can be performed by binding PEG to HP-CyD using a known method. Modification with PEG is expected to improve blood retention.

[0021] (2) Target disease The Lac-HP-CyD used in the pharmaceutical composition or treatment method of the present invention has the characteristic of being transported through the blood-brain barrier into neurons in the brain and reducing intracellular lipids. Because of this characteristic, the pharmaceutical composition of the present invention containing Lac-HP-CyD as an active ingredient can be used as a medicine for treating lipid storage diseases, preferably sphingolipidoses and Alzheimer's disease. Examples of sphingolipidoses include, but are not limited to, Niemann-Pick disease, GM1 and GM2 gangliosidosis, Fabry disease, Gaucher disease, Farber disease, Krabbe disease, and metachromatic leukodystrophy. The pharmaceutical composition of the present invention can be particularly preferably used for treating Niemann-Pick disease type C or Alzheimer's disease.

[0022] (3) Drugs In the pharmaceutical composition of the present invention, other drugs that can be used together with Lac-HP-CyD and have a medicinal effect in the brain refer to drugs that have a complementary medicinal effect when Lac-HP-CyD is used as an active ingredient in a pharmaceutical composition for treating lipid storage diseases. For example, the drug itself can be a substance that has a physiological activity in the brain (hereinafter, sometimes simply referred to as a physiologically active substance). Examples of physiologically active substances include, but are not limited to, low molecular weight compounds, polypeptides, oligopeptides, proteins, and nucleic acids. For example, drugs that can be used to treat sphingolipidosis and Alzheimer's disease can be included. Alternatively, drugs that can improve the condition in the brain in addition to reducing lipid accumulation can be included, such as drugs that have an anti-inflammatory effect to suppress inflammation in the brain, and drugs that are active ingredients of antibacterial drugs or antiviral drugs for treating intracerebral infections.

[0023] Examples of the low molecular weight compound include low molecular weight drugs that can be used to treat sphingolipidosis and Alzheimer's disease. For example, when the pharmaceutical composition of the present invention is used to treat Niemann-Pick disease type C, examples include miglustat, curcumin, vorinostat, and arimoclomol. Using a known method, complexes between these low molecular weight compounds and Lac-HP-CyD can be formed, and the complexes can be administered as one pharmaceutical composition. When the pharmaceutical composition of the present invention is used to treat Alzheimer's disease, examples include donepezil, rivastigmine, galantamine, and memantine. Using a known method, complexes between these low molecular weight compounds and Lac-HP-CyD can be formed, and the complexes can be administered as one pharmaceutical composition. Since Lac-HP-CyD, which is the active ingredient of the pharmaceutical composition of the present invention, passes through the blood-brain barrier and is transported into nerve cells in the brain, it is expected that other low molecular weight compounds used together will also be efficiently transported to nerve cells in the brain and exhibit enhanced efficacy.

[0024] The above peptides (polypeptides and oligopeptides) include, for example, physiologically active peptides that can be used to treat sphingolipidosis and Alzheimer's disease. Using known methods, these peptides can be complexed with Lac-HP-CyD and administered as a single pharmaceutical composition. Since Lac-HP-CyD, which is the active ingredient of the pharmaceutical composition of the present invention, passes through the blood-brain barrier and is transported into neurons in the brain, it is expected that other drugs, such as peptides, used together will also be efficiently transported to neurons in the brain and show enhanced efficacy.

[0025] Examples of the nucleic acid include nucleic acids used for the treatment of sphingolipidosis and Alzheimer's disease. Examples of the nucleic acid include, but are not limited to, nucleic acids for the treatment of diseases by gene knockdown or RNA interference, such as antisense nucleic acids (DNA and RNA), heteroduplex nucleic acids, siRNA and shRNA. Specifically, examples of the nucleic acid include, but are not limited to, nucleic acids used for the treatment of metachromatic leukodystrophy. Using a known method, a complex between these nucleic acids and Lac-HP-CyD can be formed, and the complex can be administered as one pharmaceutical composition. Since Lac-HP-CyD, which is an active ingredient of the pharmaceutical composition of the present invention, passes through the blood-brain barrier and is transported into nerve cells in the brain, it is expected that the nucleic acid, which is another drug used together, will also be efficiently transported to nerve cells in the brain and show enhanced efficacy.

[0026] Examples of the above proteins include proteins used for the treatment of sphingolipidosis and Alzheimer's disease. Examples include, but are not limited to, enzymes, antibodies, transcription factors, or specific parts thereof that bind to targets and exert physiological activity in the brain. For example, when the pharmaceutical composition of the present invention is used for Gaucher disease or Fabry disease, examples of the proteins include enzymes used in enzyme replacement therapy for these diseases. When the pharmaceutical composition of the present invention is used for the treatment of Alzheimer's disease, examples of the proteins include antibodies against amyloid β. Using a known method, complexes between these proteins and Lac-HP-CyD can be formed, and the complexes can be administered as one pharmaceutical composition. For example, but not limited to, the protein and / or Lac-HP-CyD may be chemically modified, and the two may be bound to each other, optionally via a spacer. Alternatively, the protein and Lac-HP-CyD may be bound to each other non-covalently, for example, electrostatically. Lac-HP-CyD, the active ingredient of the pharmaceutical composition of the present invention, passes through the blood-brain barrier and is transported into neurons in the brain, so that other drugs, such as proteins, used together with it can be expected to be efficiently transported to neurons in the brain and show enhanced efficacy.

[0027] Pharmaceutical Composition The pharmaceutical composition containing the Lac-HP-CyD of the present invention as an active ingredient can be formulated and administered according to known methods. For example, it can be administered parenterally or orally to humans as a liquid or a pharmaceutical composition in an appropriate dosage form. An example of a parenteral administration method is an injection. The pharmaceutical composition containing the complex of the present invention is preferably administered parenterally. Examples of parenteral administration methods include, but are not limited to, intravenous administration and subcutaneous administration.

[0028] The pharmaceutical composition of the present invention may contain any optional ingredient as long as it does not impair the efficacy of the composition. Examples of optional ingredients include, but are not limited to, crosslinking agents, solubilizers, emulsifiers, moisturizing agents, cooling agents, inorganic powders, antioxidants, preservatives, colorants, flavoring agents, pH adjusters, and stabilizers.

[0029] The dosage of Lac-HP-CyD, an active ingredient of the present invention, to humans is appropriately determined depending on the age, weight, condition, sex, administration method, and other conditions of the subject to be administered. For example, the dosage is about 0.1 g / kg to about 20 g / kg, preferably about 1 g / kg to about 10 g / kg per day.

[0030] The method of administration of Lac-HP-CyD, which is the active ingredient of the present invention, is appropriately determined depending on the condition of the subject, the purpose of administration, and the administration method. The administration interval is not limited to this, but can be, for example, daily, every other day, every other day, once a week, or twice a week, and is preferably once or twice a week. The administration period is not limited to this, but can be, for example, administration for several weeks to one or two months, or several months to half a year, or several years. When administering for a long period of time, it is preferable to administer once or twice a week for several weeks to one or two months, followed by a drug holiday, and then administer again. EXAMPLES

[0031] 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: Preparation of Lac-HP-β-CyD The outline of the synthesis is shown in Figure 2. 49.8 mL of ethylenediamine was added to HP-β-CyD (10 g) dissolved in DMSO, and the mixture was reacted at room temperature for 24 hours using CDI (9.8 g). 10 g of the obtained NH2-HP-β-CyD (ethylenediamine) was dissolved in DMSO, sodium cyanoborohydride (100 g) was added, and lactose monohydrate (472 g) was further added, and the mixture was stirred at 75°C for 48 hours. The molar ratio of lactose to NH2-HP-β-CyD was 1:100. After lyophilization, the product was obtained. The MALDI-TOF MS spectrum and 1 The H-NMR spectrum was measured, and the results are shown in Figure 3. 1 From the results of H-NMR, calculations were performed using the integral values, and Lac-HP-β-CyD with an average lactose substitution degree of about 8 was obtained. Similarly, Lac-HP-β-CyD with various lactose substitution degrees was produced by adjusting the molar ratio of lactose to NH2-HP-β-CyD at will between 1:50 and 1:200. The produced samples were analyzed by MALDI-TOF MS spectrum and 1 The degree of lactose substitution was determined by H-NMR spectroscopy. As a result, Lac-HP-β-CyD with lactose substitution degrees of 2, 4, and 8 was obtained. The following experiments were performed using Lac-HP-β-CyD with lactose substitution degrees of 2, 4, and 8.

[0032] (Example 2) Confirmation of permeability of Lac-HP-β-CyD in an in vitro blood-brain barrier model Using an in vitro blood-brain barrier model, the time-dependent permeation amount of Lac-HP-β-CyD was confirmed as follows. Lac-HP-β-CyD with lactose substitution degrees of 2, 4, and 8 and control HP-β-CyD were labeled with HiLyte fluorescent dye according to the manufacturer's manual. The following experiments were carried out using these. Human brain endothelial cells (hCMEC / D3 cells) were placed in the upper layer of the transwell at 1.0 × 10 5cells / well, while 1.0 × 10 SH-SY5Y cells, which are human neuroblastoma cells, were seeded in the lower layer. 5 The in vitro BBB model was prepared by seeding 1000 cells / well and culturing them for 7 days in a CO2 incubator at 37°C. The barrier function of the BBB was confirmed by measuring the transcutaneous electrical resistance (TEER value). After adding fluorescently labeled Lac-HP-β-CyD (0.1 mM) to the upper layer, the lower layer medium was sampled over time (5, 15, 30, and 60 minutes) and the fluorescence was measured to evaluate the membrane permeability of Lac-HP-β-CyD. The results of the permeability of the hCMEC / D3 cell monolayer are shown in Figure 4. As shown in the left figure, the permeation amount of Lac-HP-β-CyD (DSL8) significantly increased over time. The permeability coefficient Papp (cm / sec) was calculated as follows: Papp = (dQ / dt) / (A*Co). The results are shown in the right figure. The membrane permeability coefficient of Lac-HP-β-CyD (DSL8) was shown to have about 30 times the BBB permeability compared to HP-β-CyD.

[0033] (Example 3) Confirmation of competitive inhibition by lactose Whether or not lactose specifically and competitively inhibits the permeation of Lac-HP-β-CyD through hCMEC / D3 cell monolayers in an in vitro BBB model was confirmed as follows. Using the in vitro BBB model described in Example 2, fluorescently labeled Lac-HP-β-CyD (DSL8) was added to the upper layer together with glucose (4 mM), GLUT inhibitor phloretin (1 mM), or lactose (4 mM) at 37°C, and the lower layer medium was sampled over time (5, 15, 30, and 60 minutes) to evaluate membrane permeability by measuring fluorescence. A similar experiment was also performed at 4°C, which indicates endocytosis inhibition. The results are shown in Figure 5. The amount of permeation over time is shown in the left figure, and the permeability coefficient is shown in the right figure. The permeability coefficient was reduced to about 1 / 5 by the addition of lactose. This result suggests that Lac-HP-β-CyD permeates the monolayer of hCMEC / D3 cells via a transporter or receptor that recognizes lactose. Furthermore, the amount of membrane permeation was significantly reduced at 4°C.

[0034] (Example 4) Confirmation of Intracellular Uptake of Lac-HP-β-CyD in Neurons after BBB Permeation Using the in vitro BBB model described in Example 2, after permeating the hCMEC / D3 cell monolayer, Lac-HP-β-CyD taken up into the underlying SH-SY5Y cells was observed with a confocal laser scanning microscope and confirmed by flow cytometry. As a result of microscopic observation, significant uptake of Lac-HP-β-CyD (DSL8) was confirmed compared to HP-β-CyD and Lac-HP-β-CyD (DSL2 and DSL4). The uptake amounts were DSL2 < DSL4 < DSL8. The results of flow cytometry are shown in Fig. 6.

[0035] In the following examples, unless otherwise specified, Lac-HP-β-CyD with a lactose substitution degree of 8 (DSL8) was used as Lac-HP-β-CyD.

[0036] (Example 5) Confirmation of the Effect on Cytotoxicity and Tight Junction of the Blood-Brain Barrier The effects of Lac-HP-β-CyD (DSL8) on cytotoxicity and tight junction of the blood-brain barrier were examined as follows. Cytotoxicity was confirmed by culturing hCMEC / D3 cells in the presence of Lac-HP-β-CyD (DSL8). The results are shown in Fig. 7A. It was found that Lac-HP-β-CyD was significantly less cytotoxic than HP-β-CyD and had excellent safety. Using the in vitro BBB model described in Example 2, hCMEC / D3 cells were seeded in a transwell and cultured at 37°C in a CO2 incubator for 4 to 6 days. Before the start of the experiment, the hCMEC / D3 cell monolayer was equilibrated with EBM-2 medium (37°C, 2.0 mL on the apical side, 2.0 mL on the basal side). Labeled HP-β-CyD or Lac-HP-β-CyD was dissolved in the medium to a concentration of 10 μM, and the medium was prewarmed to 37°C and replaced with the medium in the upper layer. After 5, 15, 30, and 60 minutes, the effect on tight junctions was evaluated by measuring the transcutaneous electrical resistance (TEER) of the monolayer. As shown in Figure 7B, none of the mediums reduced the tight junctions of the cells. These results confirmed that Lac-HP-β-CyD does not cause cell damage at the blood-brain barrier and is safer than HP-β-CyD.

[0037] (Example 6) Confirmation of organ distribution of Lac-HP-β-CyD (1) The Lac-HP-β-CyD (DSL8) and HP-β-CyD produced in Example 1 were labeled with HiLyte fluorescent dye according to the manufacturer's manual and used in the following experiments. Labeled Lac-HP-β-CyD or HP-β-CyD was intravenously injected into the tail of 8-week-old male BALB / c mice at 7.2 μmol / kg. After 60 minutes, the mice were euthanized, perfused with PBS, and each organ (brain, heart, lung, liver, kidney, and spleen) was collected. Each of the excised organs was observed using an in vitro image spectrum (IVIS). The results are shown in FIG. 8. It was confirmed that Lac-HP-β-CyD accumulated in the brain and liver. It has already been reported by the present inventors that lactose-modified β-CyD accumulates in the liver, and it was confirmed that lactose-modified HP-β-CyD (DSL8) also accumulates in the liver. In addition, it was confirmed that Lac-HP-β-CyD (DSL8) significantly accumulates in the brain.

[0038] (Example 7) Confirmation of organ distribution of Lac-HP-β-CyD (2) Next, we used the Alzheimer's disease model mouse, AppNL-G-F Male mice (7 months old, 30 g) (provided by Professor Takashi Saito, Institute of Neuroscience, Graduate School of Medicine, Nagoya City University) were used. TM Lac-HP-β-CyD or HP-β-CyD labeled with Fluor647) was subcutaneously administered at 7.2 μmol / kg, and the transfer to each organ was confirmed. The results are shown in Figure 9. Similarly, in Alzheimer's disease model mice, it was confirmed that Lac-HP-β-CyD accumulated in the brain and liver, and that Lac-HP-β-CyD accumulated significantly in the brain.

[0039] (Example 8) Confirmation of brain penetration (1) Accumulation of Lac-HP-β-CyD in the brain 10 minutes after administration was confirmed in the same manner as in Example 6. The results are shown in Figure 10. A is an image observed with an IVIS, and B is the result of measuring the fluorescence intensity with a microreader. The brain migration rate of Lac-HP-β-CyD was 20 times that of HP-β-CyD, and was approximately 5.2% of the administered dose.

[0040] (Example 9) Confirmation of brain distribution (2) Accumulation of HF647-labeled Lac-HP-β-CyD in the brain was confirmed in the same manner as in Example 7. Rapid transfer to the brain was confirmed, with the transfer to the brain reaching a maximum 1 minute after administration.

[0041] (Example 10) Confirmation of localization in the brain (1) As in Example 6, the mice were euthanized 60 minutes after administration, perfused with PBS, and the brains were collected. The brains were stained with DAPI (blue: stains nuclei), NeuN (green: stains Neuron), and DyLight488 (green: stains cerebral vascular endothelial cells), and observed with HiLyte (red) using a confocal laser scanning microscope. The results of observing the fluorescence in tissues including cerebral vascular endothelial cells, cerebral cortex, and hippocampus are shown in FIG. 11. As a result, the fluorescence of HyLyte-HP-β-CyD was not observed in any tissue, but HyLyte-Lac-HP-β-CyD was confirmed in all tissues. Furthermore, with the administration of Lac-HP-β-CyD, green and red fluorescence did not colocalize, and red fluorescence was observed other than in vascular endothelial cells, confirming that Lac-HP-β-CyD significantly penetrates cerebral blood vessels and migrates to the brain parenchyma.

[0042] (Example 11) Confirmation of localization in the brain (2) HF647-HP-β-CyD or HF647-Lac-HP-β-CyD (7.2 mmol / kg each) was administered in 20 mL / kg saline solution. NL-G-FSubcutaneous administration was performed to male mice (7 months old, 30 g). After 60 minutes, the brain was perfused transcardially with PBS and 4% PFA, and then the brain was removed. The brain was immersed in 4% PFA and fixed overnight at 4°C. The next day, the brain was washed with PBS, immersed in 15% sucrose solution, and dehydrated for 3 days. After dehydration, the brain was immersed in OCT compound and rapidly frozen with dry ice. Then, the brain was frozen at -80°C. After freezing, the brain was sliced ​​to a thickness of 20 mm using a CM3050 S cryostat and immersed in PBS. The brain was washed with PBS and blocked for 1 hour with 0.3% PBST solution containing 3% Donkey Serum. In the following examples, the process up to this point may be referred to as the "brain slice preparation process". The brain was washed with PBS, and MAP2-containing 0.3% PBST solution (1:1,000) was added, and the brain was stained for 12 hours at 37°C under light shielding. After washing with PBS, secondary antibody treatment (1:1,000) was added, and staining was performed for 3 hours at 37°C in the dark. After washing with PBS, DAPI (300 nM) solution was added, and staining was performed for 10 minutes at 37°C in the dark. After washing with PBS, each section was attached to a slide glass, embedded in Marinol solution, and observed with a confocal laser microscope. The results are shown in Figure 12. The left figure shows the results for the cerebral cortex, and the right figure shows the results for the hippocampus. As a result, fluorescence of HF647-HP-β-CyD was not observed in any tissue, but HF647-Lac-HP-β-CyD was confirmed in the cerebral cortex and hippocampus.

[0043] (Example 12) Confirmation of brain transport of complexes of Lac-HP-β-CyD and other compounds Whether Lac-HP-β-CyD, the active ingredient of the pharmaceutical composition of the present invention, is efficiently transported into the brain together with other drugs was confirmed as follows. Lac-HP-β-CyD (DSL8) or HP-β-CyD prepared in Example 1 was intravenously injected into the tail of 8-week-old male BALB / c mice at 5.8 μmol / kg, together with TPPS, a fluorescent substance. After 60 minutes, the mice were euthanized, perfused with PBS, and the brains were collected. The excised brains were observed with an IVIS, and the fluorescence intensity was measured with a microreader. The results are shown in FIG. 13. Compared with HP-β-CyD, it was confirmed that Lac-HP-β-CyD migrates to the brain together with other substances. The brain migration rate of Lac-HP-β-CyD was about 9 times that of HP-β-CyD, and was about 4.0% of the administered dose.

[0044] (Example 13) Confirmation of blood biochemical test values ​​after intravenous administration of Lac-HP-β-CyD (Example 13-1) Effect on wild-type mice Lac-HP-β-CyD (DSL8) and HP-β-CyD produced in Example 1 were each intravenously injected into the tail of 8-week-old male BALB / c mice at a concentration of 0.7 mmol / kg. After 24 hours, blood was collected and various biochemical test items were measured. Saline was administered as a control. The results are shown in FIG. 14. The group administered Lac-HP-β-CyD showed no effect on blood biochemical test values ​​compared to the group administered saline, confirming the safety of the drug.

[0045] (Example 13-2) Effect on NPC model mice Physiological saline containing HP-β-CyD or Lac-HP-β-CyD was administered at 20 μL / kg to 4-week-old wild-type mice (WT) and BALB / c Nctr-Npc1 mice. m1N (Npc1 - / - ) mice, the drug was administered subcutaneously at a dose of 1.4 mmol / kg every other week until the age of 8 weeks. 72 hours after the fifth administration, blood was collected from the abdominal aorta. The blood was centrifuged to separate the plasma. Each blood biochemical parameter was quantified using a Fuji DryChem 7000 "Z". The results are shown in Figure 15.

[0046] (Example 13-3) Effect on AD model mice 14) Confirmation of blood biochemistry test values ​​after subcutaneous administration of Lac-HP-β-CyD Physiological saline containing HP-β-CyD or Lac-HP-β-CyD was administered at 20 μL / kg via the App NL-G-F The drug was administered subcutaneously twice a week to male and female mice (2 months old) until they reached 7 months of age. 20 μL / kg of saline containing the above-mentioned β-CyDs was administered to male BALB / c mice in the same manner, at a dose of 0.7 mmol / kg. One week after the final administration, blood was collected from the inferior abdominal aorta. The blood was centrifuged to separate the plasma. Each blood biochemical parameter was quantified using a Fuji Dry Chem 7000 "Z". The results are shown in Figure 16.

[0047] (Example 14) Confirmation of Lac-HP-β-CyD uptake into NPC-like neuronal cells The steroid derivative U18666A (progesterone) inhibits intracellular cholesterol transport and induces free cholesterol accumulation similar to that observed in NPC cells. Using SH-SY5Y cells treated with U18666A, the uptake of Lac-HP-β-CyD into cells was measured as follows. The Lac-HP-β-CyDs with lactose substitution degrees of 2, 4, and 8 synthesized in Example 1 and the control HP-β-CyD were labeled with HF647 fluorescent dye according to the manufacturer's manual. The following experiment was carried out using these. SH-SY5Y cells (5x10 5 SH-SY5Y cells (1x10 cells) were cultured in a medium containing U18666A (1.25 μM) at 37°C for 48 hours. 4DSL1 cells) were cultured in culture medium supplemented with HF647-Lac-HP-β-CyD (DSL2, 4, and 8) or HF647-HP-β-CyD at a concentration of 0.1 mM for 24 hours at 37°C. Fluorescence intensity in the cells was measured using a BZ-II analyzer. The results are shown in Figure 17. The right graph shows the average fluorescence intensity. The amount of uptake increased with an increase in the degree of lactose substitution, and remarkable uptake was confirmed in DSL8.

[0048] (Example 15) Confirmation of competitive inhibition by lactose Using SH-SY5Y cells treated with U18666A, we examined whether the uptake of Lac-HP-β-CyD into cells was competitively inhibited by lactose. SH-SY5Y cells were treated with U18666A in the same manner as in Example 14, and then HF647-Lac-HP-β-CyD (DSL8) or HF647-HP-β-CyD was added to the medium at a concentration of 0.1 mM in the presence or absence of lactose (4 mM), and the cells were cultured at 37°C for 24 hours to confirm their uptake into the cells. The fluorescence intensity in the cells was measured using a BZ-II analyzer. The results are shown in Figure 18. The addition of lactose significantly inhibited the uptake of Lac-HP-β-CyD into the cells.

[0049] (Example 16) Confirmation of competitive inhibition by asialofetuin (AF) Using U18666A-treated HepG cells, we examined whether the uptake of Lac-HP-β-CyD into cells was competitively inhibited by asialofetuin (AF). HePG2 cells (2x10 5 HepG2 cells (1x10 4Cells) were cultured at 37°C for 24 hours in culture medium containing HF647-labeled Lac-HP-β-CyD or HP-β-CyD (in the presence and absence of AF), and the uptake into the cells was confirmed. The fluorescence intensity in the cells was measured using a BZ-II analyzer. The results are shown in Figure 19. The addition of AF increased the intracellular uptake of Lac-HP-β-CyD.

[0050] (Example 17) Confirmation of the effect on the amount of free cholesterol in endolysosomes The effect of Lac-HP-β-CyD on intracellular accumulation of free cholesterol was examined using SH-SY5Y cells treated with U18666A. In the same manner as in Example 11, SH-SY5Y cells were treated with U18666A, and then the U18666A-treated SH-SY5Y cells (1 x 10 4 Cells) were cultured at 37°C for 24 hours in culture medium containing Lac-HP-β-CyD (DSL2, 4, and 8) or HP-β-CyD at a concentration of 0.1 mM, and then fixed with 4% PFA for 10 minutes. After washing with PBS, the cells were stained for cholesterol and endolysosomes using Filipin III and LysoTracker (registered trademark) Green DND-26, respectively. The fluorescence intensity of Filipin III in 10 randomly selected cells was measured using a confocal laser scanning microscope and a BZ-II analyzer. The results of the observation using the confocal microscope are shown in Figure 20, and the amount of cholesterol in the endolysosomes calculated from the fluorescence intensity measured using the BZ-II analyzer is shown in Figure 21. Observation using the confocal microscope confirmed that Lac-HP-β-CyD (DSL8) showed a significant decrease in the amount of accumulated cholesterol compared to Lac-HP-β-CyD (DSL2 and DSL4). In addition, the effect of reducing accumulated cholesterol is DSL2 <DSL4<DSL8であった。

[0051] (Example 18) Confirmation of competitive inhibition by lactose against reduction of free cholesterol amount in endolysosomes by Lac-HP-β-CyD Using SH-SY5Y cells treated with U18666A, we examined whether the effect of Lac-HP-β-CyD in reducing the amount of free cholesterol in endolysosomes was competitively inhibited by lactose. In the same manner as in Example 14, SH-SY5Y cells were treated with U18666A, and then the U18666A-treated SH-SY5Y cells (1 x 10 4 Cells) were cultured for 24 hours at 37°C in a culture medium containing Lac-HP-β-CyD (DSL8) or HP-β-CyD at a concentration of 0.1 mM, with or without lactose (4 mM). Thereafter, cholesterol and endolysosomes were stained, and fluorescence was measured using a confocal laser scanning microscope and a BZ-II analyzer, in the same manner as in Example 13. The results of observation using a confocal microscope are shown in Figure 22, and the amount of cholesterol in endolysosomes calculated from the fluorescence intensity measured using a BZ-II analyzer is shown in Figure 23. The addition of lactose inhibited the effect of Lac-HP-β-CyD (DSL8) in reducing the amount of accumulated cholesterol.

[0052] (Example 19) Confirmation of cytotoxicity against nerve cells Cytotoxicity was confirmed by culturing SH-SY5Y cells treated with U18666A in the presence of various concentrations of Lac-HP-β-CyD (DSL8). HP-β-CyD was used as a control. The results are shown in Figure 24A. Lac-HP-β-CyD was found to have significantly lower cytotoxicity against NPC-like nerve cells than HP-β-CyD, and was found to be safer. In addition, SH-SY5Y cells were used to confirm the effect on cholesterol leakage from the cell plasma membrane. SH-SY5Y cells treated with U18666A were cultured in the presence of 30 mM Lac-HP-β-CyD (DSL8) for 1 hour, and then cholesterol leakage was measured using Cholesterol E-Test Wako (Fujifilm Wako Pure Chemical Industries, Ltd.) according to the manufacturer's manual. As controls, saline (control) and HP-β-CyD (30 mM) were used. The results are shown in Figure 24B. Lac-HP-β-CyD was shown to have a significantly lower cholesterol leakage effect from the cell plasma membrane in NPC-like nerve cells than HP-β-CyD.

[0053] (Example 20) Confirmation of the effect of Lac-HP-β-CyD using NPC mouse model The therapeutic effect of Lac-HP-β-CyD was confirmed using an NPC mouse model as follows. NPC model mouse (Npc1 - / - ) (BALB / c, 4 weeks old, provided by Dr. Kosaku Ohno and Dr. Katsumi Higaki of Tottori University, bred at Kumamoto University) were used, and Lac-HP-β-CyD (1.4 nmol / kg, 6,079 mg / kg) or HP-β-CyD (1.4 nmol / kg, 2,000 mg / kg) was administered subcutaneously once a week from 4 to 9 weeks of age (6 administrations in total). As controls, NPC model mice and wild-type mice (BALB / c, 4 weeks old) were administered saline. Five to seven mice (2 to 4 males, 3 females) were used for each condition. Body weight was measured, a beam walking test was performed, and survival rates were evaluated. The results of body weight measurements in NPC model mice from 4 to 10 weeks of age are shown in Figure 25. It was shown that Lac-HP-β-CyD significantly suppressed weight loss in NPC model mice compared to HP-β-CyD. The results of the beam walking test in NPC model mice from 4 to 7 weeks of age are shown in Figure 26. It was shown that Lac-HP-β-CyD significantly improved the motor dysfunction of NPC model mice compared to HP-β-CyD. The results of the survival rate in the NPC model mice are shown in Figure 27. It was shown that Lac-HP-β-CyD significantly extended the survival rate of the NPC model mice compared to HP-β-CyD.

[0054] (Example 21) Effect of Lac-HP-β-CyD on intracellular accumulation of amyloid β Amyloid β accumulation in SH-SY5Y cells was measured using U18666A-treated SH-SY5Y cells, which induces cholesterol accumulation in cells. 5 SH-SY5Y cells (1x10 cells) were cultured in a medium containing HiLyte-labeled amyloid β (150 nM) and U18666A (7.5 μM) at 37°C for 24 hours, and then the cells were washed with the medium. 4 SH-SY5Y cells) were cultured at 37°C for 24 hours in culture medium supplemented with HF647-Lac-HP-β-CyD (DSL 8) or HF647-HP-β-CyD (DS 4.4) at a concentration of 0.1 mM. PBS was used as a control. Then, the cells were fixed with 4% PFA for 10 minutes. After washing with PBS, the cells were stained for cholesterol and endolysosomes using Filipin III and LysoTracker (registered trademark) Red L7528, respectively. The fluorescence of 10 randomly selected cells was observed by confocal laser scanning microscope. The results are shown in Figure 26. As shown in Figure 28, when HP-β-CyD was added, there was no difference between the fluorescence of Filipin III, HiLyte-Aβ, and LysoTracker (registered trademark) and the control. On the other hand, when Lac-HP-β-CyD was added, it was confirmed that the amount of amyloid β accumulated in SH-SY5Y cells was significantly reduced.

[0055] (Example 22) Effect of Lac-HP-β-CyD on autophagy abnormalities The effect of Lac-HP-β-CyD on intracellular accumulation of autophagosomes induced by autophagy abnormalities was examined using SH-SY5Y cells treated with U18666A. Autophagy function was detected using the Cyto-ID® Autophagy Detection Kit according to the manufacturer's protocol. Specifically, the following procedure was used to confirm the effect. 1x10 SH-SY5Y cells 4 The cells were seeded at 1000 / dish, cultured at 37°C for 24 hours, and further cultured in U18666A (7.5μM)-containing medium for 24 hours. The cells were treated with 0.1mM HP-β-CyD or Lac-HP-β-CyD (DSL 8)-containing medium for 24 hours, washed with PBS, and cultured for 30 minutes in serum-free medium containing Cyto-ID (registered trademark) and LysoTracker (registered trademark) Red DND-99 (final concentration 100nM). The cells were washed with PBS and fixed with 4% PFA for 10 minutes. The nuclei were then stained by incubating with Hoechst33342-containing PBS solution for 30 minutes. Finally, the cells were washed with PBS, and the fluorescence of Cyto-ID (registered trademark), LysoTracker (registered trademark), and Hoechst33342 was observed by a confocal laser microscope. The results are shown in Figure 29. As shown in Figure 29, the addition of HP-β-CyD did not result in any difference in autophagosome accumulation compared to the control, whereas the addition of Lac-HP-β-CyD improved autophagosome accumulation.

[0056] Example 23: Effect of Lac-HP-β-CyD on autophagic flux p62 / SQSTM1 is degraded by autophagy when cells are normal, but accumulates in cells when cells are abnormal. Using SH-SY5Y cells treated with U18666A, the effect of Lac-HP-β-CyD on autophagic flux was confirmed as follows. 1x10 SH-SY5Y cells 4The cells were seeded at 100 cells / dish, cultured at 37°C for 24 hours, and then cultured in medium containing U18666A (7.5μM) for another 24 hours. The cells were treated with medium containing 0.1mM HP-β-CyD or Lac-HP-β-CyD (DSL 8) for 24 hours, washed with PBS, and fixed with 4% PFA for 10 minutes. After washing with PBS, the cells were treated with Triton X-100 (0.1%) for 10 minutes to permeabilize the membrane. Then, immunostaining was performed using antibodies (primary antibody: anti-p62 / SQSTM1 rabbit antibody, secondary antibody (Alexa Fluor 488 goat anti-rabbit IgG antibody). Then, nuclei were stained with Hoechst33342. After staining, the fluorescence of the secondary antibody and Hoechst33342 was observed using a confocal laser microscope. The results are shown in Figure 30. As shown in Figure 30, when HP-β-CyD was added, there was no difference in the intracellular accumulation of p62 / SQSTM1 compared to the control. On the other hand, when Lac-HP-β-CyD was added, p62 / SQSTM1 was degraded and an increase in autophagic flux was confirmed. In Alzheimer's disease, it has been reported that an increase in intracellular cholesterol inhibits autophagosome-lysosome fusion and reduces the clearance of amyloid beta. Therefore, the above results confirmed the effect of Lac-HP-β-CyD on the intracellular accumulation of amyloid beta in Alzheimer's disease.

[0057] (Example 24) Confirmation of the effect of Lac-HP-β-CyD using AD mouse model AD model mouse (App NL-G-F Behavioral tests were performed on mice using the 10-mg guinea pig (Mice). The behavioral tests were measured using the Y-maze test-short-term memory test and the Barnes maze test. Each mouse (7 months old) was allowed to acclimate to the testing room for approximately 1 h. To reduce the effects of circadian rhythm, all behavioral tests were performed with two lights on at the same time intervals (09:00-18:00) every day on the testing platform (100 cm from the floor). Each test was filmed by SMART V3.0. After each test, the mouse was returned to its home cage, and the maze was wiped with 70% ethanol and then wiped dry. A total of 60–68 mice were used for the behavioral tests.

[0058] 2-month-old App NL-G-F Mice were subcutaneously administered 20 μL / kg of saline containing HP-β-CyD or Lac-HP-β-CyD (DSL8) (1.4 mmol / kg each) twice a week until the age of 7 months (5 months). NL-G-F To improve cognitive impairment in mice, HP-β-CyD or Lac-HP-β-CyD (DSL8) was administered to 7-month-old male and female mice over a long period of time, and two types of behavioral tests (Y-maze and Barnes behavioral test) were performed.

[0059] (Example 24-1) Y-maze test (short-term memory test) In the Y-maze test (short-term memory test), a mouse was placed in the center of a three-arm Y-shaped maze (22 cm long, 5 cm wide, 15 cm high), and the rate at which the mouse was able to enter different arms consecutively within 8 minutes was defined as the alternation rate (Alternation, Eq. (2)). The above procedure was repeated for the number of mice. If the mouse remembers, it will try to enter an arm that it has not entered immediately before, so the number of times the mouse entered three consecutive different arms (Alternation Triplet) was counted, and the alternation rate (%) was calculated using the following formula. Alternation behavior rate (Alternation behavior:%) =Alternation Triplet (count)x100 / (Total Arm Entries -2) The results are shown in Figure 31.

[0060] (Example 24-2) Barnes maze test (spatial learning and memory test) There were 20 holes on the circumference of the disk, and one of them was set as a target (a deep hole with a diameter of 5 cm that the mouse could enter). The disk was divided into four equal parts, and each area was designated as the Target quadrant (TQ), Opposite quadrant (OP), Left quadrant (LP), and Right quadrant (RP). By performing training for four days, the mouse memorized the location of the target and moved toward the target from the center of the disk. In addition, during the three-minute training, mice that could not find the target were returned to the starting position, guided from the center of the maze to the target, and placed in the target box and left there for one minute. If the mouse could not enter, it was not forced to enter but was allowed to stay near the target. The above was performed three times a day for four consecutive days for each mouse. On the fifth day, the deep hole of the target was removed and a three-minute probe test (measured the time spent near the target) was performed. The target area was a circle with a diameter of 12 cm centered on the target (a hole with a diameter of 5 cm). Finally, after the probe test on the 5th day, the animals were returned to the cage for 4 days, and the probe test was carried out again on the 9th day. The results are shown in Figure 312.

[0061] Example 25: Effect of Lac-HP-β-CyD on total brain cholesterol After the behavioral test of Example 24 was performed, the brains of the mice were collected and the amount of cholesterol accumulated in the brain was evaluated. Specifically, the evaluation was performed as follows. The brains of 7-month-old mice were removed and weighed. The brains were placed in a tube, Cell Lysis Buffer M (1 mL) was added, and the mixture was homogenized to prepare the sample solution. The sample solution was added in equal amounts to two test tubes, one of which contained cholesterol esterase (TC measurement) and the other contained pure water (UC measurement), and incubated at 37°C for 30 minutes. Then, 2 mL of a 2-propanol / hexane (2:3) mixture was added to each sample and shaken for 10 minutes. Further, 1 mL of hexane was added, vortexed for 30 seconds, 1 mL of pure water was added, vortexed for 30 seconds, and centrifuged. After centrifugation, 1 mL of the supernatant was evaporated to dryness at 40°C, dissolved in 200 mL of a mixture (2-propanol / polyoxyethylene alkyl ether / polyoxyethylene lauryl ether = 87 / 10 / 3), and the amount of cholesterol in the solution was measured by measuring the absorbance using a Determiner LFC. The results are shown in Figure 33.

[0062] (Example 26) Effect of Lac-HP-β-CyD on free cholesterol in the brain After the behavioral test of Example 24 was performed, the brains of the mice were collected and the amount of cholesterol accumulated in the brain was evaluated. Specifically, the evaluation was performed as follows. Brain slices were prepared from 7-month-old mice by the above-mentioned brain slice preparation process. Then, the slices were washed with PBS, and Filipin III solution (50 μg / mL) was added, followed by staining for 1 hour at 37°C in the dark. After washing with PBS, each slice was attached to a slide glass, embedded in Marinol solution, and observed under a confocal laser microscope to confirm free cholesterol. The results are shown in Figure 34. The left figure is a microscope image, and the right figure is a graph of the fluorescence intensity. The amount of free cholesterol was significantly reduced by Lac-HP-β-CyD.

[0063] (Example 27) Effect of Lac-HP-β-CyD on Aβ in the brain (1) After the behavioral test of Example 24 was performed, the brains of the mice were collected and the amount of Aβ accumulated in the brain was evaluated. Specifically, the evaluation was performed as follows. Using 7-month-old mice, the above-mentioned brain slice preparation process was carried out to prepare brain slices. Then, the slices were washed with PBS, and Aβ-containing 0.3% PBST solution (1:1,000) was added, and the slices were stained at 37°C under light for 12 hours. The slices were washed with PBS, and secondary antibody treatment (Alexa Fluor 488, 1:1,000) was added, and the slices were stained at 37°C under light for 3 hours. The slices were washed with PBS, and DAPI (300 nM) solution was added, and the slices were stained at 37°C under light for 5 minutes. The slices were washed with PBS, and each slice was attached to a slide glass, embedded in Marinol solution, and observed with a confocal laser microscope. The results of the image of Aβ expression in the brain are shown in Figure 35. The number of Aβ plaques was quantified by visually counting using the acquired tissue images. The results are shown in Figure 36. The images confirming the accumulation of Aβ in the cerebral cortex and hippocampus are shown in Figure 37. It was confirmed that Lac-HP-β-CyD significantly reduced Aβ accumulation in the cerebral cortex and hippocampus.

[0064] (Example 28) Effect of Lac-HP-β-CyD on Aβ in the brain (2) After the behavioral experiment of Example 24 was performed, the brains of the mice were collected, and the amount of Aβ accumulated in the brain was evaluated by ELISA. Specifically, the procedure was as follows. The brains of 7-month-old mice were removed and weighed. The brains were placed in a tube, Cell Lysis Buffer M (1 mL) was added, and the homogenized solution was used as the sample solution. The sample solution (10 mL) was added to a 96-well plate together with the coating buffer. The sample solution was incubated overnight at 4°C, washed with PBST, and blocking buffer was added. The sample solution was washed with PBST, and 0.05% PBST solution containing Aβ (1:1,000, 50 mL / well) was added, and incubated at 37°C in the dark for 1 hour. The sample was washed with PBST, and secondary antibody treatment (1:5,000, 50 mL / well) was added, and incubated at 37°C in the dark for 1 hour. The sample was washed with PBST, and incubated with 3,3',5,5'-tetramethylbenzidine (TMB) solution for 1 hour, and then HCl solution was added. The absorbance of the sample at 450 nm was measured using a microplate reader. The results are shown in Figure 38. It was confirmed that Lac-HP-β-CyD significantly reduced Aβ accumulation.

[0065] (Example 29) Effect of Lac-HP-β-CyD on brain glial cells After the behavioral test of Example 24 was performed, the brains of the mice were collected and the amount of glial cell accumulation in the brain was evaluated. Specifically, the evaluation was performed as follows. The brain slices were prepared by the above-mentioned brain slice preparation process using 7-month-old mice. Then, the slices were washed with PBS, and 0.3% PBST solution containing Iba1 and GFAP (1:1,000) was added, and the slices were stained at 37°C in the dark for 12 hours. The slices were washed with PBS, and secondary antibody treatment (Alexa Fluor 488, 1:1,000) was added, and the slices were stained at 37°C in the dark for 3 hours. The slices were washed with PBS, and DAPI solution was added, and the slices were stained at 37°C in the dark for 5 minutes. The slices were washed with PBS, and each slice was attached to a slide glass, embedded in Marinol solution, and observed with a confocal laser microscope. The results are shown in Figure 39. It was confirmed that the accumulation of microglia was significantly reduced by Lac-HP-β-CyD.

[0066] (Example 30) Effect of Lac-HP-β-CyD on hearing A therapeutic dose of Lac-HP-β-CyD (2.9 mmol / kg) was administered subcutaneously to mice, and the mouse auditory brainstem response (ABR) test was performed. Hearing thresholds were measured using an ABR System 3 (Tucker-Davis Technologies, FL, USA). ABR was measured under anesthesia in mice. A measurement electrode was placed just under the skin near the head, and a ground electrode was placed just under the skin on the back. Frequencies were measured at 4, 8, 12, 20, and 32 kHz, which are included in the mouse's hearing range. The hearing threshold at each frequency was defined as the lowest level at which the ABR waveform obtained by changing the sound pressure level in 5 dB intervals could be clearly detected by visual inspection. The results are shown in Figure 40. It was confirmed that Lac-HP-β-CyD does not affect hearing.

[0067] The above detailed description is merely illustrative of the objects and subject matter of the present invention and is not intended to limit the scope of the appended claims. Various modifications and substitutions to the described embodiments will be apparent to those skilled in the art from the teachings set forth herein without departing from the scope of the appended claims. [Industrial Applicability]

[0068] The lactose-modified hydroxypropyl cyclodextrins of the present invention are useful as pharmaceutical compositions for the treatment of lipid storage diseases.

Claims

1. A pharmaceutical composition for treating lipid accumulation in the brain, comprising as an active ingredient a compound selected from the group consisting of lactose-modified hydroxypropyl-β-cyclodextrin and lactose-modified hydroxypropyl-γ-cyclodextrin, wherein the compound is transported at least into the brain when administered to a living body.

2. The pharmaceutical composition according to claim 1, wherein the entry of the compound into the brain includes entry of the compound into neurons in the brain in addition to penetration of the compound through the blood-brain barrier.

3. 3. The pharmaceutical composition according to claim 1 or 2, which is administered intravenously or subcutaneously.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the degree of substitution of lactose for cyclodextrin in the compound is 4 or more.

5. The pharmaceutical composition according to any one of claims 1 to 3, wherein the degree of substitution of lactose for cyclodextrin in the compound is 8 or more.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the lipid accumulation is caused by a lipid storage disease which causes lipid accumulation in the brain.

7. The pharmaceutical composition of claim 6, wherein the lipid storage disease is a lysosomal disease.

8. The pharmaceutical composition of claim 6, wherein the lipid storage disease is Niemann-Pick disease.

9. The pharmaceutical composition of claim 6, wherein the lipid storage disease is Alzheimer's disease.

10. The pharmaceutical composition according to any one of claims 1 to 9, further comprising another drug that exhibits a pharmacological effect in the brain.

11. The pharmaceutical composition according to any one of claims 1 to 10, which is administered continuously once or twice a week for several weeks or more.

12. A pharmaceutical composition for treating lipid accumulation in the brain, comprising as an active ingredient a compound selected from the group consisting of lactose-modified hydroxypropyl-β-cyclodextrin and lactose-modified hydroxypropyl-γ-cyclodextrin, wherein the compound is administered for the purpose of reducing the amount of cholesterol in at least neurons in the brain.

13. 13. The pharmaceutical composition of claim 12, which is administered intravenously or subcutaneously.

14. The pharmaceutical composition according to claim 12 or 13, wherein the degree of substitution of lactose for cyclodextrin in the compound is 4 or more.

15. The pharmaceutical composition according to claim 12 or 13, wherein the degree of substitution of lactose for cyclodextrin in the compound is 8 or more.

16. The pharmaceutical composition according to any one of claims 12 to 15, wherein the lipid accumulation is caused by a lipid storage disease which causes lipid accumulation in the brain.

17. 17. The pharmaceutical composition of claim 16, wherein the lipid storage disease is a lysosomal disease.

18. 17. The pharmaceutical composition of claim 16, wherein the lipid storage disease is Niemann-Pick disease.

19. 17. The pharmaceutical composition of claim 16, wherein the lipid storage disease is Alzheimer's disease.

20. The pharmaceutical composition according to any one of claims 12 to 19, further comprising another drug that exhibits a pharmacological effect in the brain.

21. The pharmaceutical composition according to any one of claims 12 to 20, which is administered continuously once or twice a week for several weeks or more.

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