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

Porous molded body for removing α-synuclein oligomers, method for removing α-synuclein oligomers, and system for removing α-synuclein oligomers

[0001] The present disclosure relates to a porous molded body for removing α-synuclein oligomers, a method for removing α-synuclein oligomers, and a system for removing α-synuclein oligomers.

[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 tremor. Parkinson's disease is known to be caused by the degeneration of dopamine-containing pigmented neurons in the substantia nigra of the midbrain, which reduces the dopamine concentration in the striatum. Pathologically, Parkinson's disease is primarily characterized by the presence of Lewy bodies in the substantia nigra, and it has been found that the main component of Lewy bodies is filament composed of α-synuclein. Because patients with early-onset familial Parkinson's disease have a major mutation of two amino acids in α-synuclein, it is believed that these Lewy bodies contribute to neurodegeneration in Parkinson's disease and related disorders.

[0003] Multiple system atrophy (MSA) is a progressive neurodegenerative disease characterized by degeneration of neurons in the cerebellar cortex, pontine nuclei, olivary nuclei, striatum, substantia nigra, autonomic nuclei in the brainstem and spinal cord, as well as the motor cortex, and the accumulation of insoluble α-synuclein inclusions in the cytoplasm of oligodendroglia. Compared to Parkinson's disease, resting tremors are less frequent, progression is more rapid, and anti-Parkinson's drugs are less effective.

[0004] Lewy body dementia is a disease characterized by the appearance of Lewy bodies in the brain, and its main symptoms include cognitive impairment, hallucinations, delusions, and sleep disorders. While motor dysfunction specific to Parkinson's disease may also occur, 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 synucleinopathies, including Parkinson's disease, multiple system atrophy, and dementia with Lewy bodies, is the administration of L-dopa, a precursor molecule to dopamine. L-dopa crosses the blood-brain barrier and is converted to dopamine in the brain. While this drug alleviates symptoms of dopamine deficiency, it does not halt the progressive neurodegeneration that characterizes synucleinopathies. To prevent the progression of synucleinopathies, it is necessary to understand the mechanisms that cause neuronal loss. Currently, no fundamental treatments or drugs exist. In Japan, synucleinopathies are designated as intractable diseases (specified diseases) due to the small number of cases and the long-term disruption they cause to daily life.

[0006] Although the physiological functions of α-synuclein, which constitutes Lewy bodies, remain largely unknown, it has been reported that it plays a defensive role against viral infection, in addition to membrane fusion and synaptic vesicle maintenance and release. When these α-synuclein monomers aggregate under certain influences to form α-synuclein oligomers, they acquire pathogenic properties that cause calcium regulation disorders, mitochondrial dysfunction, and intracellular transport disorders.

[0007] Although the process by which α-synuclein monomers aggregate to form α-synuclein oligomers in vivo is unclear at the time of filing of this application, the α-synuclein oligomer transmission hypothesis has recently been proposed, which suggests that α-synuclein oligomers may propagate between cells due to their prion protein-like properties, resulting in the pathology of synucleinopathies. In vitro, a self-templating phenomenon has been observed, in which α-synuclein oligomers with a β-sheet structure serve as templates to promote the aggregation of α-synuclein monomers with a normal structure. Furthermore, animal experiments using mice have reported that intracerebral, intraperitoneal, oral, or intravenous administration of α-synuclein oligomers resulted in neurological symptoms and the formation of α-synuclein aggregates in the central nervous system (Non-Patent Document 1).

[0008] Although the pathway by which α-synuclein oligomers propagate in the α-synuclein oligomer propagation hypothesis is not fully understood, it has been reported that higher amounts of α-synuclein oligomers are present in the plasma of Parkinson's disease patients compared to healthy individuals (Non-Patent Document 2).

[0009] Patent Document 1 describes a means for removing α-synuclein monomers present free in body fluids by adsorption before they accumulate on nerve cells. The means disclosed in Patent Document 1 aims to adsorb and remove only α-synuclein monomers using a ligand-introduced porous carrier, but does not describe a means that may be able to slow the progression of Parkinson's disease, multiple system atrophy, and / or dementia with Lewy bodies by removing α-synuclein oligomers from biological fluids.

[0010] JP 2012-193112 A

[0011] LOHMANN, Stephanie, et al., “Oral and intravenous transmission of α-synuclein fibrils to mice”, Acta Neuropathologica, (2019), 138: 515-533.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.

[0012] An object of the present disclosure is to provide a porous body, a method, and a system that can remove α-synuclein oligomers from biologically derived fluids.

[0013] Examples of embodiments of the present disclosure are listed below. [1] A porous shaped body for removing α-synuclein oligomers, comprising a porous shaped body support having a positive charge due to an amine structure, wherein the porous shaped body support has the amine structure at a concentration of 30 μmol / ml-wet carrier or more and 10,000 μmol / ml-wet carrier or less. [2] The porous shaped body according to item 1, wherein the amine structure is an aliphatic amine structure. [3] The porous shaped body according to item 1 or 2, wherein the amine structure is a secondary, tertiary, or quaternary amine, or a combination thereof, and wherein the amine carbon number is 1 to 6. [4] The porous shaped body according to any one of items 1 to 3, wherein the amine structure comprises a quaternary amine. [5] The porous shaped body according to any one of items 1 to 4, wherein the porous shaped body support is cross-linked agarose, cross-linked cellulose, or a polystyrene-based polymer, or a combination thereof. [6] The porous shaped body according to any one of items 1 to 5, wherein the harmonic mean particle diameter of the porous shaped body support is 100 μm or more and 800 μm or less. [7] The specific surface area of ​​the porous shaped body support is 1 m ２ / g or more 100m ２ / g or less. [8] An α-synuclein oligomer removal system comprising: a column having a container having an inlet and an outlet for a biologically-derived fluid and a porous body filled in the container; a biologically-derived fluid inlet flow path for introducing the biologically-derived fluid into the inlet; and a biologically-derived fluid outlet flow path for discharging the biologically-derived fluid that has passed through the column from the outlet, wherein the porous body is a porous body carrier having a positive charge due to an amine structure, the porous body carrier having the amine structure of 30 μmol / ml-wet carrier or more and 10,000 μmol / ml-wet carrier or less. [9] The system according to Item 8, wherein the biologically-derived fluid is blood, and a plasma separation device is provided between the column and the biologically-derived fluid inlet flow path, and the system is configured to pass plasma separated from the blood through the column.

[10] A method for removing α-synuclein oligomers from a biologically derived fluid, the method comprising contacting the porous shaped body according to any one of items 1 to 7 with a biologically derived fluid containing α-synuclein oligomers.

[11] The method according to item 10, wherein the biologically derived fluid is blood or plasma.

[12] A method for reducing the concentration of α-synuclein oligomers in a biologically derived fluid from a patient, the method comprising: collecting the biologically derived fluid from the patient's body; contacting the collected biologically derived fluid with a porous shaped body to obtain a contacted biologically derived fluid; and returning the contacted biologically derived fluid to the patient's body, wherein the porous shaped body is a porous shaped body carrier having a positive charge due to an amine structure, the amine structure having an amine structure of 10 μmol / ml-wet carrier or more and 3000 μmol / ml-wet carrier or less.

[13] The method according to item 12, wherein the biologically derived fluid is blood or plasma.

[0014] According to the present disclosure, it is possible to provide a porous body, a method, and a system that can remove α-synuclein oligomers from biologically derived fluids.

[0015] <<Porous Molded Body>> The porous shaped body of the present disclosure is a porous shaped body for removing α-synuclein oligomers. The porous shaped body includes a porous shaped body carrier having a positive charge due to an amine structure, thereby enabling the removal of α-synuclein oligomers from biologically derived fluids. Based on the α-synuclein oligomer transmission hypothesis (Non-Patent Documents 1 and 2, etc.), the present inventors believed that removing α-synuclein oligomers from biologically derived fluids and blocking the transmission pathway of α-synuclein oligomers would be effective in inhibiting the progression of synucleinopathy, and conducted extensive research into means for removing α-synuclein oligomers. As a result, the present inventors discovered a previously unknown attribute: that a porous shaped body carrier having a positive charge due to an amine structure can adsorb and remove α-synuclein oligomers, leading to the configuration of the present invention.

[0016] [Porous shaped carrier] The porous shaped carrier has a positive charge due to an amine structure bonded to the porous shaped carrier. The amount of the amine structure possessed by the porous shaped carrier is preferably 10 μmol / ml-wet carrier or more and 10,000 μmol / ml-wet carrier or less, more preferably 20 μmol / ml-wet carrier or more and 10,000 μmol / ml-wet carrier or less, even more preferably 30 μmol / ml-wet carrier or more and 10,000 μmol / ml-wet carrier or less, still more preferably 30 μmol / ml-wet carrier or more and 5,000 μmol / ml-wet carrier or less, particularly preferably 40 μmol / ml-wet carrier or more and 3,000 μmol / ml-wet carrier or less, and particularly preferably 50 μmol / ml-wet carrier or more and 2,000 μmol / ml-wet carrier or less. When the amount of amine structures is 10 μmol / ml or more of wet carrier, the effect of removing α-synuclein oligomers is further improved. From the viewpoint of the amount of α-synuclein oligomer adsorption, it is desirable to introduce a large amount of amine structures. However, from the viewpoint of facilitating the introduction of amine structures and reducing the amount of reagents required for the introduction reaction to improve economy, the amount of amine structures is preferably 10,000 μmol / ml or less of wet carrier, and more preferably 400 μmol / ml or less of wet carrier.

[0017] The porous carrier has a porous structure with a large number of pores of an appropriate size. The size of the pores in the porous carrier is not particularly limited, but the specific surface area is preferably 0.0001 m. ２ / g or more 1000m ２ / g or less, and ２ / g or more 100m ２ / g or less is more preferable, and ２ / g or more 50m ２ / g or less is more preferable. ２ When the surface roughness is 0.0001 m / g or less, the mechanical strength of the porous molded carrier is improved. ２ / g or more, the adsorption efficiency tends to improve, resulting in a better balance between adsorption efficiency and mechanical stability. The shape of the porous shaped carrier is not particularly limited, but may be any shape such as particulate (beads), thread, sheet, hollow fiber, cylinder, hollow cylinder, etc., or may be a monolithic carrier in which at least some of the pores in the carrier are penetrated. The porous shaped carrier is preferably particulate (beads). In the case of a particulate (bead) porous shaped carrier, the particle size is not particularly limited, but the harmonic mean particle size is preferably 20 μm or more and 1500 μm or less, more preferably 50 μm or more and 1000 μm or less, and even more preferably 100 μm or more and 800 μm or less. When used in an α-synuclein oligomer removal system described below, a harmonic mean particle size of 20 μm or more improves the fluidity of biologically derived fluid, while a harmonic mean particle size of 1500 μm or less increases the specific surface area per volume, tending to improve adsorption efficiency, resulting in a better balance between fluidity and adsorption efficiency.

[0018] The material constituting the porous shaped carrier is not particularly limited as long as it is a porous body into which an amine structure can be introduced, and may be, for example, an inorganic compound or an organic compound. Organic polymers are preferred as materials for the porous shaped carrier because they produce less elution in hot water and allow for easier and more precise control of the pores of the porous body. Examples of such organic polymers include synthetic polymer carriers, such as crosslinked or non-crosslinked polyvinyl alcohol, polyacrylate, polymethacrylate, polyacrylamide, polyacrylic acid, polyamide, polyester, polyethylene, and polystyrene; copolymer carriers composed of two or more of these; crosslinked or non-crosslinked polysaccharide carriers, such as cellulose, agarose (Sepharose), and dextrin; and organic-organic and organic-inorganic composite carriers obtained by combining these. 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-based polymers, or combinations thereof.

[0019] [Amine Structure] In the present disclosure, an "amine structure" refers to a structure having one or more amino groups on a hydrocarbon group. The "amine structure" is preferably an "aliphatic amine structure." An "aliphatic amine structure" refers to a structure having one or more amino groups on an aliphatic group of a hydrocarbon group containing or consisting 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 may be 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 are in the form of ammonium ions, i.e., primary, secondary, tertiary, or quaternary ammonium, and thus can have a positive charge at the pH of the biologically derived fluid. The porous shaped carrier may have the amine structure via a covalent bond or a non-covalent bond.

[0020] When the porous shaped support has an amine structure via a covalent bond, the amino group in the form of an ammonium ion can be, for example, a group represented by the following general formula (1): In general formula (1), the bond "-" means a bond to a hydrocarbon group (also referred to as a "linker structure" in the present disclosure). １ , R ２ , and R ３ are preferably each independently a hydrogen atom or a hydrocarbon group. １ , R ２ , and R ３ Among these, it is preferred that one or more, two or more, three or more, or all four are hydrocarbon groups. １ , R ２ , and R ３ The hydrocarbon group of R may be saturated or unsaturated, may have a straight chain, branched chain 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, still more preferably 1 to 8, and particularly preferably 1 to 6. １ , R ２ , and R ３ The hydrocarbon group may have a heteroatom, and may have, for example, a hydroxyl group, a halogen group, an amino group, a carbonyl group, a sulfonyl group, an ether bond, or the like as part of its structure.

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

[0022] When the porous shaped support has an amine structure via a covalent bond and the amine structure has a plurality of amino groups, the amine structure may be, for example, a compound represented by the following general formula (2): At least one of the amino groups in general formula (2) is in the form of an ammonium ion at the pH of the biologically derived fluid and can have a positive charge. The leftmost bond "-" indicates a bond to the hydrocarbon group of the linker structure. n is an integer of 1 to 100, preferably 1 to 50, more preferably 1 to 30, even more preferably 1 to 10, and particularly preferably 1 to 5. R ４ , R ６ , and R ７ are each independently a hydrogen atom or a monovalent hydrocarbon group. ４ , R ６ , and R ７ The hydrocarbon group of R may be saturated or unsaturated, may have a straight chain, branched chain or cyclic structure, and may be aliphatic, aromatic or a combination thereof. ４ , R ６ , and R ７The 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, still more preferably 1 to 8, and particularly preferably 1 to 6. ４ , R ６ , and R ７ The hydrocarbon group R may have a heteroatom, and may have, for example, a hydroxyl group, a halogen group, an amino group, a carbonyl group, a sulfonyl group, an ether bond, or the like as part of its structure. ４ , R ６ , and R ７ It is preferable that at least one of the groups is a hydrogen atom, and all of the groups are hydrogen atoms (—NH(R ５ NH) ｎ It is more preferable to use R ５ are each independently a divalent hydrocarbon group. ５ The hydrocarbon group of R may be saturated or unsaturated, may have a straight chain, branched chain 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, still more preferably 1 to 8, and particularly preferably 1 to 6. ５ The hydrocarbon group R may have a heteroatom, and may have, for example, a hydroxyl group, a halogen group, an amino group, a carbonyl group, a sulfonyl group, an ether bond, or the like as part of its structure. ５ is preferably a divalent alkylene group, and examples thereof include a substituted or unsubstituted methylene group, ethylene group, propylene group, butylene group, etc. Examples of the structure of the above general formula (2) include -NH(CH ２ NH) ｎ H, —NH(CH ２ CH ２ NH) ｎ H, —NH(CH ２ CH ２ CH ２ NH) ｎ H and the like are preferred.

[0023] The amine structure is a hydrocarbon group (R １ , R ２ , or R ３) has fewer carbon atoms, the more readily electrostatic interaction occurs between the positive charge of the amine structure and α-synuclein oligomers, and α-synuclein oligomers tend to be removed more effectively. Also, from an economical point of view, the fewer the carbon atoms in the hydrocarbon group bonded to the amino group, the lower the cost of the reagent 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 longest hydrocarbon group (excluding the linker) in the hydrocarbon chain bonded to the amino group (R １ , R ２ , or R ３ When the number of carbon atoms in the alkyl group (N,N-dimethylbutylamine) is defined as the "amine carbon number," the number of amine carbon atoms is preferably 1 or more and 6 or less. Examples of amine compounds that give an amine structure with such an 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 amine carbon atoms. This increases the proportion of amine structures that are positively charged at the pH of the biologically derived fluid, which tends to facilitate electrostatic interaction between the positive charge of the amine structure and α-synuclein oligomers and more effectively remove α-synuclein oligomers. Examples of amine compounds that provide such a quaternary amine structure include trimethylamine, triethylamine, N,N-dimethylbutylamine, N,N-dimethylhexylamine, N-benzyl-N-methyl-ethanolamine, and N,N-dimethyl-2-phenoxyethylamine.

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

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

[0027] In an embodiment in which the porous shaped carrier has an amine structure via a non-covalent bond, for example, the porous shaped carrier may have a polymer having an amine structure on its surface. The polymer having an amine structure preferably has the above-mentioned amine structure in the main chain and / or as a side chain of the polymer. More specifically, the polymer having an amine structure may be a polyalkyleneimine, such as polyethyleneimine.

[0028] [α-Synuclein Oligomers] Examples of α-synuclein oligomers include oligomers and aggregates formed from two or more α-synuclein monomer molecules, as well as phosphorylations thereof. The porous shaped article of the present disclosure is a porous shaped article for removing α-synuclein oligomers from a biologically derived fluid. The porous shaped article of the present disclosure is suitable for contacting a biologically derived fluid with the biologically derived fluid to remove α-synuclein oligomers from the biologically derived fluid. Generally, biologically derived fluids may contain sufficient low-molecular-weight substances that can be adsorbed to the porous shaped article. Since low-molecular-weight substances quickly occupy accessible adsorption sites on the porous shaped article, removal of large molecular weight adsorbates generally tends to be difficult using the porous shaped article. Furthermore, in the case of adsorbates with significantly high molecular weights, the adsorbates do not penetrate deep into the pores of the porous shaped article, and adsorption sites are limited to the surface of the porous shaped article, making adsorption and removal even more difficult. However, the porous shaped article of the present disclosure is designed to overcome this problem and has 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 shaped article of the present disclosure is not particularly limited, but it can remove, for example, α-synuclein oligomers of 500,000 kDa or less, and in particular, it can efficiently remove α-synuclein oligomers of 100,000 kDa or less. The efficient removal of α-synuclein oligomers can slow the progression of synucleinopathies such as Parkinson's disease, multiple system atrophy, and / or dementia with Lewy bodies. Therefore, the porous shaped article of the present disclosure can be preferably used to slow the progression of synucleinopathies.

[0029] The porous shaped article of the present disclosure may also be used in pharmaceutical applications, such as producing blood products using blood from which α-synuclein oligomers have been removed, or in diagnostic applications, such as concentrating, recovering, and detecting α-synuclein oligomers in biological fluids to diagnose synucleinopathies.

[0030] [Bio-derived fluid] The bio-derived fluid is preferably blood, plasma, serum, spinal fluid, ascites, urine, saliva, tears, nasal discharge, sweat, digestive fluid, breast milk, etc., and particularly preferably blood or plasma.

[0031] <<Method for Producing a Porous Molded Body>> The method for producing a porous molded body of the present disclosure is not limited. Examples of the method for producing a porous molded body of the present disclosure include a method that includes introducing an amine structure onto the surface of a porous molded body support that does not have an amine structure. Alternatively, a method that includes synthesizing a porous polymer support from a monomer having an amine structure or a precursor structure thereof by a polymerization reaction is also included. Details of the porous molded body support and the amine structure have been described above, so a description thereof will be omitted here.

[0032] Methods for introducing amine structures onto the surface of a porous shaped carrier that does not have an amine structure include a covalent bond immobilization method and a non-covalent bond immobilization method. An example of a covalent bond immobilization method is a method in which a linker compound that imparts a linker structure is reacted with the porous shaped carrier, and then an amine compound (ligand) that imparts an amine structure is reacted to introduce a linker structure between the porous shaped carrier and the amine compound. Since the amine compound and the linker have been described in detail above, a detailed description thereof will be omitted here.

[0033] As an example of a non-immobilized introduction method via a non-covalent bond, a polymer having an amine structure (such as polyethyleneimine) is coated on the surface of a porous shaped carrier. Any coating method, such as painting, spraying, or dipping, can be used to coat the surface of the porous shaped carrier with the polymer having an amine structure. Details of the polymer having an amine structure have been described above, so a detailed description will be omitted here.

[0034] <<Method for Removing α-Synuclein Oligomers>> The method for removing α-synuclein oligomers of the present disclosure comprises contacting a porous shaped article of the present disclosure with a biologically-derived 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 biologically-derived fluid. For example, the removal method preferably comprises introducing a biologically-derived fluid containing α-synuclein oligomers into a container having an inlet and an outlet for the biologically-derived fluid and filled with a porous shaped article of the present disclosure, allowing the biologically-derived fluid to contact the porous shaped article of the present disclosure and pass through the container, and discharging the biologically-derived fluid through the outlet. The removal method may further comprise collecting the biologically-derived fluid from the patient's body and returning the biologically-derived fluid that has contacted the porous shaped article of the present disclosure back into the patient's body.

[0035] In the present disclosure, "removal" does not mean complete removal of α-synuclein oligomers from a biologically derived fluid, but rather refers to the reduction of the concentration of α-synuclein oligomers contained in the biologically derived fluid. The removal method can remove α-synuclein oligomers contained in the biologically derived fluid by preferably 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, or 30% or more.

[0036] As described above, blood or plasma is particularly preferred as the biologically derived fluid. In this case, the removal method may include contacting blood containing α-synuclein oligomers with a porous shaped article of the present disclosure to obtain blood from which α-synuclein oligomers have been removed. Alternatively, the removal method may further include previously separating plasma containing α-synuclein oligomers from the blood, contacting the separated plasma with a porous shaped article of the present disclosure to remove α-synuclein oligomers from the plasma, and combining the plasma from which α-synuclein oligomers have been removed with the separated hemocyte concentrate to obtain blood from which α-synuclein oligomers have been removed. The removal method may further include removing blood from the patient's body and / or returning the blood that has been contacted with a porous shaped article of the present disclosure back into the patient's body.

[0037] <α-Synuclein Oligomer Removal System> The porous body of the present disclosure can be incorporated into an α-synuclein oligomer removal system. The α-synuclein oligomer removal system of the present disclosure includes a column having a container having an inlet and an outlet for a biologically derived fluid and a porous body filled in the container, a biologically derived fluid introduction channel (circuit) through which the biologically derived fluid is introduced into the inlet, and a biologically derived fluid discharge channel through which the biologically derived fluid that has passed through the column is discharged from the outlet. The biologically derived fluid introduction channel and the biologically derived fluid discharge channel are preferably made of flexible tubing. During use of the system, the biologically derived fluid introduction channel may be fluidically connected to the inside of a patient's body to collect the biologically derived fluid from the patient's body. Furthermore, the biologically derived fluid discharge channel may be fluidically connected to the inside of a patient's body to return the biologically derived fluid after contacting the porous body to the patient's body.

[0038] The α-synuclein oligomer removal system can remove α-synuclein oligomers contained in a biologically derived fluid, preferably at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, or at least 30%.

[0039] As described above, blood or plasma is particularly preferred as the biologically derived fluid, and in this case, the system is used as a blood purification system. One example of a blood purification system includes a blood inlet flow path consisting of a flexible tube for removing blood from a patient's body and introducing it into a column, a column packed with a porous molded article of the present disclosure, and a blood outlet flow path consisting of a flexible tube for returning the blood leaving the column to the body. When the blood purification system is in use, the blood inlet flow path and the blood outlet flow path are connected to the patient's blood vessels.

[0040] The blood purification system described above is merely an example, and is not limited to the above configuration. Various modifications are possible by appropriately applying conventional blood purification techniques. For example, the blood purification system may include two or more α-synuclein oligomer adsorption / removal columns connected in series or parallel. As another example, the blood purification system may include a plasma separator between the α-synuclein oligomer adsorption / removal column and the bio-derived fluid introduction channel, and configured to pass plasma separated from blood through the column. More specifically, examples of the plasma separator include a membrane-type plasma separator using a plasma separation membrane and a centrifugal plasma separator. In this case, the blood purification system may be further configured to remove α-synuclein oligomers from the plasma, combine the plasma from which α-synuclein oligomers have been removed with the blood cell concentrate separated by the plasma separator, and return the resulting α-synuclein oligomer-free blood to the patient. Further, other elements that can be included in the blood purification system include elements used in conventional blood purification systems, such as an anticoagulant addition device, a pressure gauge, a flow rate detection device, an abnormality detection device, a particulate removal filter, an air chamber, and a hemolysis sensor.

[0041] Examples and comparative examples of the present disclosure will be specifically described below, but the present disclosure is not limited to these examples and comparative examples.

[0042] Evaluation and Measurement Methods [Method for Measuring the Removal Rate of Oligomers Composed of Two or More α-Synuclein Monomer Molecules] 0.18 mL of a porous molded article was placed in a 3.0 mL Libra tube (manufactured by HiPep Research Institute) and washed with phosphate-buffered saline (PBS-, manufactured by Takara Bio Inc.) and physiological saline (manufactured by Otsuka Pharmaceutical Factory, Inc.). 0.9 mL of commercially available heparin-anticoagulated bovine plasma or heparin-anticoagulated bovine blood adjusted to a concentration of approximately 32 ng / mL of α-synuclein oligomers (Recombinant Human Alpha-synuclein protein aggregate (Active), manufactured by Abcam) was then added. The mixture was stirred at 5 rpm at 37°C for 1 hour, and the α-synuclein oligomer concentration in the supernatant plasma was measured by sandwich ELISA using two antibodies (Anti-Alpha-synuclein filament MJFR14-6-4-2 Conformation specific, Abcam, and Purified Mouse Anti-α-Synuclein Clone 42 / α-Synuclein (RUO), BD Transduction Laboratories). The measured value of α-synuclein oligomers in a sample without a porous body was set to 100%, and the value obtained by subtracting the residual rate (%) of α-synuclein oligomers from 100% was defined as the α-synuclein oligomer removal rate (%) of the porous body. When a porous body adsorbs little or no α-synuclein oligomers, the removal rate may take a negative value due to the effects of increased sensitivity of ELISA measurement caused by the adsorption and removal of plasma proteins other than α-synuclein oligomers by the porous body and / or variability in ELISA measurement.

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

[0044] [Method for measuring α-synuclein monomer removal rate] 0.18 mL of the porous molded body was measured into a 3.0 mL Libra tube (manufactured by HiPep Research Institute) and washed with phosphate-buffered saline (PBS-, manufactured by Takara Bio Co., Ltd.) and physiological saline (manufactured by Otsuka Pharmaceutical Factory). Then, 0.9 mL of commercially available heparin-anticoagulated bovine plasma or heparin-anticoagulated bovine blood adjusted to a concentration of approximately 32 ng / mL of α-synuclein monomer (α-Synuclein, recombinant, human, manufactured by Cosmo Bio Co., Ltd.) 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, manufactured by R&D Systems). The measured value of α-synuclein monomer in the sample without the porous body was set to 100%, and the value obtained by subtracting the remaining rate (%) of α-synuclein monomer from 100% was defined as the α-synuclein monomer removal rate (%) of the porous body. When the porous body adsorbs little or no α-synuclein monomer, the removal rate may take a negative value due to the influence of increased sensitivity of ELISA measurement caused by the adsorption and removal of plasma proteins other than α-synuclein monomer by the porous body and / or variability in ELISA measurement.

[0045] [Method for Measuring the Amount of Allyl Groups Introduced into Porous Molded Body] The amount of allyl groups introduced into the porous molded body carrier (μmol / mL - wet carrier) was obtained by substituting the allyl groups introduced into the porous molded body carrier with carboxyl groups and then neutralizing the carboxyl groups by titration. Specifically, 1 mL of the porous molded body was measured and placed in a 5.0 mL Libra tube (manufactured by HiPep Laboratories) and mixed with 1 mL of 25 mg / mL aqueous ammonium persulfate solution and 120 μL of thioacetic acid. The reaction mixture was stirred at 50°C for 16 hours. 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 solution. The porous molded body was transferred to a 50 mL centrifuge tube, 30 mL of 1 M HCl solution was added, and the mixture was shaken and stirred for 30 minutes. The porous formed body was collected using a Libra tube and washed with 60 mL of ultrapure water (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), and then the porous formed body was transferred to a 100 mL centrifuge tube, and 100 mL of ultrapure water was added and shaken and stirred for 30 minutes. The porous formed body was again collected using a Libra tube, and after washing with 60 mL of ultrapure water, it was transferred to a centrifuge tube, and 2 mL to 500 mL of 0.01 M NaOH water was added and shaken and stirred 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 group introduced into the porous formed body (μmol / ml - wet carrier) was calculated from the amount of dropwise addition at the pH inflection point. In this case, the proportion of NaOH that was ion-exchanged with the porous formed body out of the NaOH added to the porous formed body was calculated as the NaOH consumption rate, and if the NaOH consumption rate was between 20% and 40%, it was determined that the measurement of the amount of introduced allyl groups was appropriate.

[0046] [Method for measuring the amount of amine structure introduced into the porous molded body] The amount of amine structure introduced (μmol / mL - wet carrier) was obtained by neutralization titration of the amino group introduced into the porous molded body carrier. Specifically, 0.2 mL of the porous molded body was measured into a 3.0 mL Libra tube (manufactured by HiPep Research Institute), washed with 10 mL of physiological saline, and then the porous molded body was transferred to a 25 mL centrifuge tube, 6 mL of 1 M NaOH water was added, and the mixture was shaken and stirred for 30 minutes. The porous molded body was recovered using a Libra tube, washed with 12 mL of ultrapure water (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), and then the porous molded body was transferred to a 100 mL centrifuge tube, 50 mL of ultrapure water was added, and the mixture was shaken and stirred for 30 minutes. The porous formed 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 water was added and shaken and stirred for 30 minutes. After the reaction, 0.1 mL to 5 mL of the supernatant was collected, and 0.1 M NaOH water was added dropwise while stirring. The amount of amine structure introduced into the porous formed body (μmol / ml - wet carrier) was calculated from the amount of HCl added to the porous formed body that was ion-exchanged. At this time, the proportion of HCl that was ion-exchanged with the porous formed body out of the HCl added to the porous formed body was calculated as the HCl consumption rate. If the HCl consumption rate was between 20% and 40%, the measurement of the amount of amine structure introduced was determined to be appropriate.

[0047] [Harmonic mean particle diameter of porous formed body] The harmonic mean particle diameter of the porous formed body was measured by swollen with ultrapure water using a particle size distribution measuring device (MT3300II, manufactured by Microtrackbell Co., Ltd.), and the harmonic mean particle diameter (μm) was calculated as the harmonic mean particle diameter.

[0048] [Specific surface area of ​​porous molded body] The porous molded body swollen with ultrapure water was frozen and then freeze-dried for 24 hours to dry the porous molded body. The dried porous molded body was subjected to degassing treatment (vacuum drying) at 60°C for 15 hours using a VacPrep061 (manufactured by Shimadzu Corporation-Micromeritics). Thereafter, the porous molded body was degassed using a TriStar II 3020 (manufactured by Shimadzu Corporation-Micromeritics) with N ２ Specific surface area (m ２ / g) was measured. At this time, the value obtained by BET plot was used as the specific surface area. The above measurement was carried out 10 times, and the average value of the 8 values ​​excluding the maximum and minimum values ​​was taken as the specific surface area.

[0049] Comparative Example 1 [A. Introduction of Allyl Groups into Porous Molded Body] 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 aqueous NaOH, and 9 ml of dimethyl sulfoxide. The mixture was stirred at 30°C for 18 hours. After filtering the mixture, the beads were washed sequentially with 750 ml of distilled water. Titration revealed that the amount of allyl groups introduced was 108.9 (µmol) / bead (mL)-wet carrier.

[0050] B. Activation of Sepharose 4 Fast Flow by Bromination: 14 ml of allyl-activated Sepharose 4 Fast Flow (108.9 μmol of allyl groups / ml of wet carrier) was mixed with 14 mL of 0.9 M N-bromosuccinimide (50% aqueous acetone). 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 to Activated Beads. Aliphatic amine structures were directly introduced into beads via linker structures. In a typical procedure, coupling to beads was achieved by bromination of the allyl group and nucleophilic substitution under basic conditions. 1 ml of bromo-activated gel (allyl group introduction amount: 108.9 μmol / ml—wet support) was transferred to a reaction vessel containing 100 mg of hexadecylamine as a ligand in 1 ml of 50% DMSO solution. The reaction mixture was stirred at 30°C for 16 hours. After filtering the reaction mixture, the beads were washed sequentially with 3 ml of isopropanol, 3 ml of distilled water, 3 ml of 1 M HCl, 3 ml of distilled water, 3 ml of 1 M NaOH, and finally 30 ml of saline. Hexadecylamine-introduced Sepharose 4 Fast Flow was obtained with an amine structure-introduced amount of 6.2 μmol / ml of wet carrier.

[0052] The α-synuclein oligomer adsorption performance from plasma of the obtained porous shaped article is shown in the following Table 1. The removal rate of α-synuclein oligomer by the porous shaped article was less than 30%.

[0053] Example 1 Hexadecylamine-incorporated Sepharose 4 Fast Flow was obtained in the same manner 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 to be reacted with the bromo-activated gel. The amount of amine structure introduced was 39.4 micromoles / ml-wet carrier. The α-synuclein oligomer adsorption performance of the obtained porous molded article from plasma is shown in Table 1 below. The α-synuclein oligomer removal rate of the porous molded article was 30% or more. It was revealed that the obtained porous molded article efficiently removed α-synuclein oligomers from plasma.

[0054] Comparative Example 2 A. Introduction of Allyl Groups into Porous Molded Body 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 aqueous NaOH, and 2.25 ml of dimethyl sulfoxide. The mixture was stirred at 30°C for 18 hours. After filtering the mixture, the beads were washed sequentially with 750 ml of distilled water. Titration revealed that the amount of allyl groups introduced was 73.9 (µmol) / mL of wet carrier.

[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 μmol / ml - wet carrier) was mixed with 14 mL of 0.9 M N-bromosuccinimide (50% aqueous acetone). 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 Activated Beads. Amine groups were directly introduced onto the beads via a linker structure. In a typical procedure, coupling to the beads was achieved by bromination of the allyl group and nucleophilic substitution under basic conditions. 4 ml of bromo-activated gel (allyl group loading: 73.9 μmol / ml—wet support) was transferred to a reaction vessel containing 20 mg of diethylamine as a ligand in 4 ml of 50% aqueous isopropanol. The reaction mixture was stirred at 30°C for 16 hours. After filtering the reaction mixture, the beads were washed sequentially with 8 ml of isopropanol, 4 ml of distilled water, 4 ml of 1 M HCl, 4 ml of distilled water, 4 ml of 1 M NaOH, and finally 120 ml of saline. Diethylamine-introduced Sepharose 4 Fast Flow was obtained with an amine structure-introduced amount of 5.5 μmol / ml of wet carrier.

[0057] The α-synuclein oligomer adsorption performance from plasma of the obtained porous shaped article is shown in the following Table 1. The removal rate of α-synuclein oligomer by the porous shaped article was less than 30%.

[0058] Comparative Example 3: Diethylamine-incorporated Sepharose 4 Fast Flow was obtained in the same manner 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 μmol / ml-wet carrier. The α-synuclein oligomer adsorption performance of the obtained porous molded article from plasma is shown in Table 1 below. The α-synuclein oligomer removal rate of the porous molded article was less than 30%.

[0059] Comparative Example 4: Diethylamine-incorporated Sepharose 4 Fast Flow was obtained in the same manner 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 μmol / ml-wet carrier. The α-synuclein oligomer adsorption performance of the obtained porous molded article from plasma is shown in Table 1 below. The α-synuclein oligomer removal rate of the porous molded article was less than 30%.

[0060] Example 2 Diethylamine-incorporated Sepharose 4 Fast Flow was obtained in the same manner 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 μmol / ml-wet carrier. The α-synuclein oligomer adsorption performance of the obtained porous molded article from plasma is shown in Table 1 below. The α-synuclein oligomer removal rate of the porous molded article was 30% or more. It was revealed that the obtained porous molded article efficiently removed α-synuclein oligomers from plasma.

[0061] Example 3 Diethylamine-incorporated Sepharose 4 Fast Flow was obtained in the same manner as in Comparative Example 1, except that 1 mL of 50% isopropanol aqueous solution in which 500 mg of diethylamine was dissolved was used as the amine solution to be reacted with the bromo-activated gel. The amount of amine structure introduced was 95.3 μmol / mL-wet carrier. The α-synuclein oligomer adsorption performance of the obtained porous molded article from plasma is shown in Table 1 below. The α-synuclein oligomer removal rate of the porous molded article was 30% or more. It was revealed that the obtained porous molded article efficiently removed α-synuclein oligomers from plasma.

[0062] Example 4 Ethylamine-incorporated Sepharose 4 Fast Flow was obtained in the same manner as in Comparative Example 1, except that 1 mL of 50% isopropanol aqueous solution containing 77.0 mg of ethylamine was used as the amine solution to be reacted with the bromo-activated gel. The amount of amine structure introduced was 53.2 μmol / mL-wet carrier. The α-synuclein oligomer adsorption performance of the obtained porous molded article from plasma is shown in Table 1 below. The α-synuclein oligomer removal rate of the porous molded article was 30% or more. It was revealed that the obtained porous molded article efficiently removed α-synuclein oligomers from plasma.

[0063] Example 5 Triethylamine-incorporated Sepharose 4 Fast Flow was obtained in the same manner as in Comparative Example 1, except that 1 mL of a 50% isopropanol solution containing 691.4 mg of triethylamine was used as the amine solution to be reacted with the bromo-activated gel. The amount of amine structure introduced was 53.7 μmol / mL-wet carrier. The α-synuclein oligomer adsorption performance of the obtained porous molded article from plasma is shown in Table 1 below. The α-synuclein oligomer removal rate of the porous molded article was 30% or more. It was revealed that the obtained porous molded article efficiently removed α-synuclein oligomers from plasma.

[0064] Example 6 N-benzyl-N-methyl-ethanolamine-incorporated Sepharose 4 Fast Flow was obtained in the same manner as in Comparative Example 1, except that 1 mL of 50% isopropanol aqueous solution containing 2259.5 mg of N-benzyl-N-methyl-ethanolamine was used as the amine solution to be reacted with the bromo-activated gel. The amount of amine structure introduced was 49.2 micromoles / ml-wet carrier. The α-synuclein oligomer adsorption performance of the obtained porous molded article from plasma is shown in Table 1 below. The removal rate of α-synuclein oligomers by the porous molded article was 30% or more. It was revealed that the obtained porous molded article efficiently removed α-synuclein oligomers from plasma.

[0065] Example 7 N,N-dimethyl-2-phenoxyethylamine-incorporated Sepharose 4 Fast Flow was obtained in the same manner as in Example 1, except that 1 mL of 50% isopropanol aqueous solution containing 141.2 mg of N,N-dimethyl-2-phenoxyethylamine was used as the amine solution to be reacted with the bromo-activated gel. The amount of amine structure introduced was 59.4 μmol / mL-wet carrier. The α-synuclein oligomer adsorption performance of the obtained porous molded article from plasma is shown in Table 2 below. The removal rate of α-synuclein oligomers by the porous molded article was 30% or more. It was revealed that the obtained porous molded article efficiently removed α-synuclein oligomers from plasma.

[0066] Example 8 Butylamine-incorporated Sepharose 4 Fast Flow was obtained in the same manner as in Comparative Example 1, except that 1 mL of 50% isopropanol aqueous solution containing 987.4 mg of butylamine was used as the amine solution to be reacted with the bromo-activated gel. The amount of amine structure introduced was 68.3 μmol / mL-wet carrier. The α-synuclein oligomer adsorption performance of the obtained porous molded article from plasma is shown in Table 2 below. The α-synuclein oligomer removal rate of the porous molded article was 30% or more. It was revealed that the obtained porous molded article efficiently removed α-synuclein oligomers from plasma.

[0067] Example 9 N,N-dimethylbutylamine-incorporated Sepharose 4 Fast Flow was obtained in the same manner as in Example 1, except that 1 mL of 80% isopropanol aqueous solution containing 379.4 mg of N,N-dimethylbutylamine was used as the amine solution to be reacted with the bromo-activated gel. The amount of amine structure introduced was 51.0 μmol / mL-wet carrier. The α-synuclein oligomer adsorption performance of the obtained porous molded article from plasma is shown in Table 2 below. The α-synuclein oligomer removal rate of the porous molded article was 30% or more. It was revealed that the obtained porous molded article efficiently removed α-synuclein oligomers from plasma.

[0068] Example 10 N,N-dimethylhexylamine-incorporated Sepharose 4 Fast Flow was obtained in the same manner 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 to be reacted with the bromo-activated gel. The amount of amine structure introduced was 40.4 μmol / mL-wet carrier. The α-synuclein oligomer adsorption performance of the obtained porous molded article from plasma is shown in Table 2 below. The α-synuclein oligomer removal rate of the porous molded article was 30% or more. It was revealed that the obtained porous molded article efficiently removed α-synuclein oligomers from plasma.

[0069] Example 11 A. Introduction of Allyl Groups into Porous Molded Body 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 aqueous NaOH, and 10.8 ml of dimethyl sulfoxide. The mixture was stirred at 30°C for 18 hours. After filtering the mixture, the beads were washed sequentially with 900 ml of distilled water. Titration revealed that the amount of allyl groups introduced was 132.6 (µmol) / mL of wet carrier.

[0070] [B. Activation of Cellfine GH-25 by Bromination] 17 ml of allyl-activated Cellfine GH-25 (allyl group introduction amount 132.6 μmol / ml - wet carrier) was mixed with 17 mL of 0.9 M N-bromosuccinimide (50% aqueous acetone). 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 Activated Beads. Amine groups were directly introduced onto the beads via a linker structure. In a typical procedure, coupling to the beads was achieved by bromination of the allyl group and nucleophilic substitution under basic conditions. 1 ml of bromo-activated gel (allyl group loading: 132.6 μmol / ml—wet support) was transferred to a reaction vessel containing 15.6 mg of diethylamine as a ligand dissolved in 1 ml of 50% aqueous isopropanol. The reaction mixture was stirred at 25°C for 16 hours. After filtering the reaction mixture, the beads were washed sequentially with 3 ml of isopropanol, 3 ml of distilled water, 3 ml of 1 M HCl, 3 ml of distilled water, 3 ml of 1 M NaOH, and finally 30 ml of saline. Diethylamine-loaded Sepharose 4 Fast Flow gel was obtained with an amine structure loading of 67.4 μmol / ml of wet carrier.

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

[0073] Example 12 Diethylamine-incorporated Sepharose 4 Fast Flow was obtained in the same manner as in Example 11, except that Cellfine GCL-2000F (crosslinked cellulose beads manufactured by JNC Corporation) was used as the porous shaped article, and 1 mL of 50% isopropanol 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 μmol / mL-wet carrier. The α-synuclein oligomer adsorption performance of the obtained porous shaped article from plasma is shown in Table 2 below. The removal rate of α-synuclein oligomers by the porous shaped article was 30% or more. It was revealed that the obtained porous shaped article efficiently removed α-synuclein oligomers from plasma.

[0074] Example 13 Plasova BR (polystyrene beads, manufactured by Asahi Kasei Medical Corporation) was used as the porous formed body. This porous formed body had 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 μmol / ml-wet carrier. The harmonic mean particle diameter of this porous formed body was 396 μm, and the specific surface area was 24.0 m ２ / g. The removal rate of α-synuclein oligomers from plasma using the porous shaped body was 30% or more. It was revealed that the obtained porous shaped body efficiently removes α-synuclein oligomers from plasma. Furthermore, the removal rate of α-synuclein monomers from plasma using the porous shaped body was 30% or more. It was revealed that the obtained porous shaped body efficiently removes α-synuclein monomers from plasma. Furthermore, the porous shaped body had an excellent balance between adsorption efficiency and mechanical stability, and also an excellent balance between fluidity and adsorption efficiency.

[0075] Example 14 DIAION WA20 (polystyrene beads, manufactured by Mitsubishi Chemical Corporation) was used as a porous molded body. This porous molded body contained a methylene group as a linker structure and diethylenetriamine (-NH(CH ２ CH ２ NH) ２The amount of amine structure introduced was 733.6 μmol / ml-wet carrier. The harmonic mean particle size of this porous molded body was 569 μm and the specific surface area was 2.3 m ２ / g. The removal rate of α-synuclein oligomers from plasma by the porous shaped article was 30% or more. It was revealed that the obtained porous shaped article efficiently removed α-synuclein oligomers from plasma. Furthermore, the porous shaped article 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 (polystyrene beads, manufactured by Mitsubishi Chemical Corporation) was used as the porous shaped body. This porous shaped body had 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 diameter of this porous shaped body was 582 μm. The removal rate of α-synuclein oligomers from plasma using the porous shaped body was 30% or more. It was revealed that the obtained porous shaped body efficiently removed α-synuclein oligomers from plasma. Furthermore, the porous shaped body had an excellent balance between fluidity and adsorption efficiency.

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

[0078]

[0079]

[0080] The porous molded article of the present disclosure can be suitably used for efficiently removing α-synuclein oligomers from biologically derived fluids.

Claims

1. A porous shaped body for removing α-synuclein oligomers, comprising a porous shaped body carrier having a positive charge due to an amine structure, the porous shaped body carrier having the amine structure at a concentration of 30 μmol / ml-wet carrier or more and 10,000 μmol / ml-wet carrier or less.

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

3. The porous molded body 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 or more and 6 or less.

4. The porous formed body according to claim 1 or 2, wherein the amine structure includes a quaternary amine.

5. The porous shaped body according to claim 1 or 2, wherein the porous shaped body support is cross-linked agarose, cross-linked cellulose, or a polystyrene-based polymer, or a combination thereof.

6. The porous molded body according to claim 1 or 2, wherein the porous molded body support has a harmonic mean particle size of 100 μm or more and 800 μm or less.

7. The specific surface area of ​​the porous molded carrier is 1 m ２ / g or more 100m ２ The porous formed body according to claim 1 or 2, wherein the molecular weight is 1 / g or less.

8. An α-synuclein oligomer removal system comprising: a column having a container having an inlet and outlet for a biologically derived fluid, and a porous molded body filled in the container; a biologically derived fluid inlet flow path for introducing the biologically derived fluid into the inlet; and a biologically derived fluid outlet flow path for discharging the biologically derived fluid that has passed through the column from the outlet, wherein the porous molded body is a porous molded body carrier having a positive charge due to an amine structure, the amine structure being from 30 μmol / ml-wet carrier to 10,000 μmol / ml-wet carrier.

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 flow path, and the system is configured to pass plasma separated from the blood through the column.

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