JP2013032295A - Tau protein adsorbent material, and tau protein adsorption/removal system - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a tau protein adsorption/removal system which can adsorb and remove tau protein, which is believed to be a causative substance for neurodegeneration in various types of tauopathies including Alzheimer's disease, selectively from a liquid including such as a body fluid.SOLUTION: The tau protein adsorption/removal system can remove tau protein from a liquid including such as a body fluid by treating a liquid including tau protein using the adsorbent material obtained by immobilizing a ligand, having a high affinity for tau protein, onto a porous water-insoluble carrier having an exclusion limit molecular weight of 20,000 or more.

Description

  The present invention relates to an adsorbent for tau protein, an adsorption removal system using an adsorbent column having affinity for tau protein, and a method for adsorbing and removing tau protein from a liquid.

  By treating a liquid such as a bodily fluid that can contain tau protein using the present invention, it is possible to clean the liquid, and for various diseases that are considered to be related to the accumulation of tau protein in the body. The present invention relates to a tau protein adsorption removal system that can be expected to use the present invention as a treatment or prevention method.

  Since tau protein was identified in 1975, research has been conducted on methods for evaluating tau protein, forms of abnormally phosphorylated tau protein, production of model animals such as transgenic mice overexpressing mutant tau (Patent Literature) 1). However, the detailed form of tau protein and the pathophysiology and treatment of tauopathy are still unclear, and this is a field where future research trends are expected. It is.

  Tau protein is a microtubule-binding protein having a molecular weight of about 50,000 in nerve axons, and promotes or stabilizes microtubule polymerization. Tau protein has six isoforms due to alternative splicing of exon 2, exon 3, and exon 10. Among these, those having three microtubule binding regions repeated on the C-end side are called 3 repeat tau, and those having 4 are called 4 repeat tau, and this difference is caused by whether or not exon 10 is inserted. The gene expression varies depending on the species and age, and only 3 repeat tau is expressed in the human fetal brain, but 6 types of isoforms are observed in the adult brain (Non-patent Document 1).

  Among neurodegenerative diseases that exhibit dementia, which is considered to be related to tau protein, a group of diseases having clinical symptoms like Pick's disease are frontotemporal dementia (FTD) and frontal lobe dementia (FLD). It was proposed to call it. These disease groups are preceded by behavioral abnormalities associated with personality changes and personality changes, and are characterized by the occurrence of memory impairment and spatial cognitive impairment later. It is characterized by the appearance of cerebral atrophy, neuronal loss, intracellular inclusion bodies and neurofibrillary tangles containing tau protein, and no amyloid β protein deposition or senile plaque formation. In addition, many diseases such as progressive supranuclear palsy (PSP), cortical basal ganglia degeneration, parkinsonism, Creutzfeldt-Jakob disease have been known to develop due to the accumulation of this abnormal tau protein, The entire state in which these abnormal tau proteins accumulate is called tauopathy (Non-patent Document 2).

  Among them, Alzheimer's disease is a neurodegenerative disease mainly consisting of progressive cognitive symptoms that develop from early age to old age. Its pathological feature is the emergence of neurofibrillary tangles (NFT), which is the accumulated state of paired helical filaments (PHF), which is composed of senile plaques composed of amyloid β protein and abnormally phosphorylated tau protein. . It has been shown that neurofibrillary tangles correlate well with the degree of dementia, and that the accumulation of abnormal phosphorylated tau protein, which is the main body, is closely related to neuronal degeneration and neuronal cell death (Non-patent literature) 3).

  Traditionally, tau protein accumulation tended to be perceived as a secondary change rather than a disease-specific pathological change, a result after neuronal degeneration. However, a tau gene mutation was discovered in 1998 in a family with familial frontotemporal dementia (FTDP-17) with parkinsonism linked to chromosome 17, and tau protein is responsible for neurodegeneration The possibility came to be considered (Non-Patent Document 4). On the other hand, even today, there is no fundamental treatment for many tauopathy including Alzheimer's disease caused by such accumulation of tau protein, and it is released into body fluids before it accumulates on nerve cells. There was no means to remove the existing tau protein by adsorption removal.

JP 2000-253774 A

Neuron, 1998; 21: 955-958. Clinical laboratory, 2006; 50: 1121-1129. CLINICIAN, 2001; 498 (48): 31-35. Nature, 1998; 393: 702-705.

  Provided is a tau protein adsorption / removal system capable of selectively adsorbing and removing tau protein, which is considered to be a causative agent of neurodegeneration in various tauopathy including Alzheimer's disease, from liquids containing tau protein such as body fluids. about.

  As a result of intensive studies in view of these problems, the present inventors have removed tau protein from a liquid containing body fluid by treating the liquid containing tau protein with an adsorbent having affinity for tau protein. It has been possible to provide a tau protein adsorption removal system.

  According to the present invention, a tau protein adsorption / removal system includes a tau protein adsorption / removal column in which a tau protein adsorbent is packed in a container having a liquid inlet and outlet.

  The tau protein adsorbent in the present invention is formed by immobilizing a ligand having high affinity for tau protein on a porous water-insoluble carrier having an exclusion limit molecular weight of 20,000 or more.

  The present invention also relates to a tau protein adsorbent in which the ligand having high affinity for tau protein is dextran sulfate.

  The present invention also relates to a method for adsorbing and removing tau protein, which comprises contacting a tau protein adsorbent with a liquid such as a body fluid containing tau protein.

  The present invention is a tau protein adsorption / removal system capable of treating a liquid containing tau protein, and a liquid containing tau protein such as body fluid, which is considered to be a cause of neurodegeneration of many tauopathy including Alzheimer's disease. It becomes possible to remove from.

  By using the present invention, treatment or prevention of various diseases that are considered to be related to accumulation of tau protein in the body can be expected.

  Further, according to the present invention, it is possible to clean the liquid by treating a liquid such as a body fluid that may contain tau protein in the present invention.

  Tau protein is a microtubule-binding protein that binds to microtubules and acts as a hinge between tubulin and tubulin. It is currently known and has six isoforms. Exists. There is also a protein called abnormally phosphorylated tau protein that is excessively phosphorylated by some mechanism. Tau protein may be modified with certain functional groups in the body or outside the body for the purpose of detecting it or enhancing the binding ability with other molecules. Alternatively, it is possible that abnormal tau protein may be produced due to mutations in genes and the like. The tau protein adsorbed and removed according to the present invention is at least one of the above-mentioned tau proteins, but any tau protein-like substance can be called a tau protein-like substance in light of the common general knowledge of those skilled in the art. It is not limited to the various tau proteins described above.

  The water-insoluble carrier in the present invention is made of a material that is solid at normal temperature and pressure and has extremely low solubility in water. Examples of shapes include, for example, granular, plate-like, fibrous, hollow fiber, and nonwoven fabric However, it is not limited to these, and the size is not particularly limited.

  Examples of water-insoluble carriers in the present invention include inorganic carriers such as glass beads and silica gel, synthetic polymer carriers such as crosslinked polyvinyl alcohol, crosslinked polyacrylate, crosslinked polyacrylamide and crosslinked polystyrene, synthetic polymer carriers composed of two or more types, crystals Representative examples include organic carriers composed of polysaccharides such as hydrophilic cellulose, crosslinked cellulose, crosslinked agarose, and crosslinked dextrin, and organic-organic and organic-inorganic composite carriers obtained by combinations thereof. Preferably, the water-insoluble carrier is hydrophilic, or if non-specific adsorption is relatively small and tau protein adsorption selectivity is good.

  In addition, the water-insoluble carrier in the present invention is preferably a carrier having a porous structure having a large number of pores of appropriate sizes. Or, a carrier having a three-dimensional structure such as a polymer network, a monolith carrier in which at least a part of holes formed in the carrier penetrates, and the like are not particularly limited thereto. Absent.

  Furthermore, the water-insoluble carrier in the present invention preferably has an exclusion limit molecular weight such that the substance to be adsorbed can enter the pores with a certain degree of probability. The exclusion limit molecular weight as used herein is generally defined by pores in gel permeation chromatography as described in the books (for example, Hiroyuki Hatano, Toshihiko Hanai, experimental high performance liquid chromatograph, chemical doujin etc.) This refers to the molecular weight of a molecule having the smallest molecular weight that cannot enter the inside, and is generally measured using a globular protein or the like. In the present invention, the exclusion limit molecular weight is 20,000 or more, and in order to suppress the invasion of proteins other than tau protein or modified tau protein-like substance, 40,000 or more is preferable, more preferably 6 More than ten thousand. In addition, when cerebrospinal fluid or plasma, serum or the like is used as a body fluid, there is no upper limit on the exclusion limit molecular weight, but when blood or the like is used as a body fluid, platelet adhesion tends to increase when the exclusion limit molecular weight exceeds 5 million. Therefore, it is desirable that the exclusion limit molecular weight is 5 million or less. When the exclusion limit molecular weight is 5 million or less, sufficient performance can be exhibited even when the adsorbent of the present invention is used in a direct blood perfusion (DHP) type blood purification system.

  The ligand immobilized on the porous water-insoluble carrier in the present invention is preferably one having affinity for tau protein, and is immobilized on the water-insoluble carrier by a chemical reaction mechanism using a catalyst or an immobilized reactant. That is possible. That is, examples of the ligand to be immobilized include antibodies and peptides having affinity for tau protein, and low molecular weight substances. In particular, it is preferable to use dextran sulfate as the low molecular weight substance.

  Liquids that can be treated using the present invention include cerebrospinal fluid, blood, plasma, serum, ascites, lymph fluid, intra-articular fluid, bone marrow fluid, fraction components obtained therefrom, and other biological sources This refers to a liquid such as a body fluid containing tau protein, such as a liquid component, but is not limited thereto. That is, as long as it is a liquid that may contain tau protein, it may be water or a buffer solution, and liquid components derived from living organisms are also targeted.

  As a preferred form of the tau protein adsorption removal method of the present invention, a method of adsorbing and removing tau protein from a liquid by bringing a tau protein adsorbent into contact with a liquid such as a body fluid containing tau protein is used. Examples of the method include a method in which a tau protein adsorbent and a liquid are placed in one container, left standing or shaken for a certain period of time, and then the tau protein adsorbent and the liquid are separated.

  Alternatively, a tau protein adsorption / removal system comprising at least one tau protein adsorption / removal column in which one or more tau protein adsorbents are packed in a container having an inlet and an outlet for the liquid is constructed, and the liquid is allowed to flow therein. The method of making it flow out from a system after making it contact with a tau protein adsorption material is mentioned.

  In addition, the tau protein adsorption / removal system of the present invention may incorporate a device for more effectively adsorbing and removing tau protein in addition to at least one tau protein adsorption / removal column. For example, a pump for flowing the liquid may be disposed, or one or more tau protein adsorption / removal columns made of a dialysis membrane or two kinds of tau protein adsorbents may be incorporated in parallel or in series. .

  Hereinafter, although the detail of this invention is further demonstrated in a specific Example, the usage form of this invention is not limited only to a following example.

  1 L of a porous water-insoluble carrier (average particle size 150 μm, globular protein exclusion limit molecular weight of about 5,000,000) was reacted with 0.15 L of epichlorohydrin in an aqueous sodium hydroxide solution, and then 500 g of dextran sulfate was added in the aqueous solution. The immobilization reaction was treated with monoethanolamine to obtain a tau protein adsorbent (exclusion limit molecular weight 5 million, dextran sulfate fixed amount: 0.32 μmol / g-wet weight).

  Pseudoserum (4% BSA / PBS buffer) adjusted so that the concentration of tau protein (recombinant Human Tau-441, manufactured by rPeptide) is about 400 pg / mL with respect to 0.2 mL of the obtained tau protein adsorbent. 1.6 mL was added and shaken at 37 ° C. for 2 hours, and the tau protein concentration of the supernatant was measured by a commercially available ELISA (Finoscalar hTAU, manufactured by Nipro Corporation). As a result, the tau protein concentration decreased from 400 pg / mL to 80.6 pg / mL, and the adsorption rate was 80%.

  If the tau protein adsorption / removal system of the present invention is used, it can be expected to be used as a treatment or prevention method for various diseases that are considered to be related to the accumulation of tau protein in the body. Further, by treating a liquid such as a body fluid that can contain tau protein with the tau protein adsorption / removal system of the present invention, the liquid can be purified.

Claims (4)

A tau protein adsorbent obtained by immobilizing a ligand having high affinity for tau protein on a porous water-insoluble carrier having an exclusion limit molecular weight of 20,000 or more. The tau protein adsorbent according to claim 1, wherein the ligand having high affinity for tau protein is dextran sulfate. A tau protein adsorbing material according to claim 1 or 2 and a liquid such as a body fluid containing tau protein are brought into contact with each other. A tau protein adsorption / removal system comprising a tau protein adsorption / removal column filled with the tau protein adsorbent according to claim 1 or 2 in a container having a liquid inlet and outlet.