Porous molded body for α-synuclein oligomer removal, method for α-synuclein oligomer removal, and α-synuclein oligomer removal system

Abstract

The present disclosure provides a porous molded body, method, and system enabling removal of α-synuclein oligomers from a biological fluid. The porous molded body for α-synuclein oligomer removal according to the present disclosure includes a porous molded body carrier having a positive charge due to having an amine structure. The α-synuclein oligomer removal method according to the present disclosure includes bringing the porous molded body according to the present disclosure into contact with a biological fluid containing α-synuclein oligomers. The α-synuclein oligomer removal system according to the present disclosure comprises: a column which has a container that has an inlet and outlet for biological fluid, and a porous molded body that is loaded into the container; a biological fluid introduction flow path into which the biological fluid is introduced through the inlet; and a biological fluid discharge flow path from which the biological fluid is discharged through the outlet. The porous molded body is a porous molded body carrier having a positive charge due to having an amine structure.

Description

[Technical Field]

[0001] This disclosure relates to a porous molded article for removing α-synuclein oligomers, a method for removing α-synuclein oligomers, and an α-synuclein oligomer removal system. [Background technology]

[0002] Parkinson's disease is a progressive disorder of the central nervous system characterized by decreased spontaneous movement, difficulty walking, postural instability, muscle rigidity, and tremors. Parkinson's disease is caused by the degeneration of dopamine-containing pigment neurons in the substantia nigra of the midbrain, and this degeneration is known to lead to a decrease in dopamine concentration in the striatum. Pathologically, Parkinson's disease is primarily characterized by the presence of Lewy bodies in the substantia nigra, the main component of which is known to be a filament composed of α-synuclein. Since patients with early-onset familial Parkinson's disease have major mutations in two amino acids of α-synuclein, it is believed that these Lewy bodies contribute to neurodegeneration in Parkinson's disease and related disorders.

[0003] Multiple system atrophy is a progressive neurodegenerative disease characterized by degeneration of nerve cells in the cerebellar cortex, pontine nuclei, olivary nucleus, striatum, substantia nigra, autonomic nuclei of the brainstem and spinal cord, as well as the motor cortex of the cerebral cortex, and the accumulation of insoluble α-synuclein inclusion bodies within the cytoplasm of oligodendroglia. Compared to Parkinson's disease, it exhibits less resting tremor, progresses more rapidly, and is less responsive to antiparkinsonian drugs.

[0004] Lewy body dementia is a disease characterized by the appearance of Lewy bodies in the brain, with cognitive impairment, hallucinations and delusions, and sleep disorders being the main symptoms. Although motor dysfunction, which is characteristic of Parkinson's disease, may also be seen, unlike Parkinson's disease, cognitive decline is often observed from the early stages. In Lewy body dementia, Lewy bodies are often distributed over a wide area of ​​the brain, including the cerebral cortex, thalamus, and brainstem.

[0005] Currently, the standard treatment for motor dysfunction in synucleinopathy, including Parkinson's disease, multiple system atrophy, and Lewy body dementia, is the administration of L-dopa, a precursor molecule of dopamine. L-dopa crosses the blood-brain barrier and is converted to dopamine in the brain. While this drug alleviates symptoms caused by dopamine deficiency, it cannot halt the progressive neurodegeneration that is characteristic of synucleinopathy. To prevent the progression of synucleinopathy, it is necessary to elucidate the mechanisms that cause nerve cell loss, and currently there is no fundamental cure or treatment. In Japan, synucleinopathy is designated as an intractable disease (specific disease) due to its small number of cases and the long-term impact it has on daily life.

[0006] While the physiological functions of α-synuclein, a component of Lewy bodies, remain largely unknown, it has been reported to play a role in membrane fusion, maintenance and release of synaptic vesicles, and protective functions against viral infections. When this α-synuclein monomer aggregates under some influence to form α-synuclein oligomers, it acquires pathogenicity, causing calcium regulation disorders, mitochondrial dysfunction, and intracellular transport disorders.

[0007] The process by which α-synuclein monomers aggregate to form α-synuclein oligomers in vivo is not clear at the time of filing this application. However, in recent years, the α-synuclein oligomer propagation hypothesis has been proposed, suggesting that α-synuclein oligomers propagate between cells through properties similar to prion proteins, thereby contributing to the pathological condition of synucleinopathy. In vitro, a self-template phenomenon has been observed in which the aggregation of α-synuclein monomers with a normal structure is promoted using α-synuclein oligomers with a β-sheet structure as a template. Furthermore, in animal experiments using mice, it has been reported that administering α-synuclein oligomers intracerebrally, orally, or intravenously induces neurological symptoms and that aggregates of α-synuclein are observed in the central nervous system (Non-Patent Literature 1).

[0008] In the α-synuclein oligomer propagation hypothesis, although the pathway through which α-synuclein oligomers propagate has also not been fully elucidated, it has been reported that large amounts of α-synuclein oligomers are present in the plasma of Parkinson's disease patients compared with healthy individuals (Non-Patent Document 2).

[0009] Patent Document 1 describes a means for removing α-synuclein monomers that exist free in body fluids before accumulating on nerve cells by adsorption removal. The means of Patent Document 1 aims to adsorb and remove only α-synuclein monomers using a porous carrier into which a ligand is introduced, and does not describe a means that may potentially delay the progression of Parkinson's disease, multiple system atrophy, and / or Lewy body dementia by removing α-synuclein oligomers from a biological-derived fluid. [Prior Art Literature] [Patent Literature]

[0010] [Patent Document 1] Japanese Patent Application Laid-Open No. 2012-193112 [Non-Patent Literature]

[0011] [Non-Patent Document 1] LOHMANN, Stephanie, et al., “Oral and intravenous transmission of α-synuclein fibrils to mice”, Acta Neuropathologica, (2019), 138: 515-533. [Non-Patent Document 2] EL‐AGNAF, Omar MA, et al., “Detection of oligomeric forms of α‐synuclein protein in human plasma as a potential biomarker for Parkinson's disease”, The FASEB journal, (2006), 20(3): 419-425. [Overview of the Initiative] [Problems that the invention aims to solve]

[0012] One of the objectives of this disclosure is to provide a porous molded article, a method, and a system that can remove α-synuclein oligomers from biological fluids. [Means for solving the problem]

[0013] Examples of embodiments of this disclosure are listed below. [1] A porous molded support having a positive charge due to an amine structure, wherein the porous molded support has the amine structure in a concentration of 30 μmol / ml-wet support or more and 10,000 μmol / ml-wet support or less, for the removal of α-synuclein oligomers. [2] The above amine structure is an aliphatic amine structure, as described in item 1, for the porous molded article. [3] The porous molded article according to item 1 or 2, wherein the amine structure is a secondary, tertiary, or quaternary amine, or a combination thereof, and the number of amine carbon atoms is 1 or more and 6 or less. [4] A porous molded article according to any one of items 1 to 3, wherein the above amine structure includes a quaternary amine. [5] The porous molded article according to any one of items 1 to 4, wherein the porous molded article carrier is a crosslinked agarose, a crosslinked cellulose, or a polystyrene-based polymer, or a combination thereof. [6] A porous molded body according to any one of items 1 to 5, wherein the harmonic mean particle size of the porous molded body carrier is 100 μm or more and 800 μm or less. [7] The specific surface area of ​​the above porous molded support is 1 m² 2 / g or more 100m 2 A porous molded article according to any one of items 1 to 6, which is less than or equal to / g. [8] An α-synuclein oligomer removal system, wherein the system is A column comprising a container having an inlet and outlet for a biologically derived liquid, and a porous molded body filled inside the container, A bio-derived fluid introduction channel for introducing the above-mentioned bio-derived fluid into the above-mentioned inlet, The system includes a bio-derived fluid discharge channel for discharging the bio-derived fluid that has passed through the column from the outlet, The above porous molded body is a porous molded body support having a positive charge due to an amine structure, and includes a porous molded body support having the above amine structure with a concentration of 30 μmol / ml-wet support or more and 10,000 μmol / ml-wet support or less. Alpha-synuclein oligomer removal system. [9] The system according to item 8, wherein the biological fluid is blood, and a plasma separation device is provided between the column and the biological fluid introduction channel, and the plasma separated from the blood is passed through the column.

[10] A method for removing α-synuclein oligomers from a biological fluid, wherein the above method is A method comprising contacting a porous molded body described in any one of items 1 to 7 with a biological fluid containing an α-synuclein oligomer.

[11] The above biological fluid is blood or plasma, as described in item 10.

[12] A method for reducing the concentration of α-synuclein oligomers in a patient's biological fluid, wherein the above method is Collecting biological fluids from the patient's body, The above-mentioned biological fluid, which has been collected, is brought into contact with a porous molded body to obtain the biological fluid after contact. This includes returning the biological fluid after the above contact back into the patient's body, The above-mentioned porous molded body is a porous molded body support having a positive charge due to an amine structure, and comprises a porous molded body support having the above-mentioned amine structure in a concentration of 10 μmol / ml-wetted support or more and 3000 μmol / ml-wetted support or less, and a method thereof.

[13] The above biological fluid is blood or plasma, as described in item 12. [Effects of the Invention]

[0014] According to this disclosure, a porous molded article, a method, and a system can be provided that can remove α-synuclein oligomers from biological fluids. [Modes for carrying out the invention]

[0015] 《Porous molded body》 The porous molded article of this disclosure is a porous molded article for removing α-synuclein oligomers. The porous molded article contains a porous molded article carrier having a positive charge due to its amine structure, thereby enabling the removal of α-synuclein oligomers from biological fluids. Based on the α-synuclein oligomer propagation hypothesis (Non-Patent Documents 1 and 2, etc.), the inventors of this application considered that removing α-synuclein oligomers from biological fluids and blocking the propagation pathway of α-synuclein oligomers would be effective in suppressing the progression of synucleinopathy, and diligently investigated means for removing α-synuclein oligomers. As a result, the inventors of this application discovered an unknown attribute: that a porous molded article carrier having a positive charge due to its amine structure can adsorb and remove α-synuclein oligomers, leading to the configuration of the present invention.

[0016] [Porous molded support] The porous shaped carrier has a positive charge derived from the amine structure bound to the porous shaped carrier. The amount of the amine structure contained in the porous shaped carrier is preferably not less than 10 µmol / ml-wet carrier and not more than 10000 µmol / ml-wet carrier, more preferably not less than 20 µmol / ml-wet carrier and not more than 10000 µmol / ml-wet carrier, still more preferably not less than 30 µmol / ml-wet carrier and not more than 10000 µmol / ml-wet carrier, even more preferably not less than 30 µmol / ml-wet carrier and not more than 5000 µmol / ml-wet carrier, particularly preferably not less than 40 µmol / ml-wet carrier and not more than 3000 µmol / ml-wet carrier, and most preferably not less than 50 µmol / ml-wet carrier and not more than 2000 µmol / ml-wet carrier. When the amount of the amine structure is 10 µmol / ml-wet carrier or more, the removal effect of α-synuclein oligomers is further improved. Although a larger introduction amount of the amine structure is desirable from the viewpoint of the adsorption amount of α-synuclein oligomers, from the viewpoints of facilitating the introduction of the amine structure, reducing the required amount of reagents for the introduction reaction, and improving economic efficiency, the amount of the amine structure is preferably 10000 µmol / ml-wet carrier or less, and more preferably 400 µmol / ml-wet carrier or less.

[0017] The porous shaped carrier is a carrier having a porous structure with a large number of pores of appropriate size. The pore size of the porous shaped carrier is not particularly limited, but the specific surface area is 0.0001 m 2 / g or more and 1000 m 2 / g or less, preferably 1 m 2 / g or more and 100 m 2 / g or less, more preferably 1 m 2 / g or more and 50 m 2 / g or less. When the specific surface area is 1000 m 2 / g or less, the mechanical strength of the porous shaped carrier is improved, and when the specific surface area is 0.0001 m 2When the concentration is greater than or equal to / g, the adsorption efficiency tends to improve, resulting in a better balance between adsorption efficiency and mechanical stability. The shape of the porous molded support is not particularly limited, but may be any form such as particulate (beads), thread-like, sheet-like, hollow fiber-like, cylindrical, or hollow cylindrical, or a monolithic support in which at least some of the pores in the support are through-holes. The porous molded support is preferably in the form of particulate (beads). In the case of a particulate (bead) porous molded support, the particle size is not particularly limited, but the harmonic mean particle size is preferably 20 μm to 1500 μm, more preferably 50 μm to 1000 μm, and even more preferably 100 μm to 800 μm. When used in the α-synuclein oligomer removal system described later, a harmonic mean particle size of 20 μm or more improves the fluidity of the biological solution, while a harmonic mean particle size of 1500 μm or less tends to increase the specific surface area per unit volume and improve adsorption efficiency, thus resulting in a better balance between fluidity and adsorption efficiency.

[0018] The material constituting the porous molded support is not particularly limited as long as it is a porous material into which an amine structure can be introduced, and may be an inorganic compound or an organic compound, for example. Organic polymers are preferred as the material for the porous molded support because they produce fewer elutes in hot water and allow for easier and more precise control of the pore size of the porous material. Examples of such organic polymers include synthetic polymer supports such as crosslinked or non-crosslinked polyvinyl alcohol, polyacrylate, polymethacrylate, polyacrylamide, polyacrylic acid, polyamide, polyester, polyethylene, and polystyrene; copolymer supports composed of two or more of these; polysaccharide supports such as crosslinked or non-crosslinked cellulose, agarose (Sepharose), and dextrin; and composite supports such as organic-organic and organic-inorganic obtained by combinations thereof. The cellulose may be crystalline cellulose. Among these materials, it is preferable to use a material that has good blood compatibility, relatively little nonspecific adsorption, and good adsorption selectivity for α-synuclein oligomers. Such preferred materials include, for example, cross-linked agarose, cross-linked cellulose, or polystyrene polymers, or combinations thereof.

[0019] [Amine structure] In this disclosure, "amine structure" means a structure having one or more amino groups on a hydrocarbon group. Preferably, the "amine structure" is an "aliphatic amine structure." An "aliphatic amine structure" is a structure having one or more amino groups on a hydrocarbon group that contains or consists of an aliphatic group. The hydrocarbon group may have an aromatic group in addition to the aliphatic group, and the amino group may be directly bonded to the aliphatic group or directly bonded to the aromatic group. On the other hand, in the case of an aliphatic amine structure, the amino group is directly bonded to the aliphatic group. The amino group may be an unsubstituted amino group (primary amine) or a substituted amino group (secondary, tertiary, or quaternary amine). At least some or all of the amino groups may be in the form of ammonium ions, i.e., primary, secondary, tertiary, or quaternary ammonium, thereby having a positive charge at the pH of the biological solution. The porous molded support may have the amine structure via covalent or non-covalent bonds.

[0020] When a porous molded support has an amine structure via covalent bonds, the amino group in the form of an ammonium ion is, for example, represented by the following general formula (1): [ka] It can be expressed as follows. In general formula (1), the bond "-" means a bond to a hydrocarbon group (also referred to as a "linker structure" in this disclosure). 1 , R 2 , and R 3 Each of these is preferably a hydrogen atom or a hydrocarbon group, independently of the others. 1 , R 2 , and R 3 It is preferable that one or more, two or more, three or more, or all four of these are hydrocarbon groups. 1 , R 2 , and R 3 The hydrocarbon group may be saturated or unsaturated, may have a linear, branched, or cyclic structure, and may be aliphatic, aromatic, or a combination thereof. 1 , R 2 , and R 3The number of carbon atoms in the hydrocarbon group is not limited, but is preferably 1 to 20, more preferably 1 to 15, even more preferably 1 to 10, even more preferably 1 to 8, and particularly preferably 1 to 6. 1 , R 2 , and R 3 The hydrocarbon group may have heteroatoms, and for example, it may have a hydroxyl group, halogen group, amino group, carbonyl group, sulfonyl group, and ether bond as part of its structure.

[0021] Examples of amine compounds that give an amine structure like the general formula (1) above include dimethylamine, trimethylamine, ethylamine, diethylamine, triethylamine, butylamine, N,N-dimethylbutylamine, N,N-dimethylhexylamine, N-benzyl-N-methylethanolamine, N,N-dimethyl-2-phenoxyethylamine, hexadecylamine (cetylamine), and polyethyleneimine.

[0022] When a porous molded support has an amine structure via covalent bonds, and the amine structure has multiple amino groups, the amine structure is, for example, represented by the following general formula (2): [ka] It can be represented as follows. At least one of the amino groups in general formula (2) can take the form of an ammonium ion at the pH of the biological solution and have a positive charge. The leftmost bond "-" signifies a bond to the hydrocarbon group of the linker structure. n is an integer between 1 and 100, preferably between 1 and 50, more preferably between 1 and 30, even more preferably between 1 and 10, and particularly preferably between 1 and 5. R 4 , R 6 , and R 7 Each of these is independently a hydrogen atom or a monovalent hydrocarbon group. 4 , R 6 , and R 7 The hydrocarbon group may be saturated or unsaturated, may have a linear, branched, or cyclic structure, and may be aliphatic, aromatic, or a combination thereof.4 , R 6 , and R 7 The number of carbon atoms in the hydrocarbon group is not limited, but is preferably 1 to 20, more preferably 1 to 15, even more preferably 1 to 10, even more preferably 1 to 8, and particularly preferably 1 to 6. 4 , R 6 , and R 7 The hydrocarbon group may have a heteroatom, and for example, part of its structure may have a hydroxyl group, halogen group, amino group, carbonyl group, sulfonyl group, and ether bond. 4 , R 6 , and R 7 Preferably, at least one of them is a hydrogen atom, and all of them are hydrogen atoms (-NH(R 5 NH) n H) is more preferable R 5 These are, independently, divalent hydrocarbon groups. 5 The hydrocarbon group may be saturated or unsaturated, may have a linear, branched, or cyclic structure, and may be aliphatic, aromatic, or a combination thereof. 5 The number of carbon atoms in the hydrocarbon group is not limited, but is preferably 1 to 20, more preferably 1 to 15, even more preferably 1 to 10, even more preferably 1 to 8, and particularly preferably 1 to 6. 5 The hydrocarbon group may have a heteroatom, and for example, part of its structure may have a hydroxyl group, halogen group, amino group, carbonyl group, sulfonyl group, and ether bond. 5 The group is preferably a divalent alkylene group, such as a substituted or unsubstituted methylene group, ethylene group, propylene group, butylene group, etc. An example of the structure of the above general formula (2) is -NH(CH2NH) n H, -NH(CH2CH2NH) n H, -NH(CH2CH2CH2NH) n H, etc., are preferred.

[0023] The amine structure consists of a hydrocarbon group bonded to an amino group (in general formula (1), R 1 , R 2 , or R 3The fewer carbon atoms in the amine group, the easier it is for electrostatic interactions to occur between the positive charge of the amine structure and the α-synuclein oligomer, and the more effectively the α-synuclein oligomer can be removed. Also, from an economic standpoint, the fewer carbon atoms in the hydrocarbon group bonded to the amino group, the lower the cost of reagents per amino group required for introducing the amine structure. Therefore, when the amine structure is a secondary, tertiary, or quaternary amine, or a combination thereof, the largest hydrocarbon group excluding the linker in the hydrocarbon chain bonded to the amino group (in general formula (1), R) 1 , R 2 , or R 3 If the number of carbon atoms in a given structure is defined as the "amine carbon number," then the amine carbon number is preferably between 1 and 6. Examples of amine compounds that give such an amine structure with this amine carbon number include dimethylamine, trimethylamine, ethylamine, diethylamine, triethylamine, butylamine, N,N-dimethylbutylamine, and N,N-dimethylhexylamine.

[0024] The amine structure preferably contains a quaternary amine, regardless of the number of carbon atoms in the amine. This increases the proportion of positively charged amine structures at the pH of the biological fluid, which facilitates electrostatic interactions between the positive charge of the amine structure and the α-synuclein oligomer, and tends to allow for more effective removal of the α-synuclein oligomer. Examples of amine compounds that give such a quaternary amine structure include trimethylamine, triethylamine, N,N-dimethylbutylamine, N,N-dimethylhexylamine, N-benzyl-N-methylethanolamine, and N,N-dimethyl-2-phenoxyethylamine.

[0025] The linker structure may be a divalent hydrocarbon group having a saturated or unsaturated, linear, branched, or cyclic structure. The number of carbon atoms in the hydrocarbon group as the linker structure is not limited, but is preferably 1 to 20, more preferably 1 to 15, even more preferably 1 to 10, even more preferably 1 to 8, and particularly preferably 1 to 6. The hydrocarbon group as the linker structure may have heteroatoms, for example, a hydroxyl group, halogen group, amino group, carbonyl group, sulfonyl group, and ether bond in part of its structure. More specifically, examples of linker structures include substituted or unsubstituted alkylene groups such as methylene groups, ethylene groups, propylene groups, and butylene groups.

[0026] Examples of compounds that provide a linker structure (linkers) include epichlorohydrin, epibromohydrin, allyl glycidyl ether, bis-epoxide (such as butanediol diglycidyl ether), halogen-substituted aliphatic substances (such as dichloropropanol and allyl halides), and divinyl sulfone. For example, if the porous molded support has hydroxyl groups, allyl halides such as allyl bromide can be used as linkers.

[0027] As an embodiment in which the porous molded support has an amine structure via non-covalent bonds, for example, the porous molded support may have a polymer having an amine structure on its surface. It is preferable that the polymer having an amine structure has the above-mentioned amine structure in the main chain and / or as a side chain. More specifically, examples of polymers having an amine structure include polyalkyleneimines, such as polyethyleneimine.

[0028] [α-Synuclein oligomer] α-synuclein oligomers include oligomers and aggregates formed from two or more α-synuclein monomer molecules, as well as phosphorylated forms thereof. The porous molded body of this disclosure is a porous molded body for removing α-synuclein oligomers from biological fluids. The porous molded body of this disclosure is suitable for contacting biological fluids and removing α-synuclein oligomers from them. Generally, biological fluids may contain a sufficient amount of low molecular weight substances that can be adsorbed onto porous molded bodies. Since low molecular weight substances quickly occupy accessible adsorption sites on the porous molded body, removal of adsorbed substances with large molecular weights tends to be difficult in porous molded bodies. Furthermore, in the case of adsorbed substances with extremely large molecular weights, the adsorbed substances do not penetrate into the interior of the pores of the porous molded body, and the adsorption sites are limited to the surface of the porous molded body, making adsorption removal even more difficult. However, the porous molded articles of this disclosure are designed to overcome this problem and have the ability to effectively remove even large molecular weight α-synuclein oligomers. Specifically, the molecular weight of the α-synuclein oligomers removed by the porous molded articles of this disclosure is not particularly limited, but for example, α-synuclein oligomers with a molecular weight of 500,000 kDa or less can be removed, and in particular, α-synuclein oligomers with a molecular weight of 100,000 kDa or less can be efficiently removed. By efficiently removing α-synuclein oligomers, the progression of synucleinopathy such as Parkinson's disease, multiple system atrophy, and / or Lewy body dementia can be slowed. Therefore, the porous molded articles of this disclosure can preferably be used to slow the progression of synucleinopathy.

[0029] Furthermore, the porous molded body of this disclosure may be used for pharmaceutical applications, for example, to manufacture blood products using blood from which α-synuclein oligomers have been removed. Alternatively, the porous molded body of this disclosure may be used for diagnostic applications, for example, to concentrate and recover α-synuclein oligomers in biological fluids for detection and to diagnose synucleinopathy.

[0030] [Bio-derived fluid] The biological fluids are preferably blood, plasma, serum, cerebrospinal fluid, ascites, urine, saliva, tears, nasal secretions, sweat, digestive fluids, and breast milk, with blood or plasma being particularly preferred.

[0031] Method for manufacturing porous molded articles The method for producing the porous molded article of this disclosure is not limited. Examples of methods for producing the porous molded article of this disclosure include, for example, a method involving introducing an amine structure onto the surface of a porous molded article support that does not have an amine structure. Alternatively, a method involving synthesizing a porous polymer support from monomers having an amine structure or a precursor structure thereof by polymerization reaction is also included. Details regarding the porous molded article support and the amine structure have been described above and are therefore omitted here.

[0032] Methods for introducing an amine structure onto the surface of a porous molded support that does not have an amine structure include a fixed introduction method via covalent bonds and a non-fixed introduction method via non-covalent bonds. An example of a fixed introduction method via covalent bonds is a method in which a linker compound that gives a linker structure is reacted with a porous molded support, and then an amine compound (ligand) that gives an amine structure is reacted with it, thereby introducing a linker structure between the porous molded support and the amine compound. Details about amine compounds and linkers have been described above, so they will not be described here.

[0033] As an example of a non-immobile introduction method via non-covalent bonding, a polymer having an amine structure (such as polyethyleneimine) is prepared by coating the surface of a porous molded support. Any coating method can be used to coat the surface of the porous molded support with the polymer having an amine structure, such as coating, spraying, and dipping. Details of the polymer having an amine structure have been described above, so they are omitted here.

[0034] Method for removing α-synuclein oligomers A method for removing α-synuclein oligomers according to the present disclosure includes contacting a porous molded body according to the present disclosure with a biological fluid containing α-synuclein oligomers. The manner of contact is not particularly limited as long as it can reduce the concentration of α-synuclein oligomers in the biological fluid. For example, the removal method preferably includes introducing a biological fluid containing α-synuclein oligomers into a container having an inlet and an outlet for the biological fluid and filled with the porous molded body according to the present disclosure through the inlet, passing the biological fluid through the container in contact with the porous molded body according to the present disclosure, and discharging the biological fluid from the outlet to the porous molded body according to the present disclosure. The removal method may further include collecting the biological fluid from the patient's body and returning the biological fluid that has been in contact with the porous molded body according to the present disclosure back into the patient's body.

[0035] In this disclosure, "removal" does not mean the complete removal of α-synuclein oligomers from the biological fluid, but rather it is sufficient to reduce the concentration of α-synuclein oligomers contained in the biological fluid. The removal method can preferably remove 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, or 30% or more of the α-synuclein oligomers contained in the biological fluid.

[0036] As described above, the biological fluid is particularly preferably blood or plasma. In this case, the removal method may include contacting blood containing α-synuclein oligomers with the porous molded body of this disclosure to obtain blood from which the α-synuclein oligomers have been removed. Alternatively, the removal method may further include separating plasma containing α-synuclein oligomers from blood beforehand, contacting the separated plasma with the porous molded body of this disclosure to remove the α-synuclein oligomers from the plasma, and combining the plasma from which the α-synuclein oligomers have been removed with the separated hemocytose concentrate to obtain blood from which the α-synuclein oligomers have been removed. The removal method may further include withdrawing blood from the patient's body and / or returning the blood that has been in contact with the porous molded body of this disclosure back into the patient's body.

[0037] 《α-Synuclein Oligomer Removal System》 The porous molded body of this disclosure can be incorporated into an α-synuclein oligomer removal system. The α-synuclein oligomer removal system of this disclosure comprises a column having a container with an inlet and outlet for a biological fluid, and a porous molded body filled in the container; a biological fluid introduction channel (circuit) for introducing the biological fluid into the inlet; and a biological fluid discharge channel for discharging the biological fluid that has passed through the column from the outlet. The biological fluid introduction channel and the biological fluid discharge channel are preferably made of flexible tubing. When the system is in use, the biological fluid introduction channel may be fluidly connected to the patient's body in order to collect the biological fluid from the patient's body. The biological fluid discharge channel may also be fluidly connected to the patient's body in order to return the biological fluid, after it has been in contact with the porous molded body, to the patient's body.

[0038] The α-synuclein oligomer removal system can preferably remove 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, or 30% or more of α-synuclein oligomers contained in biological fluids.

[0039] As described above, the biological fluid is particularly preferably blood or plasma, in which case the system is used as a blood purification system. The blood purification system includes, as an example, a blood introduction channel consisting of a flexible tube for drawing blood from the patient's body and introducing it into a column, a column filled with the porous molded body of this disclosure, and a blood discharge channel consisting of a flexible tube for returning the blood that has exited the column back into the body. When the blood purification system is in use, the blood introduction channel and the blood discharge channel are connected to the patient's blood vessels.

[0040] The blood purification system described above is just one example and is not limited to the configuration described above. Various modifications are possible by appropriately applying conventional blood purification technologies. For example, the blood purification system may include two or more α-synuclein oligomer adsorption removal columns, which may be connected in series or in parallel. Another example is that the blood purification system may be configured to include a plasma separation device between the α-synuclein oligomer adsorption removal column and the bio-derived fluid introduction channel, and to pass the plasma separated from the blood through the column. More specifically, examples of plasma separation devices include membrane-type plasma separators using plasma separation membranes and centrifugal plasma separators. In this case, the blood purification system may be further configured to remove α-synuclein oligomers from the plasma, then combine the plasma from which the α-synuclein oligomers have been removed with a hemocytose concentrate separated by the plasma separator, and return the blood from which the α-synuclein oligomers have been removed to the patient. Furthermore, other elements that can be included in a blood purification system include those used in conventional blood purification systems, specifically, anticoagulant dispensers, pressure gauges, flow rate detectors, anomaly detection devices, particulate filters, air chambers, and hemolysis sensors. [Examples]

[0041] The following describes specific examples and comparative examples of this disclosure, but this disclosure is not limited to these examples and comparative examples.

[0042] Evaluation and Measurement Methods [Method for measuring the removal rate of oligomers consisting of two or more α-synuclein monomer molecules] 0.18 mL of the porous molded body was measured into a 3.0 mL Libra tube (manufactured by Hypep Laboratories) and washed with phosphate-buffered saline (PBS-, manufactured by TAKARA Bio Co., Ltd.) and physiological saline (manufactured by Otsuka Pharmaceutical Co., Ltd.). Then, 0.9 mL of commercially available heparinized bovine plasma or heparinized bovine blood, adjusted to have an α-synuclein oligomer (Recombinant Human Alpha-synuclein protein aggregate (Active), manufactured by Abcam) concentration of approximately 32 ng / mL, was added. The plasma was stirred at 37°C at 5 rpm for 1 hour, and the α-synuclein oligomer concentration in the supernatant was measured by sandwich ELISA using two antibodies (Anti-Alpha-synuclein filament MJFR14-6-4-2 Comformation specific, Abcam, and Purified Mouse Anti-α-Synuclein Clone 42 / α-Synuclein (RUO), BD Transduction Laboratories). The α-synuclein oligomer measurement value of the sample without the porous molded body was set as 100%, and the α-synuclein oligomer removal rate (%) of the porous molded body was calculated by subtracting the remaining α-synuclein oligomer percentage (%) from 100%. The removal rate may take a negative value due to the increased sensitivity of ELISA measurement caused by the adsorption and removal of plasma proteins other than α-synuclein oligomers by the porous molded body when the porous molded body adsorbs little to no α-synuclein oligomers, and / or due to the variability of ELISA measurement.

[0043] The molecular weight of the α-synuclein oligomer used in the test was measured using a size exclusion chromatography-multi-angle light scattering detection system (SEC-MALS) with an Optilab rEX differential refractive index detector (Wyatt Technology), a DAWN HELEOS multi-angle light scattering detector (Wyatt Technology), a Shodex Protein KW-804 separation column (Resonac), and a 0.05M phosphate buffer (pH 7) with 0.3M sodium chloride as the eluent. The measurement results showed that the weight-average molecular weight of the α-synuclein oligomer was 82,400 kDa.

[0044] [Method for measuring the removal rate of α-synuclein monomer] 0.18 mL of the porous molded body was measured into a 3.0 mL Libra tube (Hypep Laboratories) and washed with phosphate-buffered saline (PBS-, TAKARA Bio Co., Ltd.) and physiological saline (Otsuka Pharmaceutical Co., Ltd.). Then, 0.9 mL of commercially available heparinized bovine plasma or heparinized bovine blood, adjusted to have an α-synuclein monomer (α-Synuclein, recombinant, Human, Cosmo Bio Co., Ltd.) concentration of approximately 32 ng / mL, was added. The mixture was stirred at 37°C and 5 rpm for 1 hour, and the α-synuclein concentration in the supernatant plasma was measured by sandwich ELISA using a measurement kit (Human alpha-Synuclein DuoSet ELISA, R&D Systems Co., Ltd.). The α-synuclein monomer measurement value for the sample without the porous molded body was set to 100%, and the α-synuclein monomer removal rate (%) of the porous molded body was calculated by subtracting the remaining percentage of α-synuclein monomer from 100%. The removal rate may take a negative value if the porous molded body adsorbs little to no α-synuclein monomer, due to increased sensitivity of the ELISA measurement caused by the adsorption and removal of plasma proteins other than α-synuclein monomer by the porous molded body, and / or due to variability in the ELISA measurement.

[0045] [Method for measuring the amount of allyl groups introduced into porous molded bodies] The amount of allyl groups introduced (μmol / ml - wet support) was obtained by substituting the allyl groups introduced into the porous molded support with carboxyl groups and then titrating the carboxyl groups to neutralize them. Specifically, 1 mL of the porous molded material was measured into a 5.0 mL Libra tube (manufactured by Hypep Laboratories) and mixed with 1 mL of 25 mg / mL aqueous solution of ammonium persulfate and 120 μL of thioacetic acid. The reaction mixture was kept at 50°C for 16 hours under stirring. After filtering the reaction mixture, the beads were washed five times with 10 mL of distilled water and four times with 10 mL of 1 M HCl water. The porous molded material was transferred to a 50 mL centrifuge tube, 30 mL of 1 M HCl water was added, and the mixture was shaken for 30 minutes. The porous molded body was recovered using a Libra tube, washed with 60 mL of ultrapure water (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), then transferred to a 100 mL centrifuge tube, and 100 mL of ultrapure water was added and the mixture was shaken for 30 minutes. The porous molded body was again recovered using a Libra tube, washed with 60 mL of ultrapure water, transferred to a centrifuge tube, and 2 mL to 500 mL of 0.01 M NaOH water was added and the mixture was shaken for 30 minutes. After the reaction, 5 mL of the supernatant was collected, and 0.1 M HCl water was added dropwise while stirring. The amount of allyl groups introduced into the porous molded body (μmol / ml - wet support) was calculated from the amount of addition at the pH inflection point. In this process, the proportion of NaOH that was ion-exchanged with the porous molded body out of the NaOH added to the porous molded body was calculated as the NaOH consumption rate. If the NaOH consumption rate was between 20% and 40%, it was determined that the measurement of the amount of allyl group introduced was appropriate.

[0046] [Method for measuring the amount of amine structure introduced into porous molded materials] The amount of amine structure introduced (μmol / ml - wet support) was obtained by neutralization titration of the amino groups introduced into the porous molded support. Specifically, 0.2 mL of the porous molded material was measured into a 3.0 mL Libra tube (manufactured by Hypep Laboratories), washed with 10 mL of physiological saline, and then transferred to a 25 mL centrifuge tube. 6 mL of 1 M NaOH water was added, and the mixture was shaken for 30 minutes. The porous molded material was recovered using a Libra tube, washed with 12 mL of ultrapure water (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), and then transferred to a 100 mL centrifuge tube. 50 mL of ultrapure water was added, and the mixture was shaken for 30 minutes. The porous molded body was again recovered using a Libra tube, washed with 12 mL of ultrapure water, and then transferred to a centrifuge tube. 0.4 mL to 100 mL of 0.01 M HCl solution was added, and the mixture was shaken for 30 minutes. After the reaction, 0.1 mL to 5 mL of the supernatant was collected, and 0.1 M NaOH solution was added dropwise while stirring. The amount of amine structure introduced into the porous molded body (μmol / ml - wet support) was calculated from the amount of NaOH added to the porous molded body at the pH inflection point. At this time, the HCl consumption rate was calculated as the percentage of HCl that was ion-exchanged with the porous molded body out of the HCl added to the porous molded body. If the HCl consumption rate was between 20% and 40%, the measurement of the amount of amine structure introduced was considered appropriate.

[0047] [Harmonic mean particle size of porous molded material] The harmonic mean particle size of porous molded bodies was measured using a particle size distribution analyzer (MT3300II, Microtrac-Bel) on porous molded bodies swollen with ultrapure water, and the harmonic mean of these measurements was calculated as the harmonic mean particle size (μm).

[0048] [Specific surface area of ​​porous molded material] A porous molded body swollen with ultrapure water was freeze-dried for 24 hours, and then dried. After drying, the porous molded body was degassed (reduced pressure drying) at 60°C for 15 hours using VacPrep061 (Shimadzu Corporation - Micromerities). Subsequently, the specific surface area (m²) was determined by N2 gas adsorption using TriStarII 3020 (Shimadzu Corporation - Micromerities). 2Measurements were taken for the specific surface area (per g). The specific surface area was determined using the value obtained from a BET plot. The above measurement was performed 10 times, and the average of the 8 values ​​(excluding the maximum and minimum values) was used as the specific surface area.

[0049] Comparative Example 1 [A. Introduction of allyl groups into porous molded bodies] The hydroxyl groups of Sepharose 4 Fast Flow (Cytiva, cross-linked agarose beads) were activated with allyl bromide as a linker as follows: 15 ml of Sepharose 4 Fast Flow was dried by suction and mixed with 4.2 ml of allyl bromide, 18 ml of 6 M NaOH water, and 9 ml of dimethyl sulfoxide. The mixture was stirred at 30°C for 18 hours. After filtering the mixture, the beads were sequentially washed with 750 ml of distilled water. Titration revealed that the amount of allyl groups introduced was 108.9 (micromoles) / bead (mL)-wet support.

[0050] [B. Activation of Sepharose 4 Fast Flow by bromination] 14 ml of allyl-activated Sepharose 4 Fast Flow (108.9 micromoles of allyl groups / ml - wet support) was mixed with 14 ml of 0.9 M N-bromosuccinimide (50% aqueous acetone solution). The mixture was stirred at 30°C for 3 hours. The reaction mixture was filtered, and the beads were washed with 900 ml of distilled water. The activated beads were then immediately transferred to a reaction vessel and further reacted with hexadecylamine (cetylamine) as a ligand by the following method.

[0051] [C. Introduction of hexadecylamine into activating beads] Aliphatic amine structures were directly introduced into the beads via a linker structure. In a typical procedure, coupling to the beads was performed by bromination of the allyl group and nucleophilic substitution under basic conditions. 1 ml of bromo-activated gel (108.9 micromoles / ml of allyl group introduced - wet support) was transferred to a reaction vessel containing 1 ml of 50% DMSO aqueous solution in which 100 mg of hexadecylamine as the ligand was dissolved. The reaction mixture was kept at 30°C for 16 hours with stirring. After filtering the reaction mixture, the beads were sequentially washed with 3 ml of isopropanol, 3 ml of distilled water, 3 ml of 1 M HCl solution, 3 ml of distilled water, 3 ml of 1 M NaOH solution, and finally 30 ml of physiological saline. Hexadecylamine-introduced Sepharose 4 Fast Flow was obtained with an amine structure introduced at 6.2 micromoles / ml - wet support.

[0052] The adsorption performance of the obtained porous molded material for α-synuclein oligomers from plasma is shown in Table 1 below. The removal rate of α-synuclein oligomers from the porous molded material was less than 30%.

[0053] Example 1 Sepharose 4 Fast Flow with hexadecylamine was obtained using the same procedure as in Comparative Example 1, except that 13.5 mL of 80% isopropanol aqueous solution containing 3260 mg of hexadecylamine was used as the amine solution reacted with the bromo-activated gel. The amount of amine structure introduced was 39.4 micromoles / ml-wet support. The adsorption performance of α-synuclein oligomers from plasma of the obtained porous molded body is shown in Table 1 below. The removal rate of α-synuclein oligomers from the porous molded body was 30% or more. It was revealed that the obtained porous molded body efficiently removes α-synuclein oligomers from plasma.

[0054] Comparative Example 2 [A. Introduction of allyl groups into porous molded bodies] The hydroxyl groups of Sepharose 4 Fast Flow (Cytiva, cross-linked agarose beads) were activated with allyl bromide as a linker as follows: 15 ml of Sepharose 4 Fast Flow was dried by suction and mixed with 2.1 ml of allyl bromide, 9 ml of 3 M NaOH water, and 2.25 ml of dimethyl sulfoxide. The mixture was stirred at 30°C for 18 hours. After filtering the mixture, the beads were sequentially washed with 750 ml of distilled water. Titration revealed that the amount of allyl groups introduced was 73.9 (micromoles) / - wetted support (mL).

[0055] [B. Activation of Sepharose 4 Fast Flow by bromination] 14 ml of allyl-activated Sepharose 4 Fast Flow (allyl group introduction amount 73.9 micromoles / ml - wet support) was mixed with 14 ml of 0.9 M N-bromosuccinimide (50% aqueous acetone solution). The mixture was stirred at 30°C for 3 hours. The reaction mixture was filtered, and the beads were washed with 900 ml of distilled water. The activated beads were then immediately transferred to a reaction vessel and further reacted with diethylamine as a ligand by the following method.

[0056] [C. Introduction of diethylamine to activating beads] Amine groups were directly introduced into the beads via a linker structure. In a typical procedure, coupling to the beads was performed by bromination of the allyl group and nucleophilic substitution under basic conditions. 4 ml of bromo-activated gel (allyl group introduction amount 73.9 micromoles / ml - wet support) was transferred to a reaction vessel containing 4 ml of 50% isopropanol aqueous solution in which 20 mg of diethylamine as the ligand was dissolved. The reaction mixture was kept at 30°C for 16 hours with stirring. After filtering the reaction mixture, the beads were sequentially washed once with 8 ml of isopropanol, once with 4 ml of distilled water, once with 4 ml of 1 M HCl water, once with 4 ml of distilled water, once with 4 ml of 1 M NaOH water, and finally once with 120 ml of physiological saline. Diethylamine-introduced Sepharose 4 Fast Flow was obtained with an amine structure introduction amount of 5.5 micromoles / ml - wet support.

[0057] The adsorption performance of the obtained porous molded material for α-synuclein oligomers from plasma is shown in Table 1 below. The removal rate of α-synuclein oligomers from the porous molded material was less than 30%.

[0058] Comparative Example 3 Diethylamine-introduced Sepharose 4 Fast Flow was obtained using the same procedure as in Comparative Example 2, except that 40 mg of diethylamine was used as the amine reacted with the bromo-activated gel. The amount of amine structure introduced was 16.7 micromoles / ml-wet carrier. The adsorption performance of α-synuclein oligomers from plasma of the obtained porous molded body is shown in Table 1 below. The removal rate of α-synuclein oligomers from the porous molded body was less than 30%.

[0059] Comparative Example 4 Diethylamine-introduced Sepharose 4 Fast Flow was obtained using the same procedure as in Comparative Example 2, except that 80 mg of diethylamine was used as the amine reacted with the bromo-activated gel. The amount of amine structure introduced was 28.9 micromoles / ml-wet carrier. The adsorption performance of α-synuclein oligomers from plasma of the obtained porous molded body is shown in Table 1 below. The removal rate of α-synuclein oligomers from the porous molded body was less than 30%.

[0060] Example 2 Diethylamine-introduced Sepharose 4 Fast Flow was obtained using the same procedure as in Comparative Example 2, except that 100 mg of diethylamine was used as the amine reacted with the bromo-activated gel. The amount of amine structure introduced was 51.0 micromoles / ml-wet support. The adsorption performance of α-synuclein oligomers from plasma of the obtained porous molded body is shown in Table 1 below. The removal rate of α-synuclein oligomers from the porous molded body was 30% or more. It was revealed that the obtained porous molded body efficiently removes α-synuclein oligomers from plasma.

[0061] Example 3 Diethylamine-introduced Sepharose 4 Fast Flow was obtained using the same procedure as in Comparative Example 1, except that 1 mL of a 50% isopropanol aqueous solution containing 500 mg of diethylamine was used as the amine solution reacted with the bromo-activated gel. The amount of amine structure introduced was 95.3 micromoles / ml-wet support. The adsorption performance of α-synuclein oligomers from plasma of the obtained porous molded body is shown in Table 1 below. The removal rate of α-synuclein oligomers from the porous molded body was 30% or more. It was revealed that the obtained porous molded body efficiently removes α-synuclein oligomers from plasma.

[0062] Example 4 An ethylamine-introduced Sepharose 4 Fast Flow was obtained using the same procedure as in Comparative Example 1, except that 1 mL of a 50% isopropanol aqueous solution containing 77.0 mg of ethylamine was used as the amine solution reacted with the bromo-activated gel. The amount of amine structure introduced was 53.2 micromoles / ml-wet support. The adsorption performance of the α-synuclein oligomer from plasma of the obtained porous molded body is shown in Table 1 below. The removal rate of α-synuclein oligomers from the porous molded body was 30% or more. It was revealed that the obtained porous molded body efficiently removes α-synuclein oligomers from plasma.

[0063] Example 5 A triethylamine-introduced Sepharose 4 Fast Flow was obtained using the same procedure as in Comparative Example 1, except that 1 mL of a 50% isopropanol aqueous solution containing 691.4 mg of triethylamine was used as the amine solution reacted with the bromo-activated gel. The amount of amine structure introduced was 53.7 micromoles / ml-wet support. The adsorption performance of the obtained porous molded body from plasma for α-synuclein oligomers is shown in Table 1 below. The removal rate of α-synuclein oligomers from the porous molded body was 30% or more. It was revealed that the obtained porous molded body efficiently removes α-synuclein oligomers from plasma.

[0064] Example 6 Sepharose 4 Fast Flow with N-benzyl-N-methylethanolamine was obtained using the same procedure as in Comparative Example 1, except that 1 mL of a 50% isopropanol aqueous solution containing 2259.5 mg of N-benzyl-N-methylethanolamine was used as the amine solution reacted with the bromo-activated gel. The amount of amine structure introduced was 49.2 micromoles / ml on the wetted support. The adsorption performance of the α-synuclein oligomer from plasma of the obtained porous molded body is shown in Table 1 below. The removal rate of α-synuclein oligomers from the porous molded body was 30% or more. It was revealed that the obtained porous molded body efficiently removes α-synuclein oligomers from plasma.

[0065] Example 7 Sepharose 4 Fast Flow with N,N-dimethyl-2-phenoxyethylamine was obtained using the same procedure as in Example 1, except that 1 mL of a 50% isopropanol aqueous solution containing 141.2 mg of N,N-dimethyl-2-phenoxyethylamine was used as the amine solution reacted with the bromo-activated gel. The amount of amine structure introduced was 59.4 micromoles / ml - wetted support. The adsorption performance of α-synuclein oligomers from plasma of the obtained porous molded body is shown in Table 2 below. The removal rate of α-synuclein oligomers from the porous molded body was 30% or more. It was revealed that the obtained porous molded body efficiently removes α-synuclein oligomers from plasma.

[0066] Example 8 Butylamine-introduced Sepharose 4 Fast Flow was obtained using the same procedure as in Comparative Example 1, except that 1 mL of a 50% isopropanol aqueous solution containing 987.4 mg of butylamine was used as the amine solution reacted with the bromo-activated gel. The amount of amine structure introduced was 68.3 micromoles / ml-wet support. The adsorption performance of the obtained porous molded body from plasma for α-synuclein oligomers is shown in Table 2 below. The removal rate of α-synuclein oligomers from the porous molded body was 30% or more. It was revealed that the obtained porous molded body efficiently removes α-synuclein oligomers from plasma.

[0067] Example 9 Sepharose 4 Fast Flow with N,N-dimethylbutylamine was obtained using the same procedure as in Example 1, except that 1 mL of an 80% isopropanol aqueous solution containing 379.4 mg of N,N-dimethylbutylamine was used as the amine solution reacted with the bromo-activated gel. The amount of amine structure introduced was 51.0 micromoles / ml - wetted support. The adsorption performance of the obtained porous molded body from plasma for α-synuclein oligomers is shown in Table 2 below. The removal rate of α-synuclein oligomers from the porous molded body was 30% or more. It was revealed that the obtained porous molded body efficiently removes α-synuclein oligomers from plasma.

[0068] Example 10 Sepharose 4 Fast Flow with N,N-dimethylhexylamine was obtained using the same procedure as in Example 1, except that 1 mL of 80% isopropanol aqueous solution containing 161.6 mg of N,N-dimethylhexylamine was used as the amine solution reacted with the bromo-activated gel. The amount of amine structure introduced was 40.4 micromoles / ml - wetted support. The adsorption performance of α-synuclein oligomers from plasma of the obtained porous molded body is shown in Table 2 below. The removal rate of α-synuclein oligomers from the porous molded body was 30% or more. It was revealed that the obtained porous molded body efficiently removes α-synuclein oligomers from plasma.

[0069] Example 11 [A. Introduction of allyl groups into porous molded bodies] The hydroxyl groups of Cellfine GH-25 (JNC Corporation, cross-linked cellulose beads) were activated with allyl bromide as a linker as follows: 18 ml of Sepharose 4 Fast Flow was dried by suction and mixed with 5.0 ml of allyl bromide, 21.6 ml of 6 M NaOH water, and 10.8 ml of dimethyl sulfoxide. The mixture was stirred at 30°C for 18 hours. After filtering the mixture, the beads were sequentially washed with 900 ml of distilled water. Titration revealed that the amount of allyl groups introduced was 132.6 (micromoles) / - wet carrier (mL).

[0070] [B. Activation of Cellfine GH-25 by bromination] 17 ml of allyl-activated Cellfine GH-25 (132.6 micromoles / ml of allyl groups introduced - wet support) was mixed with 17 ml of 0.9 M N-bromosuccinimide (50% aqueous acetone solution). The mixture was stirred at 30°C for 3 hours. The reaction mixture was filtered, and the beads were washed with 800 ml of distilled water. The activated beads were then immediately transferred to a reaction vessel and further reacted with diethylamine as a ligand by the following method.

[0071] [C. Introduction of diethylamine to activating beads] Amine groups were directly introduced into the beads via a linker structure. In a typical procedure, coupling to the beads was performed by bromination of the allyl group and nucleophilic substitution under basic conditions. 1 ml of bromo-activated gel (132.6 micromoles / ml of allyl group introduced - wet support) was transferred to a reaction vessel containing 1 ml of 50% isopropanol aqueous solution in which 15.6 mg of diethylamine as a ligand was dissolved. The reaction mixture was kept at 25°C for 16 hours with stirring. After filtering the reaction mixture, the beads were sequentially washed with 3 ml of isopropanol, 3 ml of distilled water, 3 ml of 1 M HCl solution, 3 ml of distilled water, 3 ml of 1 M NaOH solution, and finally 30 ml of physiological saline. Diethylamine-introduced Sepharose 4 Fast Flow gel was obtained with an amine structure introduced at 67.4 micromoles / ml - wet support.

[0072] The adsorption performance of the obtained porous molded body from plasma for α-synuclein oligomers is shown in Table 2 below. The removal rate of α-synuclein oligomers from the porous molded body was 30% or more. It was revealed that the obtained porous molded body efficiently removes α-synuclein oligomers from plasma.

[0073] Example 12 Sepharose 4 Fast Flow with diethylamine was obtained using the same procedure as in Example 11, except that Cellfine GCL-2000F (JNC Corporation, cross-linked cellulose beads) was used as the porous molded body, and 1 mL of 50% isopropanol aqueous solution containing 20.8 mg of diethylamine was used as the amine solution reacted with the bromo-activated gel. The amount of amine structure introduced was 50.6 micromoles / ml-wet carrier. The adsorption performance of α-synuclein oligomers from plasma of the obtained porous molded body is shown in Table 2 below. The removal rate of α-synuclein oligomers from the porous molded body was 30% or more. It was revealed that the obtained porous molded body efficiently removes α-synuclein oligomers from plasma.

[0074] Example 13 Plasorba BR (manufactured by Asahi Kasei Medical, polystyrene beads) was used as the porous molded body. This porous molded body has a methylene group as the linker structure and a trimethylamine group as the aliphatic amine structure. The amount of amine structure introduced was 346.8 micromoles / ml-wet support. The harmonic mean particle size of this porous molded body was 396 μm and the specific surface area was 24.0 m². 2 The concentration was / g. The removal rate of α-synuclein oligomers from plasma by the porous molded body was 30% or more. It was revealed that the obtained porous molded body efficiently removes α-synuclein oligomers from plasma. Furthermore, the removal rate of α-synuclein monomers from plasma by the porous molded body was 30% or more. It was revealed that the obtained porous molded body efficiently removes α-synuclein monomers from plasma. In addition, the porous molded body had an excellent balance between adsorption efficiency and mechanical stability, and an excellent balance between fluidity and adsorption efficiency.

[0075] Example 14 DIAION WA20 (Mitsubishi Chemical Corporation, polystyrene beads) was used as the porous molded body. This porous molded body has a methylene group as the linker structure and diethylenetriamine (-NH(CH2CH2NH)2H) as the aliphatic amine structure. The amount of amine structure introduced was 733.6 micromoles / ml-wet support. The harmonic mean particle size of this porous molded body was 569 μm and the specific surface area was 2.3 m². 2 The concentration was / g. The removal rate of α-synuclein oligomers from plasma by the porous molded body was over 30%. It was revealed that the obtained porous molded body efficiently removed α-synuclein oligomers from plasma. Furthermore, the porous molded body had an excellent balance between adsorption efficiency and mechanical stability, as well as an excellent balance between fluidity and adsorption efficiency.

[0076] Example 15 DIAION WA30 (Mitsubishi Chemical Corporation, polystyrene beads) was used as the porous molded body. This porous molded body has a methylene group as the linker structure and dimethylamine as the aliphatic amine structure. The amount of amine structure introduced was 932.3 micromoles / ml-wet carrier. The harmonic mean particle size of this porous molded body was 582 μm. The removal rate of α-synuclein oligomers from plasma by the porous molded body was 30% or more. It was revealed that the obtained porous molded body efficiently removes α-synuclein oligomers from plasma. Furthermore, the porous molded body had an excellent balance of fluidity and adsorption efficiency.

[0077] Example 16 Plasorba BR (manufactured by Asahi Kasei Medical, a polystyrene-based bead) was used as the porous molded body. This porous molded body has a methylene group as the linker and a trimethylamine group as the aliphatic amine structure. The amount of amine structure introduced was 346.8 micromoles / ml-wet support. The harmonic mean particle size of this porous molded body was 396 μm and the specific surface area was 24.0 m². 2The concentration was / g. The removal rate of α-synuclein oligomers from blood by the porous molded material was 30% or more. It was revealed that the obtained porous molded material efficiently removed α-synuclein oligomers from blood. Furthermore, the porous molded material had an excellent balance between adsorption efficiency and mechanical stability, as well as an excellent balance between fluidity and adsorption efficiency.

[0078] [Table 1]

[0079] [Table 2] [Industrial applicability]

[0080] The porous molded articles of this disclosure are suitably usable for efficiently removing α-synuclein oligomers from biological fluids.

Claims

1. A porous molded support having a positive charge due to an amine structure, wherein the porous molded support has the amine structure in a concentration of 30 μmol / ml - wetted support or more and 10,000 μmol / ml - wetted support or less, for the removal of α-synuclein oligomers.

2. The porous molded article according to claim 1, wherein the amine structure is an aliphatic amine structure.

3. The porous molded article according to claim 1 or 2, wherein the amine structure is a secondary, tertiary, or quaternary amine, or a combination thereof, and the number of amine carbon atoms is 1 to 6.

4. The porous molded article according to claim 1 or 2, wherein the amine structure comprises a quaternary amine.

5. The porous molded article according to claim 1 or 2, wherein the porous molded article carrier is a crosslinked agarose, a crosslinked cellulose, or a polystyrene-based polymer, or a combination thereof.

6. The porous molded article according to claim 1 or 2, wherein the harmonic mean particle size of the porous molded article carrier is 100 μm or more and 800 μm or less.

7. The specific surface area of ​​the porous molded support is 1 m² ２ / g or more 100m ２ A porous molded article according to claim 1 or 2, wherein the amount is less than or equal to / g.

8. An α-synuclein oligomer removal system, wherein the system is A column comprising a container having an inlet and outlet for a biologically derived liquid, and a porous molded body filled inside the container, A bio-derived fluid introduction channel for introducing the bio-derived fluid into the inlet, The column comprises a bio-derived fluid discharge channel for discharging the bio-derived fluid that has passed through the column from the outlet, The porous molded body is a porous molded body carrier having a positive charge due to an amine structure, and includes a porous molded body carrier having the amine structure in a concentration of 30 μmol / ml - wetted carrier or more and 10,000 μmol / ml - wetted carrier or less. α-synuclein oligomer removal system.

9. The system according to claim 8, wherein the biological fluid is blood, and a plasma separation device is provided between the column and the biological fluid introduction channel, and the plasma separated from the blood is passed through the column.

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