JP3246681B2 - Transthyretin adsorbent - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an adsorbent for transthyretin in body fluids, particularly blood, and a method for removing transthyretin in body fluids using the adsorbent.

[0002]

2. Description of the Related Art Familial amyloid polyneuropathy is a hereditary intractable disease that causes deposition of amyloid substances throughout the body and eventually leads to death. The causative agent of this disease is said to be atypical transthyretin. Transthyretin is a plasma protein also called prealbumin, and is known to bind to retinol binding protein in human plasma. It is known that transthyretin in plasma of patients with familial amyloid polyneuropathy is partly or wholly atypical transthyretin in which transthyretin-constituting amino acids are changed due to an abnormality in the transthyretin gene. . Since this variant transthyretin is mutated only in a very small portion of the amino acids, it cannot be easily distinguished from normal transthyretin. So, as a treatment for familial amyloid polyneuropathy,
Let's also remove atypical transthyretin by removing patient plasma and transfusing healthy human plasma instead, removing all transthyretin without distinguishing between normal and atypical transthyretin Is regularly administered to patients.

[0003] However, plasma exchange therapy has problems in economy and safety, such as the necessity of a large amount of plasma for blood transfusion and the possibility of infection of the transfused plasma with a virus or the like. In addition, plasma exchange therapy cannot be performed unless the facility is capable of stably preparing a large amount of plasma for transfusion, and the operation is complicated.

[0004]

SUMMARY OF THE INVENTION An object of the present invention is to selectively adsorb transthyretin without adsorbing other proteins in plasma, and to easily remove or remove transthyretin without the necessity of a blood transfusion or a replacement fluid. An object of the present invention is to commercialize a transthyretin adsorbent that can be collected.

[0005]

DISCLOSURE OF THE INVENTION The present inventors have conducted intensive studies to complete a transthyretin adsorbent for the above purpose, and as a result, transthyretin is one of the plasma proteins having the lowest isoelectric point. Utilizing the fact that the present invention has been made. The present invention relates to a transthyretin adsorbent having a basic functional group on the surface of a water-insoluble porous body, and a method for removing transthyretin from plasma using the adsorbent.
The gist of the present invention is as follows.
You . Diethylaminoethylstyrene, N, N-diethyl
-N-vinylphenethylamine, diethylaminoethyl
Methacrylate, dimethylaminoethyl methacrylate Rate
A hydrid containing 1 to 50% by weight of a polymer unit selected from the group consisting of
A transthyretin adsorbent, comprising a copolymer with a polymer unit having a loxyl group on the surface of a water-insoluble porous material. Diethylaminoethylstyrene, N, N-
Diethyl-N-vinylphenethylamine, diethylamido
Noethyl methacrylate, dimethylaminoethyl methacrylate
The polymer units selected from Li rate from 1 to 50 wt% free
Copolymer with a polymer unit having a hydroxyl group
A method for removing transthyretin in a body fluid, comprising flowing a body fluid to an adsorbent having a water-insoluble porous body surface, and selectively adsorbing transthyretin in the body fluid to the adsorbent.

The basic functional group referred to in the present invention includes amines and amine derivatives, and includes tertiary amines and quaternary ammoniums. Preferred examples include those having a pKa of 4.0 or more.
Further, since heparin is usually used as an anticoagulant for blood or plasma, it is more desirable that the adsorbent absorbs less heparin. From this point, a more preferable example is represented by the following equation (A).

[0007]

Embedded image

There are no particular restrictions on the substituents R1, R2 and R3, and any substituent can be provided, but any of the substituents is connected to the main chain of the water-insoluble porous body by a covalent bond. Things. For example, it may be a hydrocarbon substituent such as hydrogen, methyl group, ethyl group, propyl group, phenyl group or benzyl group, or a substituent containing an atom of a different nuclide such as methylol or ethylol. R1, R
2, R3 may be a ring having two or more,
For example, pyridine, imidazole, piperidine, pyrrole, pyrimidine and the like correspond to this.

Examples of the basic functional group by the name of a monomer include allylamine, diallylamine, N, N-dimethylallylamine, N, N-diallylpiperazine, N, N-
Diallylaniline, N, N-diallylmelamine, aminostyrene, N, N-dimethylaminostyrene, N, N-
Diethylaminostyrene, vinylpentylamine, vinylphenethylamine, N, N-dimethylvinylphenethylamine, N, N-diethylvinylphenethylamine,
N-propylvinylphenethylamine, vinylpyridine, 2-methyl-5-vinylpyridine, 2-ethyl-5
-Vinylpyridine, 2-vinylquinoline, 2-vinylimidazole, 4-vinylimidazole, vinylpyrazoline, vinylpyrazine, 4-vinylpyrimidine, vinylamine, vinylcarbazole, ethyleneimine, N-phenylethyleneimine, N, N-diethyl-N Monomers such as vinylphenethylamine, diethylaminoethyl methacrylate, dimethylaminoethyl methacrylate, and diethylaminoethylstyrene; and oligomers and polymers having any of these as a polymerization unit. Also, those obtained by quaternizing these can be used. Among these, diethylaminoethylstyrene, N, N-diethyl-N-vinylphenethylamine, diethylaminoethyl methacrylate, and dimethylaminoethyl methacrylate are more preferred.

Further, these polymerized units may be combined with other polymerizable monomers such as hydroxystyrene, hydroxymethylstyrene, vinyl alcohol, 2-hydroxyethyl acrylate, 2-hydroxyethyl methacrylate, segmented polyurethane, segmented polyester and the like. It may be a copolymer. In particular, it is preferable that the polymer has a hydroxyl group from the viewpoint of improving blood compatibility.
There is no particular limitation on the mode of bonding of the hydroxyl groups in the polymer. In particular, a copolymer with a hydroxyl-containing polymer unit containing 1 to 50% by weight of a basic functional group-containing polymer unit represented by the formula (A) is preferable. The basic functional group does not necessarily need to be present on the side chain of the polymer chain constituting the water-insoluble porous body, and may be one that forms the main chain. Such as a method of introducing a basic functional group into the water-insoluble porous body as a side chain, and a method of physically bonding the basic functional group to the surface of the water-insoluble porous body without a covalent bond when the polymer is a polymer. A method of insolubilizing a basic functional group in a water-insoluble porous material can also be selected, and these methods are easier to produce than those introduced into the main chain. The basic functional group has a high transthyretin adsorptivity, a less nonspecific adsorption of other plasma proteins, and a higher selectivity for transthyretin. Adsorbents that are insolubilized in water are more preferred. At this time, the molecular weight of the oligomer or polymer is preferably 300 or more and 1,000,000 or less, particularly preferably 1 to 1.
It was 000 or more and 100,000 or less.

The method of insolubilizing a basic functional group in a water-insoluble porous material includes a method of immersing in a liquid in which the basic functional group is dissolved or a method of coating by spraying the liquid, a method of chemically or a radiation or the like. There is a method of covalent bonding to the surface of the water-insoluble porous material by a grafting method using an electron beam, or a method of covalent bonding via a functional group on the surface of the water-insoluble porous material by a chemical method. Of these, a grafting method and a covalent bonding method via a functional group are preferable because there is no danger of elution of a basic functional group during use. When the water-insoluble porous body has a coating layer, it can be insolubilized on the surface of the coating layer.

As an example of a method for obtaining a functional group on the surface of the water-insoluble porous body or its coating layer, a cyanogen halide method, an epichlorohydrin method, a bisepoxide method, a bromoacetyl bromide method and the like are known. Specifically, an amino group,
Examples thereof include a carboxyl group, a hydroxyl group, a thiol group, an acid anhydride group, a succinylimide group, a chlorine group, an aldehyde group, an amide group, an epoxy group, and a tresyl group. Among them, an epoxy group derived by the epichlorohydrin method is particularly preferable because of its stability during heat sterilization.

If the amount of the functional group on the surface of the adsorbent is too small, the adsorbing ability is low, and if it is too large, there is a risk that blood compatibility is reduced and a basic functional group is liberated during use. It is preferably from 1 μeq / ml to 1000 meq / ml per volume including the pore volume of the water-insoluble porous body. Furthermore, a more preferable range is 10
μeq / ml or more and 10 meq / ml or less.

The water-insoluble porous material referred to in the present invention is a porous material into which a basic functional group can be introduced, regardless of whether it is an inorganic compound or an organic compound. Organic polymers are preferred because they can be controlled more easily and precisely. Examples of such polymers include polymers and copolymers of vinyl compounds such as polypropylene, polystyrene, polymethacrylate ester, polyacrylate ester, polyacrylic acid and polyvinyl alcohol, polyamide compounds such as nylon 6 or 66, and polyethylene terephthalate. And polyester-based compounds such as cellulose and plant-derived polysaccharide-based compounds such as cellulose.

The material of the porous material used in the present invention is not limited as long as it can introduce a basic functional group of 1 μeq / ml or more. In the example,
From the viewpoint of easiness of polymerization and easiness of introduction of a basic functional group, polymers and copolymers of vinyl compounds are more preferably used. Such examples include styrene, methylstyrene, diphenylethylene, ethylethylene, dimethylstyrene, vinylnaphthalene, vinylphenanthrene, vinylmesitylene, 3,4,6-trimethylstyrene,
Hydrocarbon compounds such as vinyl-2-ethylacetylene:
Chlorostyrene, methoxystyrene, bromostyrene,
Styrene derivatives such as cyanostyrene, fluorostyrene, dichlorostyrene, N, N-dimethylaminostyrene, nitrostyrene, chloromethylstyrene, trifluorostyrene, trifluoromethylstyrene and aminostyrene: acrylonitrile, α-acetoxyacrylonitrile, etc. Acrylonitrile derivatives: acrylic acid, methacrylic acid: methyl acrylate, lauryl acrylate,
Acrylic esters such as chloromethyl acrylate and ethyl acetoxy acrylate: cyclohexyl methacrylate, dimethylaminoethyl methacrylate, glycidyl methacrylate, tetrahydrofurfuryl methacrylate, hydroxyethyl methacrylate and the like: diethyl maleate, fumaric acid Diethyl: vinyl ketone such as methyl vinyl ketone and ethyl isopropenyl ketone; vinylidene compounds such as vinylidene chloride, vinylidene bromide and vinylidene cyanide: acrylamide, methacrylamide, N-butoxymethylacrylamide, N-
Phenylacrylamide, diacetone acrylamide,
Acrylamide derivatives such as N, N-dimethylaminoethylacrylamide: fatty acid vinyl derivatives such as vinyl acetate, vinyl butyrate, and vinyl caprate: thiofatty acid derivatives such as phenyl thiomethacrylate, methyl thioacrylate, and vinyl thioacetate: Vinyl succinimide, N-
Vinylpyrrolidone, N-vinylphthalimide, N-vinylcarbazolevinylfuran, 2-vinylbendfuran, vinylthiophene, vinylimidazole, methylvinylimidazole, vinylpyrazole, vinyloxazolidone, vinylthiazole, vinyltetrazole, vinylpyridine, methylvinylpyridine, There are heterocyclic vinyl compounds such as 2,4-dimethyl-6-vinyltriazine and vinylquinoline.

The crosslinkable polymerizable monomer which is a constituent unit of the adsorbent of the present invention includes divinylbenzene, divinyltoluene, divinylxylene, divinylnaphthalene, divinylethylbenzene, divinylphenanthrene, trivinylbenzene, divinyldiphenyl, divinylphenylether , Divinyldiphenylsulfide, divinyldiphenylamine, divinylsulfone, divinylketone, divinylfuran, divinylpyridine, dipyrylquinoline, di (vinylpyridinoethyl) ethylenediamine, diallyl phthalate, diallyl maleate, diallyl fumarate, diallyl succinate, carbonic acid Diallyl, diallyl oxalate, diallyl adipate, diallyl sebacate, diallyl tartrate, diallylamine, triallylamine, triallyl phosphate, tricarba Le triallyl, aconitic acid triallyl, triallyl citrate, N, N-ethylene diacrylamide, N, N-ethylene methacrylamide,
N, N-methylene dimethacrylamide, ethylene glycol methacrylate, triethylene glycol dimethacrylate, polyethylene glycol dimethacrylate,
Trimethylolpropane trimethacrylate, pentaerythritol tetramethacrylate, 1,3-butylene glycol diacrylate, 1,6-hexanediol diacrylate, trimethylpropane triacrylate, pentaerythritol tetraacrylate, triallyl isocyanurate, 1,2,5- Triacryloylhexahydro-1,3,5-triazine, diarylmelamine and the like.

Among these, particularly preferred examples are those which are chemically stable and can easily introduce a functional group, and that can form porous particles having various particle diameters and pore diameters of about 50 μm to 1000 μm. Examples include a copolymer of vinyl and triallyl isocyanurate, and a styrene-divinylbenzene copolymer. The adsorbent of the present invention has an exclusion limit molecular weight such that pores can be effectively used as a site for adsorption of transthyretin having a molecular weight of about 55,000 or transthyretin / retinol binding protein complex having a molecular weight of about 80,000. Is 10
It is preferably at least 000.

The adsorption capacity is greatly affected by the surface area. Obviously, the adsorption capacity increases when the surface area is large, and decreases when the surface area is small. Therefore, the adsorbent of the present invention preferably has a total pore surface area of 0.5 m ² / ml or more. More preferably, it is 1.0 m ² / ml or more. The surface of the adsorbent has a hydrophilic coating layer made of a polymer for controlling the adhesion of platelets on the surface of the basic adsorbent that comes into contact with blood cells in order to improve the affinity with blood. May be. The original purpose of the hydrophilic coating layer is to increase blood compatibility, and it is necessary to show the degree of blood compatibility, but it is difficult to obtain blood stably and there are differences between blood. , A common stable evaluation is difficult.
The degree of hydrophilicity can be expressed simply. If the preferred range of the degree of hydrophilicity is arbitrarily shown, the contact angle of air bubbles on a sheet-like or film-like solid surface in water at 25 ° C. is 20 ° or more.

The hydrophilic coating layer is preferably a polymer in order to prevent peeling during use. To give specific examples of the hydrophilic coating layer, if a polymer unit is exemplified by a name as a monomer, 2-hydroxyethyl acrylate, 2-hydroxypropyl acrylate, 2-hydroxyethyl methacrylate, 2-hydroxypropyl methacrylate, 2
-Hydroxybutyl methacrylate, diethylaminoethyl methacrylate, dimethylaminoethyl methacrylate, methoxydiethylene glycol methacrylate,
Acrylic acid or methacrylic acid and its derivatives such as methoxytriethylene glycol methacrylate and glycerol monomethacrylate; methoxypolyethylene glycols such as methoxytriethylene glycol; styrene derivatives such as diethylaminoethylstyrene, hydroxystyrene and hydroxymethylstyrene; vinylamine and vinyl alcohol Any monomer 1 such as a monomer having a vinyl group such as a segmented polyurethane, a segmented polyester, a mono (2-methacryloyloxyethyl) acid phosphate, and a mono (2-acryloyloxyethyl) acid phosphate; Polymers containing more than one kind, such as block copolymers, graft copolymers, and monomers having a polyethylene oxide chain and other polymerizable monomers RAFT copolymer and the like.
In particular, the polymer preferably has a hydroxyl group. There is no particular limitation on the mode of bonding of the hydroxyl groups in the polymer.

The polymer may be a homopolymer of the above polymer units or a copolymer of two or more. Further, the polymer unit having a basic functional group is 0.1 to 2
It may be a copolymer containing 0% by weight and a polymer unit having a hydroxyl group. These polymers are linear polymers,
There is no particular relation to the polymerization form such as a graft polymer or a crosslinked polymer.

The polymer coating layer can be formed by a grafting method, a precipitation method,
It may be obtained by any method such as a coating method and a covalent bonding method using a functional group on the surface of the porous body. Among them, the coating method is particularly preferable in practical use because the production operation is easy.

The amount of the coating layer thus obtained, that is, the coating amount, is 1 μg / g in terms of the weight of the coating layer per dry weight of the adsorbent in order to exhibit the effect as the coating layer.
100 mg / g or less, more preferably 10 μm
It is not less than g / g and not more than 10 mg / g. As the shape of the present adsorbent, any of a spherical shape, a granular shape, a fibrous shape, a hollow fiber shape, a flat membrane shape and the like can be effectively used, but a spherical shape or a granular shape is most preferably used from the surface of the body fluid circulation during the body fluid circulation. A spherical or granular average particle size of 10 μm to 2500 μm is easy to use, but is preferably in the range of 25 μm to 1,000 μm, and more preferably 50 μm to 600 μm.

The adsorbent of the present invention is generally used as an adsorber filled and held in a container provided with a body fluid outlet. The present adsorbent may be filled alone, mixed with another adsorbent, or laminated. When used for extracorporeal circulation, the capacity of the adsorber is suitably about 10 ml to 1000 ml. When the adsorber is used in extracorporeal circulation, there are roughly the following two directions. On the one hand, after blood taken out from the body is separated into a plasma component and a blood cell component using a centrifugal separator or a membrane-type plasma separator, the plasma component is passed through an adsorber and purified, Another method is to purify the blood taken from the body directly through the adsorber to purify the blood. Among them, the plasma purification method in which the plasma component is passed through the adsorber for purification is more preferable because the flow resistance in the adsorber is small and the total adsorption capacity can be increased. The body fluid may be passed continuously or intermittently according to the necessity of use or the condition of the equipment of the facility as necessary.

[0024]

EFFECT OF THE INVENTION Diethylaminoethylstyrene, N, N
-Diethyl-N-vinylphenethylamine, diethyla
Minoethyl methacrylate, dimethylaminoethyl meth
The polymer units selected from chestnut rate from 1 to 50 wt%
Copolymer with hydroxyl-containing polymer unit
A transthyretin adsorbent having a water-insoluble porous material on the surface thereof and a method for removing transthyretin from the body fluid using the adsorbent, thereby selecting transthyretin without adsorbing other proteins in the body fluid Adsorption of transthyretin can be performed in a versatile manner because it can be easily performed without using plasma for blood transfusion or replacement fluid.

[0025]

【Example】

Example 1 A spherical water-insoluble porous body made of polyvinyl alcohol was obtained by suspension polymerization of vinyl acetate and triallylisocyanurate. The water-insoluble porous body had an average particle diameter of 100 μm, an exclusion limit molecular weight of about 2,000,000, and a surface area by nitrogen adsorption method (BET method) of 21.7 m ² / ml. An epoxy group was introduced into this water-insoluble porous body using epichlorohydrin in dimethyl sulfoxide.
Next, in a mixed solvent of sodium hydroxide and dimethylformamide, a 20:80 (wt%) copolymer of diethylaminoethyl methacrylate and hydroxyethyl methacrylate is covalently bonded at a reaction concentration of 10% using an epoxy group, and adsorbed. Agent was obtained. The used copolymer of diethylaminoethyl methacrylate and hydroxyethyl methacrylate had a polyethylene equivalent molecular weight of about 176,000. The obtained adsorbent had an ion exchange capacity of 90.
It was 6 μeq / ml. Heparin concentration 5,000 IU
1 ml of an adsorbent was added to 6 ml of / L human plasma, and reacted at 37 ° C. for 2 hours with shaking. The concentrations of transthyretin and albumin in the plasma before and after the reaction were measured by laser nephelometry. While the concentration of transthyretin before the reaction was 28.7 mg / dl, the concentration after the reaction was 0.8 mg / dl or less, indicating a very excellent adsorptivity to transthyretin. On the other hand, the concentration of albumin before the reaction was 4.60 g / dl while that after the reaction was 4.00 g / dl, and there was almost no non-specific adsorption.

[0026]

Example 2 In the same manner as in Example 1, an adsorbent was obtained at a concentration of 1% during the insolubilization reaction using a 50:50 (wt%) copolymer of diethylaminoethyl methacrylate and hydroxyethyl methacrylate. The ion exchange capacity of the obtained adsorbent was 98.4 μeq / ml. Example 1
When the adsorption of transthyretin and albumin was measured in the same manner as described above, the concentration before adsorption was 28.7 mg /
dl, 4.61 g / dl and 1.0 mg / dl, 4.01 g / dl after adsorption, exhibiting selective adsorption to transthyretin, and The adsorption capacity was also excellent.

[0027]

Example 3 In the same manner as in Example 1, an adsorbent was obtained using a 7:93 (wt%) copolymer of diethylaminoethyl methacrylate and hydroxyethyl methacrylate. The ion exchange capacity of the obtained adsorbent is 79.5 μeq.
/ Ml. When the adsorption of transthyretin and albumin was measured in the same manner as in Example 1, the concentrations before adsorption were 30.4 mg / dl and 4.70 g / dl, respectively, whereas the concentrations after adsorption were 1.1 mg / dl. , 4.40g
/ Dl, showing selective adsorption to transthyretin, and excellent transthyretin adsorption ability.

[0028]

Example 4 In the same manner as in Example 1, an adsorbent was obtained using a 3:97 (wt%) copolymer of diethylaminoethyl methacrylate and hydroxyethyl methacrylate. When the adsorption of transthyretin and albumin was measured in the same manner as in Example 1, the concentration before adsorption was 3
0.4 mg / dl and 4.70 g / dl, whereas after adsorption it was 1.0 mg / dl and 4.30 g / dl, showing selective adsorption to transthyretin, and The transthyretin adsorption capacity was also excellent.

[0029]

[0030]

────────────────────────────────────────────────── ─── Continuation of the front page (56) References JP-A-1-135533 (JP, A) JP-A-6-7430 (JP, A) Regnault et al. , "Dye-affinity purification of transthyrthin from an unexploited by-product of human plasma chromatography, fratomography, J. graphy. 87-93 (58) Field surveyed (Int. Cl. ⁷ , DB name) A61M 1/36 545 B01J 20/22

Claims (2)

(57) [Claims] (1) diethylaminoethylstyrene, N, N-
Diethyl -N- vinyl-phenethylamine, Jiechiruami
Noethyl methacrylate, dimethylaminoethyl methacrylate
Containing 1 to 50% by weight of a polymer unit selected from
Copolymer with a polymer unit having a hydroxyl group
The transthyretin adsorbent, characterized in that it comprises water-insoluble porous surface. 2. Diethylaminoethylstyrene, N, N-
Diethyl -N- vinyl-phenethylamine, Jiechiruami
Noethyl methacrylate, dimethylaminoethyl methacrylate
Containing 1 to 50% by weight of a polymer unit selected from
Copolymer with a polymer unit having a hydroxyl group
A method for removing transthyretin in a body fluid, comprising flowing a body fluid to an adsorbent having a water-insoluble porous body surface, and selectively adsorbing transthyretin in the body fluid to the adsorbent.