JPH06142198A - Antiplatelet antibody adsorbing material - Google Patents

Abstract

PURPOSE:To provide the antiplatelet antibody-adsorbing material which can remove the antiplatelet antibodies generated from a certain cause by an exocirculation treatment method of blood, etc., and is formed by immobilizing the peptide or modified peptide having bondability with the antiplatelet antibodies to a non-woven fabric. CONSTITUTION:This antiplatelet antibody-adsorbing material and the process for production of the material are formed by and consist of immobilizing the peptide or modified peptide having the bondability with the antiplatelet antibodies with the non-woven fabric having an amino group or imino group and 1 to 30mum average fiber diameter.

Description

Detailed Description of the Invention

[0001]

The present invention relates to an antiplatelet antibody which is an autoantibody produced in blood for some reason, an organic compound (modified peptide) containing a peptide containing a specific amino acid sequence or a peptide region thereof. The present invention relates to an anti-platelet antibody adsorbent capable of removing anti-platelet antibody characterized by binding to anti-platelet antibody in blood by performing extracorporeal circulation treatment of blood using a non-woven fabric having immobilized . The present invention is mainly due to a decrease in the number of platelets due to the presence of antiplatelet antibodies, which causes idiopathic thrombocytopenic purpura (hereinafter IT).
It is very useful in the treatment of diseases such as P).

[0002]

ITP is a type of autoimmune disease characterized by shortened platelet lifespan and increased antiplatelet antibody, which is a platelet-binding immunoglobulin. In the bone marrow, the number of megakaryocytes is normal or increased. Immune thrombocytopenia that does not show findings suggesting the presence of a disease does not have a causative disease such as collagen disease, lymphoproliferative disease, or drug allergy.

ITP is divided into an acute type and a chronic type. The acute form is more common in children, and bleeding symptoms are severe but relatively easy to cure. Antecedent infections such as upper respiratory tract infection and viral infection are often found 3 weeks before the onset. Since thrombocytopenia is observed in the convalescent stage of infection, there is a strong possibility of an immune complex disease in which the formed immune complex binds to the platelet membrane Fc receptor and platelets are nonspecifically destroyed. On the other hand, the chronic type has a weak bleeding symptom but persists for a long period of time, and it is considered to be an autoimmune disease due to antiplatelet antibody, which is common among adults, especially in women in their 20s.

Although there are individual differences in the symptoms observed due to the decrease in the number of platelets due to the antiplatelet antibody, in general, bleeding symptoms are not generally observed when the number of platelets is 50,000 / μl or more,
At 30,000 to 50,000 cells / μl, easy bleeding during trauma, ecchymosis, 10,000 to 30,000 cells / μl
Then, the appearance of purpura on the exposed area, frequent menses, 10000 /
Hemouria, irregular genital bleeding, epistaxis, and gingival bleeding are observed at μl or less, and intracranial bleeding and gastrointestinal bleeding are often the direct causes of death.

In the treatment of ITP, administration of a corticosteroid agent, removal of the spleen, administration of an immunosuppressive agent and the like are applied depending on the degree of the symptoms. However, it has been confirmed that there are many cases in which the platelet count is not effectively recovered even after such drug therapy or splenectomy. The mechanism for the development of most autoimmune diseases including ITP has not yet been fully elucidated, and the absolute treatment method for such patients has not been established. There is.

As a method for treating such autoimmune diseases,
Various attempts have been made to adsorb and remove autoantibodies by extracorporeal circulation of blood using an immunoadsorption column. For example, application of a column in which protein A having a binding property with immunoglobulin is immobilized on a carrier is also under study. However, protein A is not very useful because it is expensive, non-specific adsorption of other useful components cannot be suppressed, and cases in which various side effects occur are confirmed. In addition, various columns for immobilizing antibodies against substances to be removed have been investigated, but safety, storage stability problems, immobilization that makes antigen-antibody reaction effective, and sterilization methods are limited. Therefore, there are many problems in using it as medical equipment.

[0007]

DISCLOSURE OF THE INVENTION The present invention provides a non-woven fabric on which a peptide or modified peptide that recognizes anti-platelet antibodies in blood and has binding properties is immobilized, and a column packed with the non-woven fabric is used. The main purpose is to perform extracorporeal circulation of blood. Thus, by specifically binding and removing the antiplatelet antibody, it is intended to treat diseases such as ITP.

[0008]

Means for Solving the Problems The present invention, which has achieved the above objects, uses a non-woven fabric having a peptide or modified peptide having an amino acid sequence capable of forming an antigenic determinant of an antiplatelet antibody immobilized thereon as a blood purification adsorbent. The gist is the removal of antiplatelet antibodies by use in circulation therapy.

In general, antiplatelet antibody in ITP patients is a complex of glycoproteins present on the surface of platelet membrane G
It is known that it is often an antibody against P2b / 3a or GP1b / 9. The peptide region immobilized in the present invention is obtained by predicting and screening a region that can be an antigenic determinant from the primary structure of the above-mentioned platelet membrane glycoprotein by various methods.

GP2b and GP3a form a complex in bone marrow megakaryocytes or their progenitor cells in the bone marrow. GP2b and GP3a form a 1: 1 complex by a non-covalent bond on the surface of the platelet membrane. 55 in GP2b
It is presumed that the portion of the 8th to 747th amino acid residue and the portion of the 114th to 303rd amino acid residue in GP3a are associated (medical history,
160, 681, 1992). GP2b / 3a is the most abundant glycoprotein on the surface of platelet membranes.

GP1b is composed of an α chain and a β chain, and is considered to non-covalently bind to GP9 on the surface of the platelet membrane to form a complex (Medical Ayumi, 160, 6).
77, 1992). GP1b / 9 is a von Willebrand in which inactive platelets are present in the subendothelium
It is a membrane receptor for recognizing factors and is considered to be important as a molecule that controls the very early stage of platelet thrombus formation.

GP3a is a glycoprotein consisting of a single polypeptide having a molecular weight of about 105 kilodalton, and its cDNA has already been cloned (J. Biol.
Chem. , 262, 3939, 1987). It is composed of 762 amino acid residues and 26 at the N-terminal.
There is a signal peptide consisting of amino acid residues. The extracellular portion has 689 amino acids, the transmembrane domain has 29 amino acids, and the cytoplasm has 41 amino acids. 460 to 62
33 to 38 in the region consisting of the 7th amino acid residue
There are four cysteine-rich domain repeats consisting of 4 amino acids.

GP2b has a molecular weight of about 140 kilodaltons and is composed of an H chain of about 125 kilodaltons and an L chain of about 25 kilodaltons, which are linked by SS bonds. It consists of 1039 amino acid residues and its sequence has been determined (J. Biol. Chem., 2
62, 8476, 1987). There is a signal peptide consisting of 871 in the H chain, 137 in the L chain and 30 amino acid residues at the N-terminal part. Transmembrane domain is L
It is present in the chain and is composed of 26 hydrophobic amino acids.

GP1b / 9 is GP1b α chain, β chain, GP
Von Willebr composed of 3 subunits of 9 and inactivated platelets in subendothelial tissue
It is considered to be important as a molecule receptor that recognizes and factor and a molecule that controls the very early stage of platelet thrombus formation. The α chain and the β chain are linked by SS bonds, and the molecular weights are 140 kilodalton and 24 kilodalton, respectively. GP9 is 17 to 18 kilodaltons and is considered to be non-covalently bound to GP1b (Medical Ayumi, 160, 677, 1992). The amino acid sequence of each of them was determined, and the number of α chains was 610 (Proc. Natl. Acad. Sci. USA, 8
4,5615, 1987), 181 β chains (Pro
c. Natl. Acad. Sci. USA, 85, 21
35, 1988) and 160 GP9 (Proc. Na.
tl. Acad. Sci. USA, 86, 6773, 1
989).

As an example of the most abundant peptide having immunogenicity against the antiplatelet antibody for each glycoprotein described above, GP2b / 3a, for example, Pro3 corresponding to the 68th to 78th positions in the amino acid sequence of GP3a. -Leu-Ser-Asp-Lys-Gly-
Ser-Gly-Asp-Ser-Ser, 349th to 364th Gly-Lys-Ile-Arg-Se.
r-Lys-Val-Glu-Leu-Glu-Val
-Arg-Asp-Leu-Pro-Glu, 475th to 490th Glu-Glu-Asp-Tyr-A
rg-Pro-Ser-Gln-Gln-Asp-Gl
u-Cys-Ser-Pro-Arg-Glu, 619
To 623rd Lys-Phe-Asp-Arg-
Glu, 741-744th Thr-Ala-A
Five regions of sn-Asn are regions confirmed by enzyme immunoassay (hereinafter referred to as ELISA method). All of these regions are regions having relatively strong hydrophilicity and are presumed to be regions existing on the GP3a molecule surface. For GP2b, Glu-Thr
-Arg-Asn-Val-Gly-Ser (106th to 112th), Glu-Lys-Thr-Glu-
Glu-Ala-Glu-Lys-Thr (148th to 156th), Glu-Pro-Glu-Gln-P
Three regions of ro-Ser-Arg-Leu (896th to 903th) are immunogenic to the antiplatelet antibody. These are also highly hydrophilic regions and are presumed to exist on the surface of the molecule. Also with respect to GP1b / 9, several peptides have been confirmed to have immunogenicity against antiplatelet antibodies.

The region shown above is considered to be the minimum region required for binding with antiplatelet antibody. When the antiplatelet antibody is actually removed using such a peptide, it is necessary to reproduce the state close to the reaction mode with the antiplatelet antibody present in blood. Further, it is often necessary to consider the stability and hydration of the structure when present in blood, or the increase in solubility in an aqueous solvent when immobilized on a carrier.

Therefore, one or both end regions of the above-mentioned peptide region is extended according to the amino acid sequence of each protein, or several amino acids such as lysine for increasing water solubility are added to the end. It is preferable to bind or to bind an amino acid such as cysteine to the terminal to facilitate immobilization on the carrier. The total number of amino acid residues in the peptide or modified peptide actually used is preferably 60 or less, but more preferably 30 or less considering the difficulty of synthesis, cost, stability and the like. Furthermore, considering the ease of formation of higher-order structures required to express immunogenicity, etc., 10 to 2
It can be said that up to 5 are optimal.

In consideration of the immobilization reaction and stability,
For example, it is also preferable to use an organic compound obtained by modifying the above-mentioned peptide by amidating the N-terminal or introducing an appropriate functional group at the terminal. In addition, multiple peptides
_{(CH 2) n -B- (A} , B represents NH or CO)
It is possible to further enhance the binding to the antiplatelet antibody by using a side chain as shown in (4). In this case, the value of n is preferably 1 to 20, and more preferably 3 to 10.

The method of peptide synthesis is not particularly limited, but it is easier to apply the solid phase synthesis method than the liquid phase synthesis method. In this case, an amino acid corresponding to the C-terminal of the peptide to be synthesized is bound to a support that is insoluble in an organic solvent, and corresponding amino acids with functional groups such as α-amino groups other than α-carboxyl groups protected in the N-terminal direction are sequentially condensed. After binding by reaction, the peptide chain is extended by alternately repeating the reaction of removing the protecting group after binding.

After obtaining the desired peptide, the peptide chain is cleaved from the support and deprotected. Hydrogen fluoride is often used for this, but in terms of safety and ease of handling, trifluoromethanesulfonic acid (hereinafter TFM) is used.
It is suitable to use SA). After deprotection by reacting with thioanisole and 1,2-ethanedithiol in TFMSA, trifluoroacetic acid (hereinafter referred to as TF
The peptide is recovered by cleaving from the support according to (A). The crude peptide is obtained by freeze-drying this.

The above crude peptide is a high performance liquid chromatography using a reverse phase column (hereinafter referred to as HPLC).
The product is collected and purified. The HPLC conditions are usually optimized based on the system used for protein purification. A fraction corresponding to the obtained chromatographic peak is collected and freeze-dried. Molecular weight analysis of the obtained purified peptide fraction by mass spectrometry,
Identification is performed by amino acid composition analysis or amino acid sequence analysis.

The type of non-woven fabric used for immobilizing the above peptide or modified peptide is not particularly limited, but when it is used for blood purification, it may be used as a complement system or coagulation system upon contact with components in blood. Polyethylene terephthalate (PE
T), polypropylene (PP), polyethylene (P
E), polyamide, cellulose (cotton), rayon and the like can be mentioned, but PET, PE, cotton or rayon are preferable in consideration of easiness of modification and strength retention after modification.

There are various methods for introducing an amino group or an imino group into the nonwoven fabric. For example PP, PE
In this case, there is a method for producing a nonwoven fabric by copolymerizing a vinyl compound having an amino group or an imino group in advance, for example, a compound such as dialkylethyl acrylate or dialkylaminopropyl acrylate represented by Chemical formula 1 or Chemical formula 2. Further, there is a method of irradiating these materials with an electron beam, an ultraviolet ray or ozone to generate radicals or ions, and graft polymerizing a vinyl compound having an amino group or an imino group as shown in Chemical formula 1 or Chemical formula 2.
In this case, other vinyl monomers may coexist.

[0024]

[Chemical 1]

[0025]

[Chemical 2] In Chemical formula 1 and Chemical formula 2, R ₁ is a hydrogen atom, a methyl group, or R
_2, R ₃ may be the same or different. Each represents a hydrogen atom or an alkyl group having 1 to 5 carbon atoms, and n represents an integer of 2 to 5.

In the case of cotton or rayon, it may be oxidized by periodic acid to cleave its glycol moiety to introduce an aldehyde group, and this may be oxidized to introduce a carbonyl group, or the aldehyde group may be introduced. It is preferable to react a compound having at least one primary amino group such as ammonia, ethylenediamine, hexamethylenediamine, or polyethyleneimine to form a Schiff base, and reduce the Schiff base to introduce an amino group or an imino group. . When using polyethyleneimine, the molecular weight is 100 to 10
It is preferably 0,000, more preferably 200 to 100,000.

Further, as a method for introducing an amino group or an imino group by electron beam irradiation, a vinyl compound having an amino group or an imino group as shown in Chemical formula 1 or Chemical formula 2 is dissolved directly or after dissolving in a suitable solvent and drying. There is a method of irradiating a line. At this time, if the boiling point of the vinyl compound is low, it is difficult to control the graft amount by evaporating under a dry condition. In this case, it is preferable to mix with a high-boiling compound and apply it to suppress evaporation. As the high boiling point solvent to be mixed, polyhydric alcohols such as ethylene glycol, diethylene glycol, polyethylene glycol having a degree of polymerization of 10 or less, and glycerin are particularly preferable.

Further, in order to increase the grafting efficiency of the vinyl compound having an amino group or an imino group exemplified in Chemical formulas 1 and 2, it is preferable to use it in combination with a polyfunctional crosslinkable vinyl compound. As these crosslinkable vinyl compounds, in addition to monomers having a plurality of vinyl groups such as methylenebisacrylamide, trimethylolpropane diacrylate, trimethylolpropane triacrylate, tetramethylolmethane tetraacrylate, triallyl isocyanurate, Examples thereof include compounds represented by the general formula:

Among these crosslinkable compounds, the use of the chemical formula 3 has the best introduction rate of the amino group or imino group, and the content of the amino group or imino group can be freely controlled. R ₁ , R ₂ , and R 3 in Chemical formula ₃ each represent a hydrogen atom or a methyl group. N is 1 to 3
Indicates an integer of 00.

[0030]

[Chemical 3]

Further, the chemical formula 3 and an amino group or imino group having no vinyl group such as polyethyleneimine, poly (dimethylaminopropylacrylamide) and poly (diethylaminoethyl acrylate), poly (diethylaminomethacrylate) or a derivative thereof are added. It is possible to introduce amino groups or imino groups by grafting by using a mixture with a polymer having a high concentration. The value of n in Chemical formula 3 is preferably 5 to 150, more preferably 10 to 70.

The concentration of the solution for applying these compounds is 0.1 to 20%, preferably 0.5 to 10%.
%, Any solvent can be used as long as it dissolves both the crosslinkable compound and the compound containing an amino group or an imino group, but water, methanol, ethanol,
Methylene chloride, chloroform, acetone, dioxane,
Tetrahydrofuran or a mixed solvent thereof can be used. The mixing ratio of the compound having an amino group or imino group and the crosslinkable compound is 50: 1 to 1:50, preferably 30: 1 to 1:30.

Various studies were conducted on the method of introducing an amino group or an imino group into a non-woven fabric. As a result, a polymer or polyethylene obtained by containing 10 mol% or more of the chemical formula 1 and chemical formula 2 units and copolymerizing with another vinyl compound. A method of mixing imine or a derivative thereof and the compound represented by Chemical formula 3 and applying the mixture to a nonwoven fabric and then grafting by electron beam irradiation is particularly preferable because it is simple and does not cause deterioration such as strength reduction.
The material of the non-woven fabric used in this case is PET, P
E, rayon and cotton are preferable, and PET is particularly preferable. The irradiation dose is preferably 1 to 20 Mrad, more preferably 2 to 10 Mrad.

The content of the amino group or imino group thus introduced is 1 μeq / g to 5 meq / g, preferably 10 μeq / g to 3 meq / g, and more preferably 30 μeq / g to 2 meq / g. The residual amount of the amino group or imino group thus introduced after immobilization of the peptide is 0.1 μeq / g to 1 meq / g, preferably 0.5 μeq / g in order to suppress adhesion of blood cell components such as platelets. g to 0.5 meq /
g, more preferably 1 μeq / g to 0.3 meq
/ G.

When the amino group or the imino group is introduced from the compound represented by the chemical formula 3 and the polymer having the amino group or the imino group as described above, it may be due to the synergistic effect of the amino group and polyethylene glycol, such as platelets and leukocytes. Less sticky, blood complement activity and coagulation factor activity are also suppressed.When adsorbing and removing antiplatelet antibody, plasma and blood cell components are separated, and then antibody is adsorbed from plasma to remix plasma and blood cell components. Antibodies can be adsorbed in one step by whole blood treatment without the need to return to the patient.

There are various methods for immobilizing the above peptides having the binding property to the antiplatelet antibody to the non-woven fabric into which the amino group or imino group is introduced. In this case, the peptide or modified peptide used as the ligand is a relatively stable and low-molecular weight substance, and the immobilization reaction conditions are less restricted than in the case of immobilizing a high-molecular-weight protein such as an enzyme or an antibody. That is also one of the advantageous factors. Therefore, the immobilization method is not particularly limited, but the following method is preferable.

Usually, for the purpose of improving the adsorption efficiency and binding property of the antibody by reducing the steric hindrance and suppressing non-specific adsorption, a hydrophilic spacer is introduced to immobilize the peptides at the end thereof. Preferably.
As the hydrophilic spacer, one that can utilize the introduced amino group or imino group is preferable, and it is preferable to use polyethylene glycol having a carboxyl group or a glycidyl group at both ends. In this case, the molecular weight of polyethylene glycol is 100 to 20000, preferably 200 to 10000, and more preferably 500 to 5000.

When polyethylene glycol having carboxyl groups at both ends (hereinafter referred to as PEO acid) is used,
First, an amino group or imino group of the non-woven fabric is bound with an amide bond using a condensing agent such as carbodiimide, and then the other carboxyl group and the amino group end of the peptide are condensed in the presence of a condensing agent to form an amide group. Perform the immobilization.

When polyethylene glycol having glycidyl groups at both ends is used, a spacer is first introduced by reacting the glycidyl group at one end with the amino group of the nonwoven fabric, and then the glycidyl group at the other end and the amino group of the peptide. Immobilization is performed by reacting with a group.

However, when an amino group or an imino group is introduced by using the derivative polymer obtained from Chemical Formula 1 or Chemical Formula 2 and Chemical Formula 3, the grafted derivative polymer itself plays a role of a hydrophilic spacer, This is also preferable. That is, immobilization by condensing the amino group or imino group of these grafted derivative polymers and the carboxyl group end of the peptide by the above-mentioned method is advantageous as compared with other methods.

[0041]

EXAMPLES The method for introducing the peptides of the present invention into the non-woven fabric into which amino groups have been introduced is not particularly limited as long as it is based on the above description. The present invention will be described below with reference to examples.

<Example 1> Lys-Lys-Pro-
Leu-Ser-Asp-Lys-Gly-Ser-G
Immobilization of ly-Asp-Ser-Ser on a non-woven fabric (1) Synthesis of peptide Ly in which two lysine residues are bound to the N-terminal of the 68th to 78th region having immunogenicity in GP3a
s-Lys-Pro-Leu-Ser-Asp-Lys
The peptide having the sequence of -Gly-Ser-Gly-Asp-Ser-Ser was synthesized by a solid phase synthesis method using a peptide synthesizer Model 430A (manufactured by Applied Biosystems). PAM serine (t-Boc-), which is a support having C-terminal serine bound thereto.
L-Ser (Bzl)) 0.5 mmol (manufactured by Applied Biosystems) was used to repeat the deprotection reaction and the condensation reaction in the order of the N-terminal in accordance with the synthetic program published in the above peptide synthesizer to repeat the peptide chain. Extended. That is, the protecting group t-butoxycarbonyl group was removed with TFA and dichloromethane (hereinafter referred to as DCM), washed with DCM, neutralized with diisopropylethylamine and DCC, and then washed with dimethylformamide (hereinafter referred to as DMF). , DM
The operation of carrying out the condensation reaction with F and washing with DCM was repeated. The amino acid is t-Boc-L-Lys (Cl-Z),
t-Boc-L-Ser (Bzl), t-Boc-L-
Asp (OBzl), t-Boc-L-Gly, t-B
_{oc-L-Leu · H 2} O, t-Boc-L-Pro
2.0 (both manufactured by Applied Biosystems)
A mmol cartridge was used.

(2) Deprotection group, cleavage of peptide chain 1 g of thioanisole was added to 1 g of the support on which the above reaction was completed.
Add 1,0.5 ml of 1,1,2-ethanedithiol to 10
After stirring for 10 minutes, 10 ml of TFA was added while cooling with ice water, and the mixture was stirred for 10 minutes. Further, 1 ml of TFMSA was added and the mixture was stirred at room temperature for 30 minutes. Pre-cooled diethyl ether is added to this until it does not appear, and the mixture is stirred, filtered while washing with diethyl ether using a medium-pore glass filter, and TFA is added to dissolve the peptide in ether. Collected. The peptide in ether was filtered through a fine-hole glass filter, the peptide on the glass filter was dissolved in 2N acetic acid, and freeze-dried to obtain a crude peptide.

(3) Purification of Peptide The above crude peptide was dissolved again in 2N acetic acid to give a mixture of 0.
The solution filtered with a 2 μm membrane filter is HPL
Subjected to C. For HPLC, Model 130A system (manufactured by Applied Biosystems) was used, and for the column, reverse phase Aquapore Prep-10, C8 (manufactured by Applied Biosystems) was used. As the mobile phase, water containing 0.1% TFA was used as solution A, and 70% acetonitrile / water (v / v) containing 0.1% TFA was used as solution B.
Elution was performed with a linear concentration gradient from solution to solution B. The chromatographic peak was almost single, and the corresponding fractions were collected. The fractionation was repeated several times, and this was freeze-dried to obtain a purified peptide. The obtained peptide is BIOION
It was confirmed by analysis with a 20 mass analyzer (manufactured by Applied Biosystems) that the target peptide was obtained.

(4) Modification of Nonwoven Fabric Polyethylene diacrylate (CH ₂ ═C) in which R ₁ , R ₂ and R ₃ are hydrogen atoms and n is 14 in Modification 3
_{H-COO- (CH 2 CH 2} O) 14 -OC-CH 2 = C
0.1 g of H ₂ ) and ₂ g of polyethyleneimine having a molecular weight of 10,000 are dissolved in 200 ml of methanol,
A PET non-woven fabric having a fiber diameter of 3.5 μm cut into m squares was dipped in this solution, and then 10 Mrad of electron beam was irradiated on both sides of the non-woven fabric in total of 5 Mrad. The nonwoven fabric after the electron beam irradiation was washed with water and methanol three times each and air dried. The amino group content of the obtained non-woven fabric was determined by potentiometric titration with hydrochloric acid using COMMITTE10.
It was 0.11 meq / g.

(5) Introduction of Peptide into Nonwoven Fabric 25 mg of the purified peptide obtained in (3) was dissolved in a citrate buffer solution having a pH of 4.5 and then ice-cooled to obtain a water-soluble carbodiimide reagent EDC (1-ethyl). -3- (3-Dimethylaminopropyl) carbodiimide) (10 mg) was added, and the mixture was stirred for 30 minutes while cooling with ice. Furthermore, the above-mentioned modified non-woven fabric was added and shaken at room temperature for 24 hours for reaction. The non-woven fabric after the reaction was washed with water three times to obtain the target adsorbent. The peptide immobilization rate was calculated by quantifying the nitrogen content in the reaction residual liquid by the micro Kjeldahl method, and 82% was introduced.

(6) Performance evaluation of peptide-immobilized adsorbent The adsorbent performance of the antiplatelet antibody was evaluated using the blood of the above ITP patient in the adsorbent. Assemble a column filled with 20 pieces of the adsorbent cut into the shape and size of a diamond-shaped polycarbonate module case having a side of 7 cm, and a physiological saline solution containing 100 U / ml of heparin 1
The inside of the column and the inside of the blood circuit were washed with 00 ml and then with 100 ml of physiological saline containing 1 U / ml heparin.
On the other hand, blood of ITP patients (5 types) to which citric acid was added 1
A blood circuit was set up in which 00 ml was taken in a beaker and returned to the beaker through the column, and a perfusion experiment was conducted continuously with this device at a blood flow rate of 30 ml / min for 1 hour.

The amount of antiplatelet antibody in the serum after this treatment was quantified by the ELISA method (Acta. Hae).
mat. , 66, 251, 1981). The results are as shown in Table 1, and are shown as the adsorption rate [%] calculated from the value in the serum before this treatment. The nonspecific adsorption of albumin was also quantified using Albumin B-Test Wako (manufactured by Wako Pure Chemical Industries, Ltd.). The results are shown in Table 2, and the adsorption rate [%] was shown as in Table 1. The adhesion of platelets was also examined by quantifying the number of platelets before and after this treatment using a Coulter Counter ZM type (manufactured by Coulter Electronics). The results are as shown in Table 3, and are shown by the reduction rate [%] of the platelet count.

[0049]

[Table 1]

[0050]

[Table 2]

[0051]

[Table 3]

<Example 2> Glu-Glu-Asp-
Tyr-Arg-Pro-Ser-Gln-Gln-A
sp-Glu-Cys-Ser-Pro-Arg-Gl
Immobilization of u on nonwoven fabric Glu-Glu-Asp-Tyr-Arg-Pro corresponding to the 475th to 490th regions in GP3a.
-Ser-Gln-Gln-Asp-Glu-Cys-
Regarding the synthesis of the peptide having the sequence of Ser-Pro-Arg-Glu, PAM glutamic acid (t-Boc-
L-Glu (OBzl)) (manufactured by Applied Biosystems) and t-Boc-L-Arg (Tos), t.
-Boc-L-Pro, t-Boc-L-Ser (Bz
l), t-Boc-L -Cys (4-CH 3 OBz
l), t-Boc-L-Glu (OBzl), t-Bo
c-L-Asp (OBzl), t-Boc-L-Gl
It carried out like Example 1 using n, t-Boc-L-Tyr (Br-Z) (all are the Applied Biosystems company make). Deprotection group of the support after synthesis,
Cleavage of the peptide chain, purification by reverse phase HPLC and identification were also carried out in the same manner as in Example 1.

In the chemical formula 2, R ₁ and R ₂ are hydrogen atoms and R ₃
Is a methyl group and n is 3, and 10 ml of methylaminopropylacrylamide, 40 g of N-isopropylacrylamide, 2 ml of allylamine and 0.4 g of azoisobutylnitrile are dissolved in 160 ml of ethanol under stirring in the presence of nitrogen. Nitrogen was sent for a minute to completely replace the inside of the system with nitrogen. Further, polymerization was carried out by stirring in a water bath at 70 ° C. for 2 hours in the presence of nitrogen. The precipitate was washed with distilled water at 50 to 60 ° C, water was removed by decantation, and dried under reduced pressure at 40 ° C to obtain poly N-isopropylacrylamide.

3 g of the above polymer was dissolved in 150 ml of methanol together with 0.5 g of methylenebisacrylamide, and a PET non-woven fabric having a fiber diameter of 3.5 μm, which was cut into a size of 15 cm square as in Example 1, was immersed in this solution. Both sides of the nonwoven fabric were irradiated with an electron beam of 10 Mrad in total of 5 Mrad. The nonwoven fabric after the electron beam irradiation was washed with water and methanol three times each and air dried.
The amino group content of the obtained non-woven fabric was determined by potentiometric titration in the same manner as in Example 1 and found to be 0.083 meq / g.

50 mg of the above-mentioned peptide was dissolved in a citrate buffer solution having a pH of 4.5 in the same manner as in Example 1 and ice-cooled, 10 mg of EDC was added, and the mixture was stirred for 30 minutes while ice-cooling. Add the modified non-woven fabric above and add 2
The reaction was carried out by shaking for 4 hours. The non-woven fabric after the reaction was washed with water three times to obtain the target adsorbent. The peptide immobilization rate was determined in the same manner as in Example 1 and 85% was introduced. The performance of the obtained adsorbent was evaluated in the same manner as in Example 1 by assembling a column and then performing a perfusion experiment using blood of an ITP patient. The amount of antiplatelet antibody and the amount of albumin in the serum after the treatment were quantified in the same manner as in Example 1. The results are shown in Table 1 and Table 2 as the adsorption rate [%]. The platelet adhesion was also examined in the same manner as in Example 1, and the results are shown in Table 3 as the platelet count reduction rate [%].

Example 3 Arg-Ala-Lys-
Trp-Asp-Thr-Ala-Asn-Asn-P
Immobilization of ro-Leu-Tyr-Lys-Glu on a nonwoven fabric 741 to 744th Thr-A in GP3a
Arg-Ala-Lys-Trp-Asp-Thr in which both ends of the sequence of la-Asn-Asn are extended by 5 residues each.
-Ala-Asn-Asn-Pro-Leu-Tyr-
Regarding the synthesis of the peptide having the Lys-Glu sequence, PAM glutamic acid (t-Boc-L-Glu (OB
zl)) (manufactured by Applied Biosystems) and t
-Boc-L-Lys (Cl-Z), t-Boc-L-
Tyr (Br-Z), t-Boc-L-Leu.H
₂ O, t-Boc-L-Pro, t-Boc-L-As
n, t-Boc-L-Ala, t-Boc-L-Thr
(Bzl), t-Boc-L-Asp (OBzl), t
-Boc-L-Trp (CHO), t-Boc-LA
rg (Tos) (all manufactured by Applied Biosystems) was used in the same manner as in Example 1. For the deprotecting group of the support after the synthesis, t-Bo is added in the sequence.
Since it contains c-L-Trp (CHO), 1g first
1,2-ethanedithiol 0.
2 ml, m-cresol 0.8 ml, dimethyl sulfide 3 ml, TFA 5 ml, TFMSA 1 ml were added,
The reaction was carried out by stirring for 3 hours while cooling with ice water. The reaction product was filtered through a glass filter (medium pores) while being washed with diethyl ether that had been cooled beforehand, and then the deprotecting group reaction carried out in Example 1 was carried out. Hereinafter, the peptide chain cleavage, purification by reverse phase HPLC and identification were carried out in the same manner as in Example 1.

PH of 30 g of PEO acid having a molecular weight of 1000
After dissolving in 500 ml of a citrate buffer solution of 4.5, the mixture was ice-cooled, 10 mg of EDC was added, and the mixture was stirred for 30 minutes while ice-cooling. The amino group obtained in the same manner as in Example 1 was introduced (0.
12 meq / g) of 30 modified non-woven fabrics
The reaction was carried out for 24 hours by shaking in an acid solution at room temperature. After the reaction was completed, the non-woven fabric was washed with water three times to obtain a non-woven fabric into which PEO acid was introduced. The carboxyl group content was quantitatively determined by titration with an aqueous sodium hydroxide solution.
It was 074 meq / g.

In the same manner as in Example 1, 25 mg of the above peptide was dissolved in a citrate buffer solution having a pH of 4.5, followed by cooling with ice, 10 mg of EDC was added, and the mixture was stirred for 30 minutes while cooling with ice. Further, the above-mentioned PEO amine-introduced nonwoven fabric was added, and the mixture was shaken at room temperature for 24 hours for reaction. The non-woven fabric after the reaction was washed with water three times to obtain the target adsorbent. The peptide immobilization rate was determined in the same manner as in Example 1, and 86% was introduced. The performance of the obtained adsorbent was evaluated in the same manner as in Example 1 by assembling a column and then performing a perfusion experiment using blood of an ITP patient. The amount of antiplatelet antibody and the amount of albumin in the serum after the treatment were quantified in the same manner as in Example 1. The results are shown in Table 1 and Table 2 as the adsorption rate [%]. Example 1 for platelet adhesion
The results of the same examination as in Table 3 show the reduction rate of platelet count [%].
Indicated by.

Example 4 Lys-Gly-Lys-
Ile-Arg-Ser-Lys-Val-Glu-L
eu-Glu-Val-Arg-Asp-Leu-Pr
Immobilization of o-Glu to a non-woven fabric Lys-Gly-Lys in which one Lys residue was bound to the N-terminus of the 349th to 364th sequences in GP3a
-Ile-Arg-Ser-Lys-Val-Glu-
Leu-Glu-Val-Arg-Asp-Leu-P
PAM glutamate (t-Boc-L-Glu (OBz
l)) (manufactured by Applied Biosystems) and t-
Boc-L-Lys (Cl-Z), t-Boc-L-G
ly, t-Boc-L- Ile · 1 / 2H 2 O, t-B
oc-L-Arg (Tos), t-Boc-L-Ser
(Bzl), t-Boc-L-Val, t-Boc-L
-Glu (OBzl), t-Boc -L-Leu · H 2
O, t-Boc-L-Asp (OBzl), t-Boc
-L-Pro (all manufactured by Applied Biosystems) was used and carried out in the same manner as in Example 1. Deprotection group of support after synthesis, peptide chain cleavage, reverse phase H
Purification and identification by PLC were performed in the same manner as in Example 1.

6 g of PEO acid having a molecular weight of 5000 was added to pH 4.
Dissolved in 500 ml of citrate buffer solution of 5 and then cooled with ice.
DC 10 mg was added, and the mixture was stirred for 30 minutes while cooling with ice.
Introduction of an amino group obtained in the same manner as in Example 2 (0.080
30 sheets of modified non-woven fabric (meq / g) were reacted in the PEO acid solution by shaking at room temperature for 24 hours. After the reaction was completed, the non-woven fabric was washed with water three times to obtain a non-woven fabric into which PEO acid was introduced. The amino group content was determined in the same manner as in Example 3 and was 0.055 meq / g.

50 mg of the above peptide was dissolved in a citrate buffer solution having a pH of 4.5 as in Example 1 and ice-cooled, 10 mg of EDC was added, and the mixture was stirred for 30 minutes while ice-cooling. Further, the above-mentioned PEO amine-introduced nonwoven fabric was added, and the mixture was shaken at room temperature for 24 hours for reaction. The non-woven fabric after the reaction was washed with water three times to obtain the target adsorbent. The peptide immobilization rate was determined in the same manner as in Example 1, and 81% was introduced. The performance evaluation of the obtained adsorbent was carried out by assembling the same column as in Example 1 and then conducting a perfusion experiment using blood of an ITP patient. The amount of antiplatelet antibody and the amount of albumin in the serum after the treatment were quantified in the same manner as in Example 1. The results are shown in Table 1 and Table 2 as the adsorption rate [%]. The platelet adhesion was also examined in the same manner as in Example 1, and the results are shown in Table 3 as the platelet count reduction rate [%].

[0062]

[Comparative example]

<Comparative Example 1> Glu-Glu-Asp-Tyr-Ar
g-Pro-Ser-Gln-Gln-Asp-Glu
Immobilization of -Cys-Ser-Pro-Arg-Glu on the nonwoven fabric Glu-Glu-Asp-Tyr-Ar obtained in Example 2
g-Pro-Ser-Gln-Gln-Asp-Glu
Introduction of -Cys-Ser-Pro-Arg-Glu into a cotton nonwoven fabric was carried out as follows. 30 pieces of cotton nonwoven fabric with a fiber diameter of 12 μm cut into 15 cm square size
Sodium periodate having a concentration of wt% was added to 500 ml of a solution prepared by dissolving 1N sulfuric acid, and the mixture was reacted by shaking for 22 hours. After the completion of the reaction, the nonwoven fabric was washed with water three times to obtain an aldehyde group-introduced nonwoven fabric. The aldehyde content was determined by the oxime method and was 0.48 meq / g.

25 mg of the above peptide was dissolved in 500 ml of a carbonate buffer having a pH of 9.5, the above aldehyde group-introduced non-woven fabric was added, and the Schiff base reaction was carried out for 24 hours by shaking. After the reaction was completed, the nonwoven fabric was washed with water three times. The peptide immobilization rate was determined in the same manner as in Example 1 and 88% was introduced. Then 1 g of sodium borohydride (NaB
H ₄ ) was dissolved in 500 ml of a carbonate buffer solution having a pH of 9.0, the above non-woven fabric was added, and the mixture was reacted by shaking for 20 hours. After the reaction was completed, the non-woven fabric was washed with water three times to obtain a target adsorbent. The performance of the obtained adsorbent was evaluated in the same manner as in Example 1 by assembling a column and then performing a perfusion experiment using blood of an ITP patient. The amount of antiplatelet antibody and the amount of albumin in the serum after the treatment were quantified in the same manner as in Example 1. The results are shown in Table 1 and Table 2 as the adsorption rate [%].

<Comparative Example 2> Thr-Thr-Arg-
Thr-Asp-Thr-Cys-Met-Ser-S
Immobilization of er-Asn-Gly-Leu on a nonwoven fabric Thr-Thr-Arg-Thr-Asp-Thr corresponding to the 561 to 673th regions in GP3a
-Cys-Met-Ser-Ser-Asn-Gly-
For the synthesis of peptides having the sequence of Leu, PAM
Leucine (t-Boc-L-Leu) (manufactured by Applied Biosystems) and t-Boc-L-Gly, t.
-Boc-L-Asn, t-Boc-L-Ser (Bz
l), t-Boc-L-Met, t-Boc-L-Cy.
_{s (4-CH 3 OBzl)} , t-Boc-L-Thr
(Bzl), t-Boc-L-Asp (OBzl), t
-Boc-L-Arg (Tos) (all manufactured by Applied Biosystems) was used in the same manner as in Example 1. This peptide region is also a relatively large hydrophilic region. Deprotection of the support after the synthesis, cleavage of the peptide chain, purification by reverse phase HPLC and identification were also carried out in the same manner as in Example 1.

Using the same conditions as in Comparative Example 1, the above-mentioned peptide was introduced into a cotton non-woven fabric into which an aldehyde group was introduced (0.53 meq / g) under the same reaction conditions as in Comparative Example 1. The peptide immobilization rate was determined in the same manner as in Example 1 and 86% was introduced. The performance of the obtained adsorbent was evaluated in the same manner as in Example 1, and after the column was assembled I
It was carried out by a perfusion experiment using blood of a TP patient. The amount of antiplatelet antibody and the amount of albumin in the serum after the treatment were quantified in the same manner as in Example 1. The results are shown in Table 1 and Table 2 as the adsorption rate [%]. The platelet adhesion was also examined in the same manner as in Example 1, and the results are shown in Table 3 as the platelet count reduction rate [%].

[0066]

INDUSTRIAL APPLICABILITY According to the present invention, antiplatelet antibodies in blood can be effectively bound, and the present invention can be applied to the treatment of patients with severe ITP. The blood purification therapy using the anti-platelet antibody adsorbent in the present invention is safe without any risk of side effects and is excellent in stability upon storage. In addition, by using a non-woven fabric, extracorporeal circulation can be performed by whole blood treatment that does not require plasma separation as in the past. Further, the presence of an amino group or an imino group makes it easy to introduce peptides that are ligands. Furthermore, the fact that the activity as a ligand is hardly reduced even if steam sterilization by autoclave treatment is performed is another advantageous factor for medical equipment.

─────────────────────────────────────────────────── ───

[Procedure amendment]

[Submission date] December 22, 1992

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be corrected] 0022

[Correction method] Change

[Correction content]

Immobilize the above peptide or modified peptide
The types of non-woven fabrics used when converting into
However, when used for blood purification, contact with blood components
It is necessary to consider the effect on the complement system and coagulation system when touched.
Polyethylene terephthalate (PE
T), polypropylene (PP), polyethylene (P
E), polyamide, cellulose (cotton) and rayon
Examples include the ease of modification and strength retention after modification.
Considering that, PET, PE, cotton or rayon are preferred.
Yes. In order to suppress the adhesiveness of blood cell components, the fiber diameter should be
1 to 30 μm is preferable, and 3 to 20 μm is more preferable.
Good As a non-woven fabric composed of these fibers,
For the purpose of the present invention, the density (filling rate) of the nonwoven fabric is
0.6 g / cm ³Or less, more preferably 0.4 g / cm
³It is as follows. The lower limit is the contact efficiency.
From the point of 0.01g / cm³Or more, more preferably 0.
02 g / cm³That is all.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Masakazu Tanaka 2-1-1 Katata, Otsu City, Shiga Prefecture Toyobo Co., Ltd.

Claims (1)

[Claims] 1. An anti-platelet antibody adsorbing material, characterized in that a peptide or modified peptide having a binding property to an anti-platelet antibody is immobilized on a nonwoven fabric containing amino groups and / or imino groups and having an average fiber diameter of 1 to 30 μm. .