JP2795747B2 - Peptide and adsorbent immobilized on a carrier - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a peptide capable of binding to interleukin 6, and an interleukin 6 adsorbent obtained by individualizing the peptide on a carrier.

It is known that interleukin 6 (hereinafter, abbreviated as IL-6) acts on lymphocytes having an antibody-producing ability and significantly enhances the antibody-producing ability, and is known as a causative substance of autoimmune diseases such as rheumatism. It is considered one. Therefore, the peptides and adsorbents of the present invention are useful in treating autoimmune diseases such as rheumatism.

BACKGROUND ART Science, vol. 241, pp. 825-828 (19
In 1988, a human interleukin 6 receptor (hereinafter referred to as
It is reported that the precursor of this (abbreviated as IL-6 receptor) is composed of 468 amino acids, and that the primary configuration could be elucidated. According to this report, mature IL-
The primary structure of the 6 receptor is In addition, Medical Immunolo
gy), Vol. 15, pp. 195-201 (1988), reports on the association between IL-6 and autoimmune diseases.

In the treatment of autoimmune diseases such as rheumatism, it is desired to establish means for removing IL-6, which is considered to be a main disease-causing substance, but a practical method has not yet been confirmed.

One object of the present invention is to provide a novel peptide having the ability to bind to IL-6. Another object of the present invention is to provide an IL-6 adsorbent obtained by immobilizing the novel peptide on a carrier.

DISCLOSURE OF THE INVENTION According to the present invention, according to the general formula H-X-A-Y-Z [I], wherein A represents a peptide fragment having 6 to 50 amino acids bonded, and X and Y each represent Represents a single bond or Asp, Glu, Lys, Ala and the formula (Wherein, n represents an integer of 1 to 17), wherein the amino acid residue is selected from the group consisting of divalent groups represented by the following formulas, or 2 to 10 of the amino acid residues selected from the group are peptide-bonded. Represents a peptide fragment, and Z represents a hydroxyl group or an amino acid]. Further, according to the present invention, there is provided an adsorbent obtained by immobilizing the peptide on a carrier.

In this specification, various amino acid residues are described by the following abbreviations.

Ala: L-alanine residue Arg: L-arginine residue Asn: L-asparagine residue Asp: L-aspartic acid residue Cys: L-cysteine residue Gln: L-glutamine residue Glu: L-glutamic acid residue Gly: glycine residue His: L-histidine residue Ile: L-isoleucine residue Leu: L-leucine residue Lys: L-lysine residue Phe: L-phenylalanine residue Pro: L-proline residue Ser : L-serine residue Thr: L-threonine residue Trp: L-tryptophan residue Tyr: L-tyrosine residue Val: L-valine residue Also, in this specification, the amino acid sequence is N-terminal Are described on the left side, and the amino acid residue at the C-terminal is on the right side.

Examples of the peptide fragment represented by X and Y in the general formula (I) include the following peptide fragments.

In the general formula (I), a peptide in which X and / or Y is a peptide fragment in which 11 or more of the amino acid residues selected from the above group are bound by a peptide bond has no ability to bind to a desired IL-6. There are cases.

Preferred examples of the peptide fragment represented by A in the general formula (I) are shown below. The amino acid residue of each peptide fragment is
It may have undergone homologous substitution.

In the general formula (I), the peptide in which A is a peptide fragment in which 5 or less amino acids are bound does not have the ability to bind to IL-6, or the ability to bind to IL-6 is practical. Is not enough. Also, the desired IL-
It is not practical to synthesize a peptide fragment comprising 51 or more amino acids having the ability to bind to 6.

The peptide represented by the general formula (I) is synthesized by a method usually used in peptide synthesis, for example, a solid phase synthesis method or a liquid phase synthesis method such as a stepwise extension method or a fragment condensation method. It is operationally convenient to carry out by a phase synthesis method [for example, Journal of the American Chemical Society (Journal of the A
American Chemical Society), Vol. 85, pp. 2149-2154 (1963); edited by The Biochemical Society of Japan, "Course for Biochemical Experiments, Chemical IV Chemical Modification of Proteins and Peptide Synthesis" (November 1977)
15th, published by Tokyo Chemical Co., Ltd.), pp. 207-495; edited by The Biochemical Society of Japan, "Seizoku Chemistry Experiment Course 2: Protein Chemistry (bottom)", published by Tokyo Chemical Co., Ltd. on May 20, 1987) See pages 641-694. The peptide represented by the general formula (I) is produced by a solid phase synthesis method using a polymer insoluble in a reaction solvent such as a styrene-divinylbenzene copolymer as a solid phase carrier. They have an amino acid or amino acid amide corresponding to the C-terminus of the peptide of interest. An α-COOH group or α-CONH ₂ group is used to bind to a solid support. Next, the corresponding amino acid or peptide fragment is sequentially peptide-bonded to the amino acid or amino acid amide toward the N-terminus of the target peptide. At this time, the amino acid or peptide fragment to be bound is usually C-terminal α-
It is used by protecting functional groups other than COOH group. In addition, the amino acid or amino acid amide or peptide fragment on the solid support is usually after removing the protecting group of only α-NH ₂ group,
It is subjected to a peptide bond formation reaction. The formation of the peptide bond is performed by a known method such as a dehydration condensation method using carbodiimide. After forming a peptide chain corresponding to the peptide of interest on the solid support, by removing from the solid support, and removing the protecting group from the protected functional group, if necessary, by purification, The desired peptide is obtained. Here, the elimination of the peptide chain from the solid support and the removal of the protecting group can be carried out by a known method, but it is preferable to carry out these simultaneously using hydrogen fluoride from the viewpoint of suppressing side reactions. Further, it is effective that the obtained peptide is purified by reversed-phase liquid chromatography.

Since the peptide represented by the general formula (I) has an ability to bind to IL-6, it can inhibit the binding between IL-6 and the IL-6 receptor. Therefore, by administering the peptide represented by the general formula (I) to a patient with an autoimmune disease such as rheumatism in which the production of autoantibodies caused by the binding between IL-6 and its receptor is increased, Antibody production can be suppressed.

The dosage for effective expression of the peptide represented by the general formula (I) is 2 g / kg or less, preferably 1 μg
/ kg or more and 200 mg / kg or less. As a preferred administration form, a physiologically acceptable salt solution such as an aqueous solution or a physiological saline solution obtained by dissolving the peptide represented by the general formula (I) [for example, 1 mg of the peptide represented by the general formula (I)]
Or a solution obtained by dissolving the compound in 100 ml of a 5% glucose solution], intravenously, subcutaneously, or intraperitoneally.

Oral administration is also possible by encapsulating the peptide represented by the general formula (I) or the above-mentioned aqueous solution or salt solution into liposomes. Also, transdermal administration as an oil agent is possible. The peptide represented by the general formula (I) does not exhibit significant acute toxicity at the above dose.

The peptide represented by the general formula (I) is immobilized on a carrier and used as an adsorbent for IL-6. The peptide represented by the general formula (I) used for immobilization is 1
The number of types may be two or more.

The carrier used for immobilizing the peptide represented by the general formula (I) includes an amino group and a carboxyl group which have a hydrophilic surface and can be used for forming a covalent bond with the peptide. And those having a reactive functional group such as a hydroxyl group. When the peptide is used as an adsorbent for adsorbing IL-6 in a body fluid of a patient with an autoimmune disease, the carrier is preferably insoluble in a body fluid and porous. A porous carrier having a large effective area on which IL-6 can be adsorbed has an exclusion limit protein molecular weight in the range of about 10 ⁶ to 10 ⁹ or an average pore diameter in the range of about 50 to 1000 nanometers. It is preferred to use some. The carrier is particulate, fibrous,
Any shape such as a sheet shape and a hollow fiber shape can be used. Such carriers include, for example, CM-cellulofine
Cellulose-based carriers such as CH (exclusion limit protein molecular weight: about 3 × 10 ⁶ ; sold by Seikagaku Corporation); TSK-gel CM-
Polyvinyl alcohol carrier such as Toyopearl 650C (exclusion limit protein molecular weight 5 × 10 ⁶ ; manufactured by Tosoh Corporation); C
M-Trisacryl M (exclusion limit protein molecular weight: 1 × 10 ⁷ ; Pharmacia, Sweden)
Polyacrylamide carrier such as LKB (Pharmacia-LKB); Sepharose CL-4B
Organic carriers such as agarose-based carriers such as [exclusion limit protein molecular weight: 2 × 10 ⁷ ; manufactured by Pharmacia-LKB, Sweden], and CPG-10-1000 [exclusion limit protein molecules: 1 × 10 ⁸ ; average pore diameter: 100 nm; manufactured by Electro-nucleonics (USA)].

Immobilization of the peptide represented by the general formula (I) on a carrier is performed according to a method generally employed when immobilizing a peptide or protein on a carrier. As an immobilization method, for example, a carboxyl group of a carrier is converted into a succinimideoxycarbonyl group by reacting the carboxyl group with N-hydroxysuccinimide, and the peptide represented by the general formula (I) is converted to an amino group (Active ester method), condensation of the peptide represented by the general formula (I) with the carboxyl group or amino group at the amino group or carboxyl group of the carrier in the presence of a condensing reagent such as dicyclohexylcarbodiimide A reaction method (condensation method), a method of cross-linking a carrier and a peptide represented by the general formula (I) using a compound having two or more functional groups such as glutaraldehyde (carrier cross-linking method), and the like. An adsorbent obtained by immobilizing the peptide represented by the general formula (I) on a carrier by the active ester method has the highest IL-6 adsorption ability. The amount of the peptide represented by the general formula (I) immobilized on a carrier is usually about 3 × 10 ⁻⁸ mol / g (carrier) so that the obtained adsorbent can adsorb a significant amount of IL-6. The above is necessary, and the peptide represented by the general formula (I) immobilized on a carrier is IL-
In order to be effectively used for adsorption of 6, about 1 × 10 ^-7 to 2 ×
It is preferably in the range of 10 ^-6 mol / g (carrier).

To remove IL-6, an adsorbent obtained by immobilizing the peptide represented by the general formula (I) on a carrier is brought into contact with a body fluid such as blood, plasma, or serum containing IL-6, and IL is added to the adsorbent. −
6 is adsorbed. The adsorbent is used, for example, by filling in a column. The column used for this purpose has an inlet part and an outlet part which can be easily connected to the blood circuit, and is made of polyester or the like between the inlet part and the adsorbent layer and between the outlet part and the adsorbent layer. It is desirable to have a filter made of a material. Examples of the material of the column include polyethylene, polypropylene, polycarbonate, polyester, and polymethyl methacrylate. Among them, polypropylene and polycarbonate are particularly preferable in that the column filled with the adsorbent can be subjected to sterilization such as autoclave sterilization and γ-ray sterilization before use.

The removal of IL-6 from the body fluid of a patient using the column packed with the adsorbent is performed, for example, in an extracorporeal blood circulation system. The following two can be exemplified as the extracorporeal blood circulation system.

(1) Blood drawn from a patient's blood vessel is sent to a column filled with an adsorbent, where IL-6 is removed from the blood by adsorption, and then the processed blood passing through the column is circulated to the patient's blood vessel.

(2) First, blood extracted from a patient's blood vessel is separated into a blood cell component and a plasma component, and the separated plasma component is sent to a column filled with an adsorbent, where IL-6 is removed from the plasma component by adsorption. The processed plasma component that has passed through is mixed with the separated blood cell component, and the resulting mixture is then circulated into the patient's blood vessels.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described specifically with reference to examples, but the present invention is not limited to the examples.

Example 1 Formula H-Thr-Ser-Leu-Pro-Gly-Asp-Ser-Val-Th
r-Leu-Thr-Cys-Pro-Gly-Val-Glu-Pro-Glu-As
A peptide represented by p-Lys-OH was converted to an automatic peptide synthesizer [Applied Biosys.
430A (Model 430A), manufactured by T.S.

That is, 4- [N ^α- (t-butoxycarbonyl)
N ^ε- (chlorobenzyloxycarbonyl) -L-lysyloxymethyl] phenylacetamidomethyl Having a group of 0.78 mmol / g (resin)
Granular resin composed of divinylbenzene copolymer [constituent molar ratio of styrene and divinylbenzene: 99: 1] [PAM lysine (Lysin, manufactured by Applied Biosystems, USA)
e), 0.13 g of t-Boc-L-Lys (CI-Z)], and according to the series of operations shown in Table 1, the corresponding L- Aspartic acid,
L-cysteine, glycine, L-glutamic acid, L-leucine, L-serine, L-proline, L-threonine and L-valine were sequentially linked. In the condensation reaction,
Additional amino acids are N-(t-butoxycarbonyl) -O ^beta - benzyl -L- aspartic anhydride, N
- (t-butoxycarbonyl) -S- (p-methoxybenzyl) -L- cysteine anhydride, N-(t-butoxycarbonyl) glycine anhydride, N-(t-butoxycarbonyl) -O ^gamma - benzyl -L -Glutamic anhydride,
N- (t-butoxycarbonyl) -L-leucine anhydride, N- (t-butoxycarbonyl) -O-benzyl-
L-serine anhydride, N- (t-butoxycarbonyl)-
L-proline anhydride, N- (t-butoxycarbonyl)
-O ^beta - benzyl -L- threonine anhydride and N-
They were used as (t-butoxycarbonyl) -L-valine anhydride, and their usage was about 5 times the molar amount of the substrate. The condensation reaction was performed at room temperature. The reaction time varied depending on the type of amino acid to be condensed, but was in the range of 10 to 20 minutes.

After the reaction operation for all amino acids is completed,
The obtained resin was washed sequentially with diethyl ether, dichloromethane and methanol on a glass filter, and then dried under vacuum to obtain 0.41 g of a dried resin. Reaction vessel made of polytrifluoromonochloroethylene (HF-reactor type I, manufactured by Peptide Laboratories Inc.)
In the above, 0.41 g of the obtained dry resin was mixed with 0.6 ml of anisole and 0.1 ml of ethyl methyl sulfide, and 4 ml of hydrogen fluoride was added to the mixture at a temperature of -20 ° C, and the mixture was stirred at the same temperature for 30 minutes and then at 0 ° C. Stirred at a temperature of 30 ° C. for 30 minutes. Hydrogen fluoride, anisole and ethyl methyl sulfide were removed from the resulting reaction mixture under reduced pressure, and the residue was sufficiently washed on a glass filter using diethyl ether. The obtained residue was extracted with a 2N aqueous acetic acid solution, and the extract was lyophilized to obtain 0.2 g of a crude peptide.

A crude product of the obtained peptide is subjected to preparative reversed-phase high-performance liquid chromatography [column: octadecylated silica gel (particle size: 5 μm) packed column (inner diameter: 10 mm, length: 300 m)
m), manufactured by Chemco, Develosil ODS;
Mobile phase: mixed solvent of acetonitrile and water containing 0.05% by volume of trifluoroacetic acid (acetonitrile concentration: 20
The mixture was gradually changed from 20% to 30% by volume in 20 minutes) to obtain 50 mg of a purified product of the target peptide.

The obtained purified peptide is analyzed by reversed-phase high performance liquid chromatography for analysis [column: octadecylated silica gel (particle size: 5 μm) packed column (inner diameter: 4 mm, length 150 mm),
TSK gel ODS-80TM, manufactured by Tosoh Corporation; mobile phase: mixed solvent of acetonitrile and water containing 0.05% by volume of trifluoroacetic acid (the concentration of acetonitrile is 5% by volume in 30 minutes)
From 50 to 50% by volume); flow rate: 1 ml
/ Min; detection method: absorbance at a wavelength of 210 nm], showed a single sharp peak at 17.5 minutes. The molecular weight of the purified product determined by a high-speed atom bombardment (hereinafter abbreviated as FAB method) mass spectrum was 2046 (theoretical value: 2045.22). The product obtained by hydrolyzing the purified product with hydrochloric acid was subjected to amino acid composition analysis, and the results were as follows (the numbers in parentheses indicate theoretical values).

Lysine: 1.04 (1), Aspartic acid: 2.09 (2), Glutamic acid: 2.02 (2), Proline: 3.10 (3), Valine: 1.90 (2), Glycine: 1.95 (2), Cysteine: 0.4
4 (0.5), threonine: 3.11 (3), leucine: 1.99
(2), serine: 1.98 (2).

Examples 2 to 96 By performing solid phase synthesis and purification of the peptide in the same manner as in Example 1, Tables 2, 3 and 4,
Table 5, Table 6, Table 7, Table 8, Table 9, Table 10, Table
The peptides shown in Tables 11, 12 and 13 were obtained. However, as the resin for the solid phase, Example 2, Example 5, Example
In Example 41 and Example 45, 4- [N- (t-butoxycarbonyl) -0 ^β -benzyl-α-L-aspartyloxymethyl] phenylacetamidomethyl group was used in an amount of 0.78 mmol.
/ g (resin) / styrene-divinylbenzene copolymer [constituent molar ratio of styrene and divinylbenzene: 9
9: 1) granular resin [manufactured by Applied Biosystems, USA, PAM Aspartic acid,
t-Boc-L-Asp (OBzl)], Example 3, Example
11, Example 19, Example 27, Example 35, Example 43, Example
51, Example 59, Example 67, Example 75, Example 83 and Example 91 show 4- [N- (t-butoxycarbonyl) -O
styrene-divinylbenzene copolymer having [ ^γ- benzyl-α-L-glutamyloxymethyl] phenylacetamidomethyl group at a ratio of 0.78 mmol / g (resin) [99: 1 molar ratio of styrene to divinylbenzene]. Granular resin [PA, manufactured by Applied Biosystems, USA
M glutamic acid (Glutamicacid), t-Boc-L-Glu (O
Bzl)], using Example 4, Example 12, Example 20, Example 28, Example 36, Example 44, Example 52, Example 60, Example 68, Example 78, Example 84, In Example 92, 4- [N
-(T-butoxycarbonyl) glycyloxymethyl]
A granular resin comprising a styrene-divinylbenzene copolymer having a phenylacetamidomethyl group ratio of 0.78 mmol / g (resin) [constituent molar ratio of styrene and divinylbenzene: 99: 1] [manufactured by Applied Biosystems, USA , PAM glycine (Glycine), t-Boc-Gly] using Example 9, Example 10, Example 13, Example 17, Example 25, Example 33, Example 34, Example 37, Example 41, Example 49, Example 57, Example 58, Example 61, Example 65, Example 66, Example 69, Example 73, Example 74, Example 77, Example 81, Example 82, example 85, example 89, example 90 and example 93 4- ^[N α - (t- butoxycarbonyl)
-N ^epsilon - 0.78 chloro benzyloxycarbonyl) -L- lysyl oxymethyl] phenylalanine acetamidomethyl group
A granular resin comprising a styrene-divinylbenzene copolymer [constituent molar ratio of styrene and divinylbenzene: 99: 1] having a ratio of mmol / g (resin) [Applied.
Biosystems, PAM lysine, t-Boc-L-Lys
In Example 18, Example 21, Example 50 and Example 53, 4- [N- (t-butoxycarbonyl) -L-seryloxymethyl] phenylacetamidomethyl group was used in Example 18, Example 21, Example 50 and Example 53. Granular resin consisting of a styrene-divinylbenzene copolymer [constituent molar ratio of styrene and divinylbenzene: 99: 1] having a ratio of mmol / g (resin) [PAM serine (Ser, manufactured by Applied Biosystems, USA)
ine), t-Boc-L-Ser], and in Example 26 and Example 29, 4- [N- (t-butoxycarbonyl) -L
Styrene having [alanyloxymethyl] phenylacetamidomethyl group at a ratio of 0.76 mmol / g (resin)
Granular resin composed of divinylbenzene copolymer [constituent molar ratio of styrene and divinylbenzene: 99: 1] [PAM alanine (Alanin, manufactured by Applied Biosystems, USA)
e), t-Boc-L-Ala], using Example 6, Example 7, Example 8, Example 14, Example 15, Example 16, Example
22, Example 23, Example 24, Example 30, Example 31, Example
32, Example 38, Example 39, Example 40, Example 46, Example
47, Example 48, Example 54, Example 55, Example 56, Example
62, Example 63, Example 64, Example 70, Example 71, Example
72, Example 78, Example 79, Example 80, Example 86, Example
In Example 87, Example 88, Example 94, Example 95 and Example 96, the α-amino-p-methylbenzyl group was added at 0.78 mmol / g.
(Resin), a granular resin comprising a styrene-divinylbenzene copolymer [constituent molar ratio of styrene and divinylbenzene of 99: 1] [p-Methyl BHA resin, manufactured by Applied Biosystems, USA. Resi
n)] and L-alanine, L-
-Arginine, L-asparagine, L-glutamine, L
-Histidine, L-isoleucine, L-lysine, L-phenylalanine, L-tryptophan, L-tyrosine,
12-aminododecanoic acid and 18-aminooctadecanoic acid are N- (t-butoxycarbonyl) -L-alanine anhydride and N- (t-butoxycarbonyl)-(2,4,
6-trimethyl) benzenesulfonyl-L-arginine hydroxybenzotriazyl ester, N- (t-butoxycarbonyl) -L-asparagine hydroxybenzotriazyl ester, N- (t-butoxycarbonyl)
-L-glutamine hydroxybenzotriazyl ester, N ^α- (t-butoxycarbonyl-N ^Im -dinitrophenyl-L-histidine hydroxybenzotriazyl ester, N- (t-butoxycarbonyl) -L-isoleucine anhydride, N ^α -(T-butoxycarbonyl)-
N ^ε- 2-chlorobenzyloxycarbonyl-L-lysine anhydride, N- (t-butoxycarbonyl) -L-phenylalanine anhydride, N- (t-butoxycarbonyl)
-N ^Im -formyl-L-tryptophan anhydride, N-
(T-butoxycarbonyl) -O- (p-bromo) benzyloxycarbonyl-L-tyrosine anhydride, 12- (t
-Butoxycarbonylamino) dodecanoic anhydride and
Used as 18- (t-butoxycarbonylamino) octadecanoic anhydride.

The obtained purified peptide is analyzed by reversed-phase high performance liquid chromatography [column: octadecylated silica gel (particle size: 5 μm) packed column (inner diameter: 4 mm, length: 150 mm),
TSK gel ODS-80TM, manufactured by Tosoh Corporation; mobile phase: mixed solvent of acetonitrile and water containing 0.05% by volume of trifluoroacetic acid (the concentration of acetonitrile is 5% by volume in 30 minutes)
From 50 to 50% by volume); flow rate: 1 ml
/ Min; detection method: absorbance at a wavelength of 210 nm], all showed a single peak. Table 14 shows the molecular weights of the purified products determined by the FAB method mass spectrum and the amino acid composition analysis values of the products obtained by hydrolysis using hydrochloric acid.

Example 97 A suspension obtained by suspending 10 g of cellulose particles (CM-Cellulofine CH, sold by Seikagaku Corporation) in 50 ml of anhydrous dioxane (a commercially available dioxane distilled in the presence of metallic sodium). To the mixture were added 0.5 g of N-hydroxysuccinimide and 1.0 g of dicyclohexylcarbodiimide, and the mixture was shaken and stirred at room temperature overnight. The resulting mixture was washed with 0.02 mol / phosphate buffer (pH: 7.4) and suctioned off. The particles obtained are mixed with 20 ml of a 0.02 mol / phosphate buffer (pH: 7.4) containing 20 mg of the peptide obtained in Example 1, and the mixture is added at a temperature of 4 ° C. to 1 ml.
Stirred overnight. The resulting mixture was suctioned off. The solution was subjected to reverse phase high performance liquid chromatography for analysis, but no unreacted peptide remained (immobilization ratio of peptide on carrier: about 100%). Thus, about 10 g of an adsorbent in which 20 mg of the peptide obtained in Example 1 was immobilized on cellulose particles was obtained.

Example 98 Instead of 10 g of the cellulose particles in Example 97, polyvinyl alcohol particles (TSK-gel C manufactured by Tosoh Corporation) were used.
M-Toyopearl 65C) 10 g of peptide obtained in Example 9 instead of 20 mg of peptide obtained in Example 1
By the same method except that 20 mg was used, about 10 g of an adsorbent in which 18.4 mg of the peptide obtained in Example 9 was immobilized on polyvinyl alcohol particles was obtained (immobilization ratio of the peptide on the carrier: about 92%). .

Example 99 10 g of porous glass particles (CPG-10-1000, manufactured by Electro-Nucleonics, USA) were reacted under heating and reflux for 24 hours in 100 ml of a toluene solution containing 5 ml of γ-aminopropyltriethoxysilane. I let it. The resulting mixture is
Washed with anhydrous dioxane and filtered off with suction. The obtained particles were suspended in 100 ml of anhydrous dioxane, 3 g of succinic anhydride was added to the obtained suspension, and the mixture was stirred at room temperature overnight. The resulting mixture was washed with anhydrous dioxane and suction filtered. The obtained particles were suspended in 50 ml of anhydrous dioxane, and the obtained suspension was treated with 0.1 ml of N-hydroxysuccinimide.
5 g and 1.0 g of dicyclohexylcarbodiimide were added,
The mixture was stirred overnight at room temperature. The resulting mixture was washed with 0.02 mol / phosphate buffer (pH: 7.4) and suctioned off. The resulting particles were obtained from the peptide 20 obtained in Example 17.
0.02 mol / mg phosphate buffer (pH: 7.4)
20 ml and the mixture was stirred overnight at a temperature of 4 ° C. The resulting mixture was suction-filtered to obtain about 10 g of an adsorbent in which 20 mg of the peptide obtained in Example 17 was immobilized on porous glass particles (immobilization rate of the peptide on the carrier: about 100).
%).

Examples 100-192 Example 97 except that 20 mg of the peptides shown in Table 10 were used.
To 99, an adsorbent in which the peptide was immobilized on the particulate carrier was obtained. The immobilization rate of the particulate carrier used and the peptide on the carrier
It is shown in Table 15.

Test Example 1 Acquisition of IL-6 receptor-expressing cells Ficoll-Paqu from human peripheral blood
e) A human lymphocyte fraction was obtained by (Pharmacia-LKB). This includes the Epstein-Barr virus (Epstei
Human lymphocytes were transformed by the action of the culture supernatant of the n-Barr Virus-producing cell line B95-8 to obtain IL-6 receptor-expressing cells.

Preparation of FITC-labeled anti-IL-6 antibody Anti-human IL-6 antibody [Rabbit Anti-Human
Interleukin-6 (Rabbit Anti-human Interleuk
in-6), manufactured by Genzyme Co.] 1 mg was dissolved in 0.05 M carbon salt buffer (pH 9.5), and FITC was added to the resulting solution.
[Fluorosein isothiocyanate] (Fluorocein i
sothiocyanate) (manufactured by Sigma) 10 μg and stirred at 4 ° C. overnight. The obtained solution was passed through a PD-10 column (Pharmacia-LKB) to obtain a FITC-labeled anti-IL-6 antibody as a fraction eluted first.

0.5% BSA (bovine serum albumin, Sigma) IL-6 receptor binding inhibitory activity IL-6 receptor-expressing ¹⁰⁵ cells with the peptide -PBS (phosphate buffer containing 0.15M sodium chloride, pH 7 .4), and the resulting dispersion was added to IL-6 5
0 ng [Human recombinant interleukin-6, manufactured by Zenzyme] and 5 μg of the peptide obtained in any of Examples 1 to 96 were added, and the mixture was allowed to stand at 4 ° C. for 1 hour. Next, the cells were centrifugally washed three times with 0.5% BSA-PBS (1200 rpm, 5 min), and then 1 μg of a FITC-labeled anti-IL-6 antibody was added, followed by standing at 4 ° C. for 30 minutes. After washing three times with 0.5% BSA-PBS, the fluorescence intensity of the cells was measured. The binding inhibitory activity of the peptide of each Example was evaluated by the following formula using the case where no peptide was added as a control and the case where IL-6 was not added as a blank.

The results obtained for the peptides of Examples 1 to 96 are summarized in Table 16.

Test Example 2 Preparation of Biotin-Labeled Anti-IL-6 Antibody 200 μg of the same anti-IL-6 antibody used in Test Example 1 was added to 0.1 M
Was dissolved in 0.2 ml of an aqueous solution of NaHCo ₃ and NHS-LS
-A solution of bition [Pierce] in DMF (1 mg / m
l) 20 μg was added and reacted at room temperature for 4 hours. PBS at 4 ° C
The dialysis was carried out to obtain a bition-labeled anti-IL-6 antibody.

Adsorption of IL-6 in serum 50 mg of the adsorbent obtained in any of Examples 97 to 192 and IL
500 µm of rheumatic patient serum containing -6 at 37 ° C
After shaking for a time, the supernatant was used as a test solution.

Measurement of IL-6 concentration in test solution and evaluation of adsorption capacity of adsorbent 2.5 μg / wel of the same anti-IL-6 antibody as used in Test Example 1
Each well of a 96-well plate was fixed with 1% BSA-PBS immobilized on a flat bottom 96-well plate (Falcon Rigid-Assay Plate, Becton-Dickinson) at a ratio of 1 l. After blocking, 50 μl of the test solution was dispensed into each well. After allowing to stand at 4 ° C. overnight and washing each well, 0.5 μg / well of a biothion-labeled anti-IL-6 antibody was dispensed, and further allowed to stand at 37 ° C. for 1 hour. After washing each well, HRP-labeled streptavidin [1500-fold dilution, Kirkegaard & Perry Lab Inc. (Kirkegaard & Perry Lab. In
c.)) was dispensed into each well, and left still at 37 ° C. for 30 minutes. After washing each well, ABTS was added in the presence of H ₂ O ₂ to develop color, and the difference between the absorbance of each well at 409 nm and 501 nm was measured. A calibration curve was prepared from the absorbance of wells to which a solution containing human IL-6 at a known concentration was added instead of the test solution, and the IL-6 concentration in each test solution was determined using the calibration curve. Using the adsorbent obtained by immobilizing glycine in place of the peptide on the cellulose particles and using the IL-6 concentration in the serum as a control value, the IL-6 adsorption removal rate was calculated by the following equation. It is shown in the table.

Industrial Applicability According to the present invention, a peptide represented by the general formula (I) which is advantageous for treating an autoimmune disease is provided. General formula (I)
Is inhibited by binding of IL-6 to its receptor by administering the peptide to an autoimmune disease patient because the peptide inhibits the binding of IL-6 to the IL-6 receptor. Is effective in suppressing the production of autoantibodies that

Further, according to the present invention, there is provided an adsorbent in which the peptide represented by the general formula (I) is immobilized on an insoluble carrier. The adsorbent is used to remove IL-6 from body fluids of patients with autoimmune disease by extracorporeal blood circulation using the adsorbent.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI // A61K 38/00 ABG B01J 20/22 B B01J 20/22 G01N 33/53 P G01N 33/53 A61K 37/02 ABG (58 6) Fields investigated (Int.Cl. ⁶ , DB name) C07K 14/715 C07K 14/54 C07K 17/00-17/14 CA (STN) CAOLD (STN) REGISTRY (STN) BIOSIS (DIALOG)

Claims (2)

(57) [Claims] (1) A compound represented by the general formula: H—X—A—Y—Z Wherein X and Y each represent a single bond, or Asp, Glu, Lys, Ala and the formula (Wherein, n represents an integer of 1 to 17), wherein the amino acid residue is selected from the group consisting of divalent groups represented by the following formulas, or 2 to 10 of the amino acid residues selected from the group are peptide-bonded. Represents a peptide fragment, and Z represents a hydroxyl group or an amino acid.] A peptide having an ability to bind to interleukin 6. 2. An adsorbent obtained by immobilizing the peptide according to claim 1 on a carrier.