Drug carrier agent and its manufacturing method

Abstract

To provide a drug carrier useful for a drug delivery system by utilization of cyclodextrin.SOLUTION: Provided a drug carrier agent comprising a heat condensate of lactose and cyclodextrin. The heat condensate is preferably obtained by heating a mixture of lactose and cyclodextrin in the presence of a catalyst. This drug carrier agent is preferably used for delivering a drug into the brain and allowing it to be absorbed by cerebrovascular endothelial cells. It is also suitably used to form a pharmaceutical composition in combination with a drug.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a drug carrier agent for drug delivery to target organs, tissues, cells, etc., and a method for producing the same. [Background technology]

[0002] It is known that there is a blood-brain barrier (BBB) ​​between the blood and the brain that restricts the transfer of substances. This is thought to be due to the tight junction of cerebral vascular endothelial cells, which physically narrows the gap between the cells, and the selective uptake and excretion of substances by the cells themselves. The permeability selectivity of the blood-brain barrier is very high, and even if the central nervous system is damaged, drugs cannot be delivered to the target area of ​​the central nervous system.

[0003] Several methods for delivering drugs to the brain have been reported so far. For example, Patent Document 1 describes a technique for delivering antibodies to the brain by glycosylation. Patent Document 2 describes a technique for delivering drugs to the brain by modifying the surface of a nanocarrier formed by self-organization of a high molecular weight polymer in a solution with glucose. Patent Document 3 describes a technique for transporting drugs to the brain by attaching a peptide that can pass through the BBB.

[0004] On the other hand, cyclodextrin (hereinafter sometimes simply referred to as "CD") is a cyclic oligosaccharide in which glucose residues are linked in an α-1,4 bond, and from a three-dimensional perspective, it has a bottomless bucket-like structure, with the outside of the cavity being hydrophilic, while the inside of the cavity is hydrophobic. Due to this characteristic, CD is known to exhibit the phenomenon of incorporating specific organic molecules (guest molecules) into the cavity by wrapping them around them (inclusion).

[0005] Due to the inclusion action of CDs, they are used in various applications in the pharmaceutical field, such as solubilization and stabilization of drugs, improvement of bioavailability, and improvement of taste. In addition, CD derivatives with improved biocompatibility and directionality have been developed by adding sugars, amino acids, functional groups, etc. to CDs, and research on their application to drug delivery systems (DDS) and active pharmaceutical ingredients (APIs) is ongoing. For example, Non-Patent Document 1 describes a DDS carrier that targets cancer cells via folate receptors by modifying the primary hydroxyl group of β-CD with folic acid using two molecules of caproic acid as a spacer. Non-Patent Document 2 describes the use of methylated β-CD as an anticancer drug that targets cancer cells via folate receptors by modifying folic acid. Non-Patent Document 3 suggests that CDs can be used as DDS carriers for drug delivery to specific tissues, since the interaction with various lectins can be improved by modifying CDs with glycans that serve as ligands. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Special table 2009-520691 [Patent Document 2] WO2017 / 002979 [Patent Document 3] Patent Publication 2018-165253 [Non-patent literature]

[0007] [Non-Patent Document 1] The Science and Technology of Cyclodextrin, CMC Publishing Co., Ltd., December 2013, pp. 153-169 [Non-Patent Document 2] The Science and Technology of Cyclodextrin, CMC Publishing Co., Ltd., December 2013, pp. 145-146 [Non-Patent Document 3] Science and Technology of Cyclodextrin, CMC Publishing Co., Ltd., December 2013, pp. 70-78 Summary of the Invention [Problem to be solved by the invention]

[0008] Although various cyclodextrin derivatives have been developed for application as DDS carriers and active pharmaceutical ingredients (APIs), there have been no reports on cyclodextrin derivatives for delivery to the brain.

[0009] An object of the present invention is to provide a drug carrier agent that utilizes cyclodextrin and is useful for a drug delivery system, and to provide a pharmaceutical composition containing the drug carrier agent. [Means for solving the problem]

[0010] Means of the Invention The present inventors have conducted extensive investigations to achieve the above object and have found that a heat condensation product of lactose and cyclodextrin is useful as a drug carrier agent, thus completing the present invention.

[0011] That is, the present invention provides the following configuration. [1] A drug carrier containing a heat condensation product of lactose and cyclodextrin. [2] The drug carrier agent described in [1], wherein the thermal condensation product is obtained by heating a mixture of the lactose and the cyclodextrin in the presence of a catalyst. [3] A drug carrier agent according to [1] or [2], which is used to deliver a drug to the brain. [4] A drug carrier agent according to any one of [1] to [3], which is used to absorb a drug into cerebrovascular endothelial cells. [5] A pharmaceutical composition comprising the drug carrier agent according to any one of [1] to [4] and a drug, either together or separately. [6] A method for producing a drug carrier agent comprising a thermal condensation product of lactose and cyclodextrin, the method comprising the steps of mixing lactose and cyclodextrin to obtain a mixture, and heating the mixture to obtain a thermal condensation product of the lactose and cyclodextrin. [7] The method for producing a drug carrier agent described in [6], wherein the heating to obtain the thermal condensation product is carried out in the presence of a catalyst. [8] The method for producing a drug carrier agent according to [6] or [7], further comprising a step of purifying the heat-condensation product after the step of obtaining the heat-condensation product. Effect of the Invention

[0012] According to the present invention, a drug carrier agent useful for a drug delivery system can be provided by utilizing cyclodextrin, and a pharmaceutical composition containing the drug carrier agent can be provided. [Brief description of the drawings]

[0013] [Figure 1] 1 is a graph showing the results of observation by a confocal laser scanning microscope of the uptake of the fluorescent substance TPPS complexed with β-CD or various sugar-modified β-CDs in Test Example 1 into human cerebrovascular endothelial cells (hCMEC / D3 cells). [Diagram 2] This is a graph showing the results of investigating the uptake of the fluorescent substance TPPS, which was complexed with β-CD or various sugar-modified β-CDs, into human cerebrovascular endothelial cells (hCMEC / D3 cells) by relative fluorescence intensity in Test Example 1. The * in the graph indicates that there is a significant difference (p<0.05) compared to the fluorescence intensity when β-CD was added. [Diagram 3] 1 is a graph showing the results of observation of the uptake of fluorescently labeled lactose-modified β-CD into human cerebrovascular endothelial cells (hCMEC / D3 cells) using a confocal laser scanning microscope in Test Example 2. [Figure 4]This is a graph showing the results of investigating the uptake of fluorescently labeled lactose-modified β-CD into human cerebrovascular endothelial cells (hCMEC / D3 cells) by relative fluorescence intensity in Test Example 2. The * in the graph indicates a significant difference (p<0.05) compared to the fluorescence intensity when lactose or glucose was not added. [Diagram 5] This is a graph showing the results of measuring the electrical resistance of the cell layer over time for human cerebrovascular endothelial cells (hCMEC / D3 cells) seeded on the upper side of a Transwell when examining the permeability of fluorescently labeled β-CD or various sugar-modified β-CD through a human BBB model in Test Example 4. [Figure 6] This is a graph showing the results of measuring the concentration in the medium on the lower side of a Transwell over time when examining the permeability of fluorescently labeled β-CD or various sugar-modified β-CD through a human BBB model in Test Example 4. [Figure 7] This is a graph showing the results of observation by a confocal laser scanning microscope of each cell (upper side of Transwell: hCMEC / D3 cells, and lower side of Transwell: SH-SY5Y cells) after 60 minutes of incubation when examining the permeability of fluorescently labeled β-CD or various sugar-modified β-CD through a human BBB model in Test Example 4. [Figure 8] This is a graph showing the results of measuring the concentration in the medium on the lower side of the Transwell over time in Test Example 5, when examining the inhibitory conditions that affect the permeability of fluorescently labeled lactose-modified β-CD through a human BBB model. [Figure 9] FIG. 13 is a graph showing the results of measuring the concentration of galectin-1 in the medium on the lower side of a Transwell over time in Test Example 6, when investigating the effect of galectin-1 on the permeability of fluorescently labeled β-CD or various sugar-modified β-CD through a human BBB model. [Figure 10]This is a graph showing the results of imaging the fluorescence intensity of each organ recovered 60 minutes after intravenous administration of fluorescently labeled β-CD or various sugar-modified β-CD to mice in Test Example 7, using a luminescence / fluorescence imaging system (IVIS Lumina XRMS In Vivo Imaging System). [Figure 11] This is a graph showing the results of quantifying the relative fluorescence intensity from images taken with a luminescence and fluorescence imaging system (IVIS Lumina XRMS In Vivo Imaging System) for each organ collected 60 minutes after intravenous administration of fluorescent-labeled β-CD or various sugar-modified β-CD to mice in Test Example 7. * and † in the figure indicate a significant difference (p<0.05) compared to the fluorescence intensity when fluorescent-labeled β-CD or fluorescent-labeled mannose-modified β-CD was added, respectively. [Figure 12] This is a graph showing the results of determining the brain-localization rate per dose of fluorescent-labeled β-CD or various sugar-modified β-CD per dose, which was determined by homogenizing and extracting the brain tissue collected 60 minutes after intravenous administration to mice in Test Example 7. * and † in the figure indicate a significant difference (p<0.05) compared to the brain-localization rate when fluorescent-labeled β-CD or fluorescent-labeled mannose-modified β-CD was added, respectively. [Figure 13] FIG. 13 is a graph showing the results of observing the localization of fluorescently labeled β-CD or various sugar-modified β-CD in cerebral blood vessels, hippocampus, and cerebral cortex by confocal laser scanning microscope 60 minutes after intravenous administration to mice in Test Example 8. [Figure 14] This is a graph showing the results of measuring the concentration in the medium on the lower side of the Transwell over time when examining the permeability of the fluorescent substance TPPS complexed with β-CD or various sugar-modified β-CDs through a human BBB model in Test Example 9. [Figure 15]This is a graph showing the results of determining the brain distribution rate per dose by measuring the amount of brain distribution in the brain collected 60 minutes after intravenous administration of the fluorescent substance TPPS formed with β-CD or various sugar-modified β-CD in Test Example 10. *, †, and ‡ in the figure indicate a significant difference (p<0.05) compared to the brain distribution rate when TPPS alone, β-CD / TPPS complex, or mannose-modified β-CD / TPPS complex was added, respectively. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] The present invention relates to a drug carrier agent comprising a heat condensation product of lactose and cyclodextrin (hereinafter, sometimes simply referred to as "heat condensation product").

[0015] The above-mentioned heat condensation product is a composition containing, as a main component, lactose-modified cyclodextrin in which lactose is dehydrated and condensed with cyclodextrin. Since the condensation reaction (dehydration condensation between hydroxyl groups) caused by heat treatment proceeds randomly, the bond site of lactose to each cyclodextrin (which hydroxyl group in cyclodextrin is dehydrated and condensed with), the bond type (α-bond, β-bond), and the degree of substitution (how many lactose molecules are bonded per cyclodextrin molecule) are random, and lactose-modified cyclodextrins of various structures may be mixed. In addition, the above-mentioned heat condensation product may contain unreacted raw materials (lactose, cyclodextrin) and by-products of the heat condensation reaction (heat condensation product of lactose, monosaccharides and their condensates produced by decomposition of lactose, sugar heat decomposition products, etc.). Furthermore, the above-mentioned heat condensation product may contain a small amount of a heat condensation product of lactose condensation further condensed into cyclodextrin, or a product of glucose or galactose produced by decomposition of lactose and their condensation products condensed into cyclodextrin. In addition, when the raw material contains carbohydrates other than lactose and cyclodextrin, the above-mentioned heat condensation product may contain unreacted raw materials, condensation products / decomposition products of the carbohydrates, modified cyclodextrins in which the carbohydrates are condensed into cyclodextrins, etc.

[0016] As shown in the examples below, lactose-modified cyclodextrin obtained by dehydration condensation of lactose with the cyclodextrin contained in the above-mentioned heat condensation product is useful as a drug carrier (e.g., a carrier for brain delivery). In addition to the heat condensation reaction, lactose-modified cyclodextrin can also be produced by a conventional chemical synthesis method. However, by adopting the heat condensation reaction, lactose-modified cyclodextrin can be obtained more cheaply and with less environmental impact.

[0017] The conditions for the heat condensation reaction to obtain the heat condensate are not particularly limited and can be appropriately selected or determined under any conditions. For example, the heat treatment may be performed at 100°C to 300°C for 1 to 180 minutes, more preferably at 150°C to 250°C for 1 to 180 minutes.

[0018] The raw material may be any material containing lactose and cyclodextrin. There are no particular limitations on the nature of the raw material, and crystalline products, amorphous powder products, liquid products, etc. may be used. In consideration of the efficiency of the condensation reaction, crystalline products and amorphous powder products are preferred. In addition, the raw material may be either a pure product or a mixture with other carbohydrates (carbohydrate composition), but in consideration of the efficiency of the condensation reaction, it is preferable to use a pure product.

[0019] There is no particular limitation on the type of cyclodextrin, and α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, etc. can be used. Any suitable type may be selected taking into consideration the interaction with the drug that serves as the guest molecule, and a mixture of multiple cyclodextrins may also be used.

[0020] The above-mentioned heat condensation reaction may be carried out under catalyst-free conditions, but from the viewpoint of reaction efficiency of the condensation reaction, it may be carried out in the presence of a catalyst. There are no particular limitations on the catalyst as long as it catalyzes the sugar condensation reaction, and inorganic acids (hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, etc.), organic acids (citric acid, fumaric acid, tartaric acid, succinic acid, acetic acid, etc.), mineral substances (diatomaceous earth, activated clay, acid clay, bentonite, kaolinite, talc, etc.), activated carbon (steam carbon, zinc chloride carbon, sulfonated activated carbon, oxidized activated carbon, etc.), etc. can be used. Considering the coloration and safety of the obtained heat condensation product, as well as the taste and odor, it is preferable to use activated carbon as a catalyst. In addition, two or more catalysts can be used in combination.

[0021] In the present invention, the above-mentioned heat condensation product may be used as it is obtained by the heat condensation reaction, but since there is a possibility that the effect as a drug carrier agent may be weakened by unreacted raw materials (lactose, cyclodextrin) and by-products of the heat condensation reaction (heat condensation products of lactose, monosaccharides and their condensates produced by the decomposition of lactose, sugar heat decomposition products, etc.) competing with the lactose-modified cyclodextrin, it is preferable to use a product that has been purified to increase the purity of the lactose-modified cyclodextrin. That is, it is preferable to remove substances other than the lactose-modified cyclodextrin, such as raw materials, by-products, and even catalysts used as necessary, from the product obtained by the heat condensation reaction by purification. In addition to the treatment of removing the above-mentioned impurities, in the purification process, a lactose-modified cyclodextrin of a specific structure may be separated from a lactose-modified cyclodextrin composition of a different structure. As a purification means, a general method may be used and there is no particular limitation. For example, fractionation using a column packed with silica gel or activated carbon modified with octadecylsilyl groups, fractionation using a membrane with a pore size suitable for the substance to be removed / retained, fractionation by recrystallization utilizing the solubility of the substance to be removed / retained, etc. can be used.

[0022] The drug carrier agent of the present invention is preferably used in combination with any drug to efficiently deliver the drug to tissues, organs, cells, etc. in the body. Specifically, for example, it is preferably used to efficiently deliver the drug to the brain by passing through the blood-brain barrier. Alternatively, it is preferably used to efficiently absorb the drug into the cerebrovascular endothelial cells that constitute the blood-brain barrier.

[0023] The drugs to which the drug carrier agent of the present invention is applied are not particularly limited as long as they form an inclusion complex with lactose-modified cyclodextrin, and examples thereof include substances whose molecules themselves are physiologically active in the brain (hereinafter sometimes simply referred to as "biologically active substances") or substances that exert their functions in the brain (hereinafter sometimes simply referred to as "brain functional substances").

[0024] The physiologically active substance includes, but is not limited to, low molecular weight compounds, polypeptides, oligopeptides, proteins, nucleic acids, and the like.

[0025] Examples of low molecular weight compounds as the above-mentioned physiologically active substances include, but are not limited to, compounds contained as active ingredients in pharmaceuticals used for the treatment and / or prevention of various diseases related to the brain or central nervous system, such as active ingredients in therapeutic agents for central nervous system diseases, or compounds used for the treatment and / or prevention of brain diseases, such as compounds having an anti-inflammatory effect for suppressing inflammation in the brain, compounds exhibiting anti-cancer effects, and compounds that are active ingredients in antibacterial agents or antiviral agents for the treatment of intracerebral infections.

[0026] Examples of the above-mentioned bioactive substances include peptides (polypeptides and oligopeptides), such as bioactive peptides, and peptides used for treating and / or preventing diseases related to the brain and central nervous system. Specific examples include, but are not limited to, somatostatin, which regulates the expression of enzymes involved in the degradation of amyloid beta peptide in the brain, insulin, which controls the function of neurons in the brain, or other peptides related to the functions of the brain and central nervous system, and derivatives thereof.

[0027] Examples of the physiologically active substance include, but are not limited to, protein molecules that have physiological activity in the brain, and include proteins used for treating and / or preventing diseases, etc. Examples include enzymes, antibodies, transcription factors, or specific parts that constitute these.

[0028] Examples of the nucleic acid as the above-mentioned physiologically active substance include nucleic acids used for the treatment and / or prevention of diseases related to the brain or central nervous system. Examples include, but are not limited to, nucleic acids for the treatment of various diseases by gene knockdown method or using RNA interference, such as antisense nucleic acids (DNA or RNA), heteroduplex nucleic acids, siRNA and shRNA. Specific examples include, but are not limited to, gene therapy for amyotrophic lateral sclerosis (ALS) and Parkinson's disease.

[0029] The drug to which the present invention is applied may be a known drug containing the above-mentioned physiologically active substances as active ingredients, and examples thereof include, but are not limited to, anticancer drugs, anti-Parkinson's disease drugs, anti-dementia drugs, psychotropic drugs, etc.

[0030] On the other hand, the above-mentioned intracerebral functional substance includes, but is not limited to, for example, a molecule that serves as a marker in the brain, a molecule that can be used to image the brain or a target in the brain, etc. The intracerebral imaging molecule is not particularly limited, but examples thereof include compounds that can visualize targets in vivo, such as fluorescent dyes, quantum dots, nanomagnetic materials, nanogold, intracellular molecule visualization reagents, and labeled molecules that can be detected by PET.

[0031] In any embodiment not limited by the present invention, the drug carrier agent containing the above-mentioned heat-treated lactose and cyclodextrin may be combined with any drug to form a pharmaceutical composition. In this case, a known method can be used to form an inclusion complex between any drug and the above-mentioned lactose-modified cyclodextrin, etc., to provide a pharmaceutical composition containing them as one agent. Alternatively, it may be made into a pharmaceutical composition containing them in the form of a separate agent that is mixed when used.

[0032] In the drug carrier agent of the present invention or the pharmaceutical composition using the same, the type of cyclodextrin, the degree of lactose substitution, the amount of cyclodextrin, etc. can be appropriately selected and used according to the drug to be transferred into the brain and according to various conditions such as the purpose of transfer, for example, the transfer rate and the transfer amount. In addition, it can be formulated according to a known method and administered parenterally or orally to mammals including humans as a legally acceptable pharmaceutical in an appropriate dosage form such as a liquid. Examples of parenteral administration methods include injections and patches (transdermal administration). The drug carrier agent of the present invention or the pharmaceutical composition using the same is preferably administered parenterally.

[0033] The drug carrier agent of the present invention or the pharmaceutical composition using the same can be appropriately mixed with any component within a range that does not impair its effect.The optional components include, but are not limited to, crosslinking agents, dissolving agents, emulsifiers, moisturizing agents, cooling agents, inorganic powders, antioxidants, preservatives, colorants, flavoring agents, pH adjusters, stabilizers, etc.

[0034] The drug carrier provided by the present invention or the pharmaceutical composition using the same can be suitably used as a pharmaceutical for preventing or treating, for example, a brain or nervous system disease or disorder. The brain or nervous system disease or disorder can include, but is not limited to, Alzheimer's disease, malignant brain tumor, Parkinson's disease, Niemann-Pick disease type C, stroke, cerebral hematoma, dementia, muscular dystrophy, multiple sclerosis, amyotrophic lateral sclerosis, cystic fibrosis, Angelman syndrome, Liddle syndrome, myasthenia gravis, spinal muscular atrophy, Down's syndrome, Huntington's disease, schizophrenia, depression, taupathy disease, Pick's disease, Paget's disease, lysosomal disease accompanied by brain damage, cancer, prion disease, traumatic brain injury, viral and bacterial central nervous system diseases, etc. EXAMPLES

[0035] The present invention will be described in more detail below with reference to examples, although the scope of the present invention is not limited to these examples.

[0036] (Preparation Example 1) Preparation of lactose-modified β-CD (Lac-modified β-CD) 100 parts by mass of β-CD (Celdex B-100, Nippon Shokuhin Kako) were mixed with 100 parts by mass of lactose monohydrate and 3 parts by mass of activated carbon until homogeneous, placed in a stainless steel tray, and heated at 180 ° C. for 1 hour in a hot air dryer. The resulting heated reaction product was dissolved in ultrapure water, and the activated carbon was removed by filtration. The solution was then fed to an activated carbon column made by mixing activated carbon and Celite, and impurities were eluted by passing ultrapure water and a 10% ethanol aqueous solution through the column. Subsequently, a lactose-modified β-CD crude fraction was obtained by passing a 25% ethanol aqueous solution through the column. The lactose-modified β-CD crude fraction was subjected to preparative high-performance liquid chromatography using an ODS column multiple times to remove the remaining impurities and β-CD, and lactose-modified β-CD was obtained. 1H-NMR analysis confirmed that the number of lactose bonds (substitution degree) per β-CD molecule of this lactose-modified β-CD was 1.7.

[0037] (Preparation Example 2) Preparation of glucose-modified β-CD (Glc-modified β-CD) 100 parts by mass of β-CD (Celdex B-100, Nippon Shokuhin Kako) were mixed with 100 parts by mass of glucose and 3 parts by mass of activated carbon until homogeneous, placed in a stainless steel tray, and heated in a hot air dryer at 180°C for 1 hour. The resulting heated reaction product was dissolved in ultrapure water, and the activated carbon was removed by filtration. The solution was then fed to an activated carbon column made by mixing activated carbon and Celite, and impurities were eluted by passing ultrapure water and a 10% ethanol aqueous solution through the column. Subsequently, a glucose-modified β-CD crude fraction was obtained by passing a 25% ethanol aqueous solution through the column. The glucose-modified β-CD crude fraction was subjected to preparative high-performance liquid chromatography using an ODS column multiple times to remove the remaining impurities and β-CD, and glucose-modified β-CD was obtained. 1H-NMR analysis confirmed that the number of glucose bound per β-CD molecule (substitution degree) of this glucose-modified β-CD was 3.3.

[0038] (Preparation Example 3) Preparation of mannose-modified β-CD (Man-modified β-CD) 100 parts by mass of β-CD (Celdex B-100, Nihon Shokuhin Kako) were mixed with 100 parts by mass of mannose and 3 parts by mass of activated carbon until homogeneous, placed in a stainless steel tray, and heated at 180°C for 1 hour in a hot air dryer. The resulting heated reaction product was dissolved in ultrapure water, and the activated carbon was removed by filtration. The solution was then passed through an activated carbon column made by mixing activated carbon and Celite, and impurities were eluted by passing ultrapure water and a 10% ethanol aqueous solution through the column. Subsequently, a mannose-modified β-CD crude fraction was obtained by passing a 25% ethanol aqueous solution through the column. The mannose-modified β-CD crude fraction was subjected to preparative high-performance liquid chromatography using an ODS column multiple times to remove the remaining impurities and β-CD, and mannose-modified β-CD was obtained. 1H-NMR analysis confirmed that the number of mannose bound per β-CD molecule (substitution degree) of this mannose-modified β-CD was 3.6.

[0039] (Preparation Example 4) Preparation of galactose-modified β-CD (Gal-modified β-CD) 100 parts by mass of β-CD (Celdex B-100, Nippon Shokuhin Kako) were mixed with 100 parts by mass of galactose and 3 parts by mass of activated carbon until homogeneous, placed in a stainless steel tray, and heated at 180°C for 1 hour in a hot air dryer. The resulting heated reaction product was dissolved in ultrapure water, and the activated carbon was removed by filtration. The solution was then passed through an activated carbon column made by mixing activated carbon and Celite, and impurities were eluted by passing ultrapure water and a 10% ethanol aqueous solution through the column. Subsequently, a galactose-modified β-CD crude fraction was obtained by passing a 25% ethanol aqueous solution through the column. The galactose-modified β-CD crude fraction was subjected to preparative high-performance liquid chromatography using an ODS column multiple times to remove remaining impurities and β-CD, and galactose-modified β-CD was obtained. 1H-NMR analysis confirmed that the number of galactoses bound per β-CD molecule of this galactose-modified β-CD (substitution degree) was 1.8.

[0040] (Preparation Example 5) Preparation of N-acetylglucosamine-modified β-CD (GlcNAc-modified β-CD) 100 parts by mass of β-CD (Celdex B-100, Nihon Shokuhin Kako) was mixed with 100 parts by mass of N-acetylglucosamine and 3 parts by mass of activated carbon until homogeneous, placed in a stainless steel tray, and heated at 180°C for 1 hour in a hot air dryer. The resulting heated reaction product was dissolved in ultrapure water and the activated carbon was removed by filtration. The solution was then fed to an activated carbon column made by mixing activated carbon and Celite, and impurities were eluted by passing ultrapure water and a 10% aqueous ethanol solution through the column. Subsequently, a crude fraction modified with N-acetylglucosamine was obtained by passing a 25% aqueous ethanol solution through the column. The crude fraction modified with N-acetylglucosamine was subjected to preparative high-performance liquid chromatography using an ODS column multiple times to remove the remaining impurities and β-CD, and N-acetylglucosamine-modified β-CD was obtained. 1H-NMR analysis confirmed that the number of N-acetylglucosamines bound per β-CD molecule (substitution degree) of this N-acetylglucosamine-modified β-CD was 3.7.

[0041] [Test Example 1] Cellular uptake of glycosylated β-CD / fluorescent substance complex The uptake into human cerebrovascular endothelial cells (hCMEC / D3 cells) was confirmed by using a complex of various sugar-modified β-CD and the fluorescent substance tetraphenylporphine tetrasulfonic acid (TPPS).

[0042] hCMEC / D3 cells were cultured at 1 × 10 per glass-bottom culture vessel. 4 The cells were seeded and incubated in EBM-2 medium at 37°C for 24 hours. Separately, unmodified β-CD or various sugar-modified β-CDs (125μM) prepared in Preparation Examples 1 to 5, and TPPS (10μM) were dissolved in 200μL of EBM-2 medium to prepare complexes, and 200μL of the complex solution was added and incubated at 37°C for 3 hours. After washing three times with phosphate-buffered saline (PBS), the cells were fixed by treatment with 150μL of 4% paraformaldehyde for 10 minutes. After washing three times with PBS, 200μL of Hoechst staining solution was added and incubated at 37°C for 30 minutes. After washing three times with PBS, fluorescence was observed with a confocal laser scanning microscope (Leica TCS SP5, Leica). Fluorescence intensity was calculated using BZ-II analysis application (KEYENCE).

[0043] FIG. 1 shows an example of an image captured by a confocal laser scanning microscope for each example.

[0044] Figure 2 shows the results of relative fluorescence intensity. The analysis results are shown as the mean ± standard error, and the standard error was calculated by repeating the experiment 4 to 5 times. The * in the figure indicates a significant difference (p<0.05) compared to the fluorescence intensity when β-CD was added.

[0045] As a result, as shown in Figures 1 and 2, the lactose-modified β-CD / TPPS complex showed a significantly higher uptake into human cerebrovascular endothelial cells (hCMEC / D3 cells) than the complex with unmodified β-CD. Also, the uptake into human cerebrovascular endothelial cells (hCMEC / D3 cells) was significantly higher than the complex with other sugar-modified β-CD.

[0046] [Test Example 2] Intracellular uptake of fluorescently labeled lactose-modified β-CD and the effect of the presence of sugar The uptake into hCMEC / D3 cells was confirmed by using lactose-modified β-CD fluorescently labeled with 5-(4,6-dichlorotriazinyl)aminofluorescein (5-DTAF).

[0047] The lactose-modified β-CD and 5-DTAF prepared in Preparation Example 1 were dissolved in sodium bicarbonate buffer (pH 9.0), incubated at room temperature for 24 hours, and then washed three times with acetone to prepare fluorescently labeled lactose-modified β-CD.

[0048] hCMEC / D3 cells were cultured at 1 × 10 per glass-bottom culture vessel. 4 The cells were seeded and incubated in EBM-2 medium at 37°C for 24 hours. After washing three times with PBS, 150μL of a mixture of fluorescently labeled lactose-modified β-CD (10μM) and lactose (0-40mM) or glucose (0-40mM) was added and incubated at 37°C for 3 hours. After washing three times with PBS, the cells were fixed by treatment with 150μL of 4% paraformaldehyde for 10 minutes. After washing three times with PBS, 200μL of Hoechst staining solution was added and incubated at 37°C for 30 minutes. After washing three times with PBS, the fluorescence was observed with a confocal laser scanning microscope. The fluorescence intensity was calculated using a BZ-II analysis application (KEYENCE).

[0049] FIG. 3 shows an example of an image captured by a confocal laser scanning microscope for each example.

[0050] Figure 4 shows the results of relative fluorescence intensity. The analysis results are shown as mean ± standard error, and the standard error was calculated by repeating the experiment three times. * in the figure indicates a significant difference (p<0.05) compared to the fluorescence intensity when lactose or glucose was not added.

[0051] As a result, as shown in Figures 3 and 4, it was confirmed that fluorescently labeled lactose-modified β-CD was taken up by human cerebrovascular endothelial cells (hCMEC / D3 cells). In addition, the amount of fluorescently labeled lactose-modified β-CD taken up decreased as the amount of competing lactose increased, but was not affected by the amount of glucose present. Therefore, it was revealed that the uptake into human cerebrovascular endothelial cells (hCMEC / D3 cells) occurs through a mechanism dependent on lactose residues.

[0052] [Test Example 3] Interaction between lactose-modified β-CD and drugs The interaction between lactose-modified β-CD and memantine, a drug used to treat Alzheimer's disease, was confirmed by isothermal titration calorimetry (ITC). Specifically, the cell side of the ITC was filled with β-CD dissolved in PBS or lactose-modified β-CD (14 mM) prepared in Preparation Example 1, and the syringe side was filled with memantine (70 mM) dissolved in PBS. The ITC was performed according to the usual method at a stirring speed of 300 rpm, a temperature of 25°C, and a drop rate of 2.5 μL / 5 min to measure the binding constant.

[0053] [Table 1]

[0054] As a result, as shown in Table 1, the binding constants of lactose-modified β-CD and memantine were similar to those of unmodified β-CD, indicating that the drug inclusion efficiency of lactose-modified β-CD was not reduced by its sugar modification.

[0055] [Test Example 4] Evaluation of the permeability of fluorescently labeled glycosylated β-CD in a human BBB model The permeability through the human BBB model was confirmed by using various sugar-modified β-CD labeled with fluorescent substances.

[0056] Fluorescently labeled sugar-modified β-CD was prepared as follows. 72 μmol of various sugar-modified β-CDs, 604 μmol of 1,1-carbonyldiimidazole, and 0.14 μmol of triethylamine were dissolved in 1.2 mL of dehydrated dimethyl sulfoxide (DMSO) and stirred overnight at room temperature. 1.0 μmol of ethylenediamine was added and stirred overnight at room temperature in nitrogen. The reaction solution was then added to acetone to produce a precipitate, which was then dried to obtain sugar-modified β-CD (NH 2 -glycosylated β-CD) was obtained. 2 -Sugar-modified β-CD was dissolved in 5 mL of dehydrated DMSO, 384 nmol of HiLyte™ Fluor 647 (HF647) acid was added, and the mixture was stirred for 24 hours at room temperature in the dark. The reaction solution was added to acetone to produce a precipitate, which was then dried to obtain fluorescently labeled sugar-modified β-CD.

[0057] 2×10 5 hCMEC / D3 cells were seeded on the upper side of a Transwell (Corning) and incubated at 37°C for 5 days. The electrical resistance was measured to be 125 Ω×cm 2 After confirming that the above is true, add 1×10 5 Human neuroblastoma cells (SH-SY5Y cells) were seeded on the lower side of the Transwell and incubated at 37°C for 1 day. Then, 1.5 mL of EBM-2 medium in which fluorescently labeled sugar-modified β-CD was dissolved to a final concentration of 100 μM was added to the upper side, and 2.6 mL of EBM-2 medium was added to the lower side. After incubation at 37°C, the medium in the lower side was sampled over time and the electrical resistance was measured. The fluorescence intensity of the sampled medium was measured using a microplate reader (Infinite M1000 PRO, TECAN) (excitation wavelength 649 nm, fluorescence wavelength 672 nm). After 60 minutes from the start of incubation, the lower side was washed three times with PBS and fixed by treating with 150 μL of 4% paraformaldehyde for 10 minutes. After washing three times with PBS, 200 μL of Hoechst staining solution was added and incubated at 37°C for 30 minutes. After washing three times with PBS, the fluorescence was observed using a confocal laser scanning microscope.

[0058] In addition, the permeability coefficient P app was calculated.

[0059] P app =(dQ / dt) V / (A C 0 )...Formula 1 P app (cm / min) is the apparent permeability coefficient, dQ / dt (mol / L / min) is the amount of fluorescently labeled glycosylated β-CD that migrates to the lower side per minute, V (mL) is the initial volume of the medium in the lower side of the Transwell, and A (cm 2 ) is the cell surface area on the human BBB model, C 0 (mol / L) indicates the initial concentration of fluorescently labeled sugar-modified β-CD in the medium contained in the upper side of the Transwell.

[0060] In addition, unmodified β-CD was also fluorescently labeled and tested in the same manner as sugar-modified β-CD.

[0061] The results of measuring the electrical resistance up to 60 minutes after incubation are shown in Figure 5. The analysis results are expressed as the mean value ± standard error, and the standard error was calculated by repeating the experiment three times.

[0062] Figure 6 shows the concentration of fluorescently labeled β-CD or various sugar-modified β-CDs on the lower side of the Transwell up to 60 minutes after incubation. The analytical results are shown as the mean ± standard error, and the standard error was calculated by repeating the experiment four times.

[0063] FIG. 7 shows an example of an image taken by a confocal laser scanning microscope of each of the cells seeded in the Transwell (upper side of the Transwell: hCMEC / D3 cells, and lower side of the Transwell: SH-SY5Y cells) after 60 minutes of incubation.

[0064] [Table 2]

[0065] As a result, as shown in Figure 5, fluorescently labeled β-CD and various sugar-modified β-CDs did not affect the electrical resistance value, confirming that they did not affect the barrier properties of the BBB.

[0066] Furthermore, as shown in FIG. 6 and Table 2, fluorescently labeled lactose-modified β-CD passed through the cell layer of human cerebrovascular endothelial cells (hCMEC / D3 cells) seeded on the upper side of the Transwell and migrated to the lower side, confirming that its permeability was higher than that of fluorescently labeled β-CD and fluorescently labeled mannose-modified β-CD.

[0067] Furthermore, as shown in Figure 7, it was confirmed that the fluorescently labeled lactose-modified β-CD that passed through the cell layer of human cerebrovascular endothelial cells (hCMEC / D3 cells) seeded on the upper side of the Transwell was taken up by human neuroblastoma cells (SH-SY5Y cells) seeded on the lower side.

[0068] [Test Example 5] Effects of various inhibitory conditions on the permeability of fluorescently labeled glycosylated β-CD in a human BBB model The effects of various inhibitory conditions on the permeability of the human BBB model were examined using lactose-modified β-CD labeled with a fluorescent substance.

[0069] Preparation of fluorescently labeled lactose-modified β-CD and evaluation of its permeability to the human BBB model were performed in the same manner as in Test Example 4. As inhibition conditions, the addition of glucose (molar ratio 1:40 relative to 1 molar amount of fluorescently labeled sugar-modified β-CD), lactose (same molar ratio 1:40), phloretin (glucose transporter inhibitor, same molar ratio 1:10), and incubation at low temperature (4°C) were examined.

[0070] Figure 8 shows the results of measuring the concentration of fluorescently labeled lactose-modified β-CD on the lower side of the Transwell up to 60 minutes after incubation under each condition. The analysis results are shown as the mean ± standard error, and the standard error was calculated by repeating the experiment three times.

[0071] [Table 3]

[0072] As a result, as shown in FIG. 8 and Table 3, it was confirmed that the permeability of the fluorescently labeled lactose-modified β-CD through the human BBB model was not affected by the addition of glucose or phloretin, but was decreased by the addition of lactose or at low temperatures.

[0073] [Test Example 6] Effect of galectin-1 on the permeability of fluorescently labeled glycosylated β-CD in a human BBB model The effect of galectin-1 on the permeability of the human BBB model was confirmed by using various sugar-modified β-CD labeled with fluorescent substances.

[0074] Preparation of fluorescently labeled glycosylated β-CD and evaluation of its permeability through the human BBB model were performed in the same manner as in Test Example 4, except that hCMEC / D3 cells lacking the galectin-1 gene (hCMEC / D3LGALS1(-) cells) were used instead of hCMEC / D3 cells. Unmodified β-CD was also fluorescently labeled and tested in the same manner as glycosylated β-CD.

[0075] Figure 9 shows the results of measuring the concentration of fluorescently labeled β-CD or various sugar-modified β-CDs on the lower side of the Transwell up to 60 minutes after incubation. The analytical results are shown as the mean ± standard error, and the standard error was calculated by repeating the experiment three times.

[0076] [Table 4]

[0077] As a result, as shown in Figure 9 and Table 4, it was confirmed that the deletion of the galectin-1 gene from human cerebrovascular endothelial cells (hCMEC / D3 cells) reduced the permeability of fluorescently labeled lactose-modified β-CD through the human BBB model to the same level as that of fluorescently labeled β-CD and fluorescently labeled mannose-modified β-CD. This suggests that galectin-1 contributes to the BBB permeation of lactose-modified β-CD.

[0078] [Test Example 7] Evaluation of brain penetration of fluorescently labeled glycosylated β-CD in mice The intravenous delivery of β-CD to the brain in mice was confirmed by using various glycosylated β-CD labeled with fluorescent substances.

[0079] Fluorescently labeled sugar-modified β-CD was prepared in the same manner as in Test Example 4.

[0080] 7.2 μmol / kg body weight (100 μL) of fluorescently labeled glycosylated β-CD was administered intravenously to BALB / c mice (8 weeks old, Japan LSC). After 60 minutes, the mice were perfused with PBS and each organ was collected. The collected organs were imaged using a luminescence and fluorescence imaging system (IVIS Lumina XRMS In Vivo Imaging System, Perkin Elmer), and the relative fluorescence intensity was measured from the images. The brain of each collected organ was homogenized in 1 mL of cell lysis buffer, centrifuged at 4°C and 14,000 rpm for 20 minutes, and the supernatant was collected. Next, after ultrafiltration at 14,000 rpm at 4°C for 20 minutes using Amicon Ultra-0.3 (MWCO: 30,000, Merck Millipore), the fluorescence intensity was measured using a microplate reader (excitation wavelength 649 nm, fluorescence wavelength 672 nm) to determine the amount of fluorescently labeled glycosylated β-CD transported into the brain, and the brain transport rate per dose was calculated. Unmodified β-CD was also fluorescently labeled in the same manner as glycosylated β-CD and tested.

[0081] Figure 10 shows an example of an image captured by a luminescence / fluorescence imaging system (IVIS Lumina XRMS In Vivo Imaging System).

[0082] Figure 11 shows the relative fluorescence intensity calculated from images captured by a luminescence and fluorescence imaging system (IVIS Lumina XRMS In Vivo Imaging System). The analysis results are shown as the mean ± standard error, and the standard error was calculated by repeating the experiment three times. * and † in the figure indicate a significant difference (p<0.05) compared to the fluorescence intensity when fluorescently labeled β-CD or fluorescently labeled mannose-modified β-CD was added, respectively.

[0083] Figure 12 shows the brain transfer rate per dose, which was determined by measuring the amount of brain transfer after homogenization and extraction from the brain following intravenous administration. The analysis results are shown as mean ± standard error, and the standard error was determined by repeating the experiment three times. * and † in the figure indicate a significant difference (p<0.05) compared to the brain transfer rate when fluorescent-labeled β-CD or fluorescent-labeled mannose-modified β-CD was added, respectively.

[0084] As a result, as shown in Figures 10 and 11, it was confirmed that the brain-transferability of fluorescently labeled lactose-modified β-CD was higher than that of fluorescently labeled β-CD and fluorescently labeled mannose-modified β-CD.

[0085] Furthermore, as shown in FIG. 12, the brain localization rate per dose of fluorescently labeled lactose-modified β-CD 60 minutes after administration was 4.6%, which was 18.0-fold and 6.9-fold higher than the brain localization rates per dose of fluorescently labeled β-CD and fluorescently labeled mannose-modified β-CD, respectively.

[0086] [Test Example 8] Confirmation of intracerebral localization of fluorescently labeled glycosylated β-CD using mice The localization in the mouse brain was confirmed by using various sugar-modified β-CD labeled with fluorescent substances.

[0087] Fluorescently labeled sugar-modified β-CD was prepared in the same manner as in Test Example 4.

[0088] 7.2 μmol / kg body weight (100 μL) of fluorescently labeled glycosylated β-CD was administered intravenously to BALB / c mice (8 weeks old). After 60 minutes, the mice were perfused with PBS and 4% paraformaldehyde, and the brains were collected. The collected brains were then immersed in 4% paraformaldehyde for 1 day at 4°C, and then immersed in 15% sucrose for 3 days at 4°C, and 20 μm-thick frozen sections were prepared using a cryostat (Leica CM3050 S, Leica). After labeling the frozen sections, the fluorescence was observed using a confocal laser microscope. For immunolabeling of neurons, Anti-NeuN Antibody, clone A60 was used as the primary antibody, and Donkey Anti-Mouse IgG H&L (Alexa Fluor 488) was used as the secondary antibody. In addition, DyLight 488 labeled Lycopersicon Esculentum (Tomato) Lectin (Vector Laboratories) and 4',6-Diamidino-2-phenylindole (DAPI) were used to label cerebrovascular endothelial cells and cell nuclei, respectively.

[0089] FIG. 13 shows an example of an image captured by a confocal laser scanning microscope for each example.

[0090] As a result, as shown in Figure 13, fluorescence was confirmed in the cerebral blood vessels, hippocampus, and cerebral cortex of mice administered fluorescently labeled lactose-modified β-CD. On the other hand, no fluorescence was confirmed in any part of the brain for fluorescently labeled β-CD and fluorescently labeled mannose-modified β-CD. From the above, it was confirmed that lactose-modified β-CD permeates cerebral blood vessel endothelial cells, migrates into the brain, and reaches the nerve cells of the hippocampus and cerebral cortex.

[0091] [Test Example 9] Evaluation of the permeability of the glycosylated β-CD / fluorescent substance complex in a human BBB model In order to confirm the usefulness of the conjugates of various sugar-modified β-CD and the fluorescent substance TPPS as drug carriers for the brain, the permeability through a human BBB model was evaluated.

[0092] The evaluation of the permeability to the human BBB model was performed in the same manner as in Test Example 4. Specifically, the sugar-modified β-CD / TPPS complex was prepared by dissolving sugar-modified β-CD (100 μM) and TPPS (8 μM) in 1.5 mL of EBM-2 medium, and the entire amount was added to the upper side of the Transwell. In addition, the fluorescence intensity was measured using a microplate reader at an excitation wavelength of 424 nm and a fluorescence wavelength of 656 nm. In addition, a complex with TPPS was prepared and tested for unmodified β-CD in the same manner as sugar-modified β-CD. In addition, the permeability to the human BBB model was evaluated using only TPPS that did not form a complex.

[0093] The measurement results of the TPPS concentration on the lower side of the Transwell up to 60 minutes after incubation are shown in Figure 14. The analysis results are shown as the mean value ± standard error, and the standard error was calculated by repeating the experiment three times.

[0094] [Table 5]

[0095] As a result, as shown in Figure 14, it was confirmed that the TPPS concentration on the lower side of the Transwell of the lactose-modified β-CD / TPPS complex was higher than that of the β-CD / TPPS complex and the mannose-modified β-CD / TPPS complex. In addition, as shown in Table 5, the permeability coefficient of the lactose-modified β-CD / TPPS complex was 8.9 times and 4.4 times that of the β-CD / TPPS complex and the mannose-modified β-CD / TPPS complex. From the above, it was confirmed that lactose-modified β-CD can permeate the human BBB model.

[0096] [Test Example 10] Evaluation of brain transport of glycosylated β-CD / fluorescent substance complex in mice To confirm the usefulness of the conjugates of various sugar-modified β-CD and the fluorescent substance TPPS as a drug carrier for the brain, we evaluated their intravenous delivery to the brain in mice.

[0097] The glycosylated β-CD / TPPS complex was prepared by dissolving the glycosylated β-CD in 100 μL of physiological saline so that the dosage of the glycosylated β-CD was 7.2 μmol / kg body weight and the dosage of the TPPS was 4.6 μmol / kg body weight, and the entire amount was administered intravenously to BALB / c mice (8 weeks old). After 60 minutes, the brain was perfused with PBS and collected. The collected brain was then homogenized in 1 mL of cell lysis buffer and centrifuged at 4 °C and 14,000 rpm for 20 minutes to collect the supernatant. After ultrafiltration at 4 °C and 14,000 rpm for 20 minutes using Amicon Ultra-0.3 (MWCO: 30,000), the fluorescence intensity was measured using a microplate reader (excitation wavelength 424 nm, fluorescence wavelength 656 nm) to determine the amount of TPPS transferred into the brain, and the brain transfer rate per dose was calculated. In addition, unmodified β-CD was also tested by preparing a complex with TPPS in the same manner as sugar-modified β-CD.

[0098] Figure 15 shows the brain localization rate per dose of TPPS. The analysis results are shown as mean ± standard error, and the standard error was calculated by repeating the experiment three times. *, †, and ‡ in the figure indicate a significant difference (p<0.05) compared to the brain localization rate when TPPS alone, β-CD / TPPS complex, or mannose-modified β-CD / TPPS complex were added, respectively.

[0099] As a result, as shown in Figure 15, the brain transfer rate after administration of the lactose-modified β-CD / TPPS complex was 3.5%, which was 11.3 times and 6.1 times that of the β-CD / TPPS complex and the mannose-modified β-CD / TPPS complex, respectively. From the above, it was confirmed that lactose-modified β-CD is useful as a drug carrier for the brain.

Claims

1. A drug carrier comprising a heat condensation product of lactose and cyclodextrin for delivering drugs into the brain.

2. A drug carrier agent comprising a heat condensation product of lactose and cyclodextrin, for enabling drugs to be absorbed into cerebrovascular endothelial cells.

3. 3. The drug carrier according to claim 1, wherein the heat condensation product is obtained by heating a mixture of the lactose and the cyclodextrin in the presence of a catalyst.

4. A pharmaceutical composition for delivering a drug to the brain, comprising the drug carrier agent according to claim 1 and a drug, either together or separately.

5. A pharmaceutical composition for absorbing a drug into cerebrovascular endothelial cells, comprising the drug carrier agent according to claim 1 and a drug, either together or separately.

6. A method for producing a drug carrier agent for delivering a drug into the brain, comprising a heat condensation product of lactose and cyclodextrin, the method comprising the steps of mixing lactose and cyclodextrin to obtain a mixture, and heating the mixture to obtain a heat condensation product of the lactose and cyclodextrin.

7. A method for producing a drug carrier agent, which contains a heat condensation product of lactose and cyclodextrin and is intended for absorption by cerebrovascular endothelial cells, comprising the steps of mixing lactose and cyclodextrin to obtain a mixture, and heating the mixture to obtain a heat condensation product of the lactose and cyclodextrin.

8. The method for producing a drug carrier agent according to claim 6 or 7, wherein the heating to obtain the thermal condensation product is carried out in the presence of a catalyst.

9. The method for producing a drug carrier agent according to any one of claims 6 to 8, further comprising a step of purifying the heat condensate after the step of obtaining the heat condensate.

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