JPH02209890A - New peptide - Google Patents

Abstract

NEW MATERIAL:A peptide expressed by the formula (A and B represent Phe or Tyr; X and Y represent single bond, amino acid residue such as Asp or Glu, etc.; Z represents OH or amino group). USE:Useful for therapy of myasthenia gravis caused by an autoantibody to a nicotinic acetylcholine receptor (hereinafter referred to as 'receptor'). An adsorbing agent which effectively adsorbs and removes a human antibody to the receptor from a body is efficiently formed by immobilizing it on a carrier and heat-treating the peptide. PREPARATION:For example, a styrene-divinylbenzene copolymer bound with an acyloxy group formed by eliminating hydrogen atom from an alpha-carboxyl group contained in an amino acid corresponding to a C-terminus of the objective peptide is successively bound to corresponding amino acids toward the N- terminus of the objective peptide to form the peptide chain. Then the peptide chain is removed from the polymer and the protecting group thereof is eliminated.

Description

[Detailed description of the invention] [Industrial application fields] The present invention relates to novel peptides.

The peptide provided by the present invention is easily immobilized on a carrier and then heat-treated, thereby exhibiting the ability to specifically react with human antibodies against nicotinic acetylcholine receptors and selectively adsorb the antibodies. do.

Therefore, the peptide provided by the present invention can be used to treat severe muscle pain, in which neuromuscular transmission disturbances caused by autoantibodies against nicotinic acetylcholine receptors, which are present on the postsynaptic membrane of the neuromuscular junction, are said to be central to the pathology. Useful in the treatment of asthenia.

[Prior Art] Nature, Volume 299, No. 793
~797! (1982) described Torbet caliornica, a species of ray ray.
It has been reported that the α-subunit precursor of the nicotinic acetylcholine receptor obtained from the electric organ of A. According to this report, the amino acid sequence at positions 183 to 200 in the primary structure of the α-subunit precursor has the formula -Gly-Trp-Lys-H4
s -Trp -Vat -Tyr -Tyr -Th
It has been reported that it binds to r-Cys-su antibody and rabbit antibody. Biochemical and Biophysical Research Communications (Bio
chemical and biophysical
Research Communications),
Vol. 135, pp. 82-89 (1986) describes how the α-subunit of the nicotinic acetylcholine receptor obtained from Torbet californii can be decomposed with proteases (Proceedings
or the National Aca
demy ofSciences of the
United 5tates or Ame
rica), Vol. 84, pp. 3633-3637 (
(1987) reported that the primary structure of the α-subunit of the nicotinic acetylcholine receptor obtained from the electric organ of the Torbet-Californi force was 182nd to 198th.
A peptide corresponding to the amino acid sequence at
It has been reported that an adsorbent formed by immobilization on 5epharose CL-4B) binds to a mouse monoclonal antibody directed to the ligand binding site of the mouse receptor for nicotinic acetylcholine receptors and α-bungarotoxin.

[Problem to be solved by the invention] In the treatment of myasthenia gravis, it is desired to establish a means to remove human autoantibodies against nicotinic acetylcholine receptors, which are said to be the main causative agent of myasthenia gravis. However, the reality is that practical means have not yet been established.

Therefore, an object of the present invention is to provide a novel peptide useful for efficiently producing an adsorbent capable of effectively adsorbing human antibodies against nicotinic acetylcholine receptors.

[Means for Solving the Problems] According to the present invention, the above-mentioned object is achieved by - formula H-X-Gly
-A -Lys -t(is -B -!/al -
Tyr-Tyr-Thr-Cys-Cys-Pr
o −Asp −Thr −Pro −Tyr= Le
u -Asp -Y -Z
(1) [wherein A and B each represent Phe or Tyr, X and Y each represent a single bond, or 2 to loI [i represents a peptide residue formed by a peptide bond, Z represents a hydroxyl group or an amine 7 group, and in the above amino acid sequence Cys - Cys, the mercapto groups possessed by each Cys are bonded to each other to form a disulfide bond. Good too. ] This is achieved by providing a peptide shown in the following.

Various amino acid residues are described herein using conventional abbreviations. Although the abbreviations are well known in the technical field of the present invention, those related to the present invention are listed below.

Asp: L-aspartic acid residue Cys:
L-cystine residue Glu: L-glutamic acid residue GLy: glycine residue
Base: L-histidine residue Leu: L-leucine residue Lys: L-lysine residue Phe: L-phenylalanine residue Pro:
L-proline residue Thr: L-) leonine residue Tyr: L-thyronine residue Vat': L-valine residue In addition, in this specification, the amino acid sequence is determined according to a conventional method so that the N-terminal amino acid is located on the left side. , are written so that the C-terminal amino acid is located on the right.

Peptides in which X and Y in general formula ([) are defined as above, but both X and Y are single bonds, and amino acid residues or peptides in which either X or Y is different from those defined above The peptide residue may not be immobilized efficiently on the carrier, but also may not fully develop its ability to adsorb human antibodies against nicotinic acetylcholine receptors even if it is immobilized on the carrier and heat-treated. It may not. - Examples of the peptide residues represented by X and Y in the general formula (+) include the following peptide residues.

1^sp-^ps-, -Glu-Glu-, -Lys-
Lys --Gly-Lys- NH(CH,) , C-Glu- -N)I(Cut)ttc Lys-■ -Lys-Lys-Gly- (Asp)s, +Glu)s, (Lys)s,
4G1y)s.

-Lys -Asp -Glu -Gly -NH(C
H,) t tc -Lightning-Gly-Lys-Glu-Glu-^5p--Asp
-Glu -NH(Cut) ttc -Lys -
Gly −Lys −+As1))+o,4Glu)+
o, (Lys)to, 4GIV)to.

-Asp-Glu +, -Asp-Gly +,
-Glu-Asp--Lys -Glu-Gly
-NH(CH,) + rc -Asp -Asp
-Lys -Lys -Glu -Gly --Lys
-Glu-Glu-Gly-^sp-^5p-Lys-
Lys-Gly-Gly- The synthesis of the peptide represented by the general formula ([) involves the reaction of a styrene-divinylbenzene copolymer bonded with an acyloxy group or an acylamino group obtained by removing a hydrogen atom from a peb group, respectively. The corresponding amino acid was added to the solvent-insoluble polymer in the direction of the N-terminus of the peptide of interest.
Journal of the American'C
'chemical 5ociety), Vol. 85, pp. 2149-2154 (1963); Japanese Society of Chemical Chemistry l
s "Biochemistry Experiment Lecture 1 Protein Chemistry - Chemical Modification and Peptide Synthesis" (Published by Tokyo Kagaku Doujin Co., Ltd., 15th May 1975), pp. 207-495; Japan Biochemistry Golden Line "Biochemistry Experiment Lecture" 2 Chemistry of Proteins (Part 2)” (Showa 6)
Published by Tokyo Kagaku Doujin Co., Ltd. on May 20, 2015), No. 641
See pages 694, etc.].

In the production of the peptide represented by the general formula (1) by solid-phase synthesis, for example, a protecting group having an α-carboxyl group or an α-carbamoyl group possessed by an amino acid or an amino acid amide corresponding to the C-terminus of the target peptide is used. By sequentially repeating the removal operation, the peptide chain is elongated to form a peptide chain corresponding to the desired peptide, and then the peptide chain is removed from the polymer, and the protecting group is removed from the protected functional group. The target peptide is obtained by removing the peptide, and then purified. Here, it is preferable to simultaneously remove the peptide chain from the polymer and remove the protecting group using hydrogen fluoride from the viewpoint of suppressing side reactions. Furthermore, it is effective to purify the obtained peptide by reverse-phase liquid chromatography.

The peptide represented by general formula (1) can be efficiently immobilized on a carrier. General formula (
When the peptide shown in 1) is subjected to heat treatment, it significantly exhibits the ability to adsorb human antibodies against nicotinic acetylcholine receptors in body fluids such as autopsy. The carrier used for immobilizing the peptide represented by the general formula (1) has a hydrophilic surface and is compatible with the peptide or has an average pore diameter within the range of about 50 to 1000 nanometers. It is preferable to use The carrier can be in any shape such as particulate, fibrous, sheet, or hollow fiber. Such carriers include, for example, C
M-Cellulofine CH (exclusion limit protein molecular weight:
Cellulose-based carriers such as CM-Toyovar 650C (exclusion limit protein molecular weight: 5X to'; manufactured by Tosoh Corporation); CM-h Lisacrylic M (CM-TrisacrylM) [Exclusion limit protein molecule 1: IX 10'; Pharmacia LKB, Sweden
When used as an adsorbent for adsorbing human antibodies against a human antibody, the carrier is preferably porous and insoluble in body fluids. Porous carriers have a large effective surface area that can adsorb human antibodies against nicotinic acetylcholine receptors, so the exclusion limit protein molecular weight for such carriers is approximately 106 to 10.
Organic carriers such as agarose-based carriers such as 1-manufactured by LKB); and c p c -lo-
+ooo [Exclusion limit protein molecular weight: 1X1(1'
;Average pore diameter: lQOnm''; American Electronucreonics (Electr).

Examples include inorganic carriers such as porous glass (manufactured by Nucleonics).

The peptide represented by the general formula (+) is immobilized on a carrier according to a method generally employed for immobilizing peptides or proteins on carriers. As for its immobilization method, for example, the carboxyl group of the carrier is reacted with N-hydroxysuccinimide to convert it into a succinimide oxy7carbonyl group, and then a peptide represented by the general formula ([) is added to the amine group. partial reaction method (active ester method), in which the amino group or carboxyl group of the carrier is subjected to a condensation reaction with the carboxyl group or amine group of the peptide represented by general formula (1) in the presence of a condensation reagent such as synchhexylcarbodiimide. The method (condensation method) and the immobilization amount of the peptide represented by the general formula (1) on the carrier are such that the resulting adsorbent can absorb a significant amount of human antibodies against nicotinic acetylcholine receptors. For adsorption, Jg usually needs to be about 3X IQ" mol/g (carrier) or more, and the peptide shown by the general formula (1) immobilized on the carrier is effectively used for adsorption of human antibodies. For about IX 10-' to 2X to-' mol/g (carrier)
It is preferable that it is within the range of .

The peptide represented by general formula (1) immobilized on a carrier exhibits high activity for adsorbing human antibodies against nicotinic acetylcholine receptors when subjected to heat treatment as described above. The heat treatment temperature is preferably 60°C or higher, but if the heat treatment temperature is too high, the peptide and/or carrier may decompose.
It is preferable to suppress the temperature of the heat treatment to 180°C or less.

Further, the heat treatment time is preferably about 5 minutes or more, but if the heat treatment time is too long, the peptide and/or carrier may decompose, so the heat treatment time is about 1 minute or more.
It is preferable to do so within hours. Heat treatment is preferred from the standpoint of suppressing the decomposition of the peptide.

Removal of antibodies against nicotinic acetylcholine receptors can be carried out by: - Contacting an adsorbent obtained by immobilizing the peptide represented by formula (1) on a carrier with body fluids such as blood, plasma, and pure blood containing the antibody; This is carried out by adsorbing the antibody to. For example, the adsorbent is used by filling a column.

The column used for this purpose has an artificial part and an outlet part shaped to be easily connected to the blood circuit, and a material such as polyester between the artificial part and the adsorbent layer and between the outlet part and the adsorbent layer. It is desirable to have a filter made of the same material. Examples of the material for the column include polyethylene, polypropylene, polycarbonate, polyester, and polymethyl methacrylate. Among these, polypropylene and polycarbonate are particularly preferred in that the column filled with the adsorbent can be subjected to sterilization such as autoclave sterilization or γ-ray sterilization before use.

can be done.

(1) Blood taken out from a patient's blood vessel is coated with an adsorbent at -4
, to a packed column where antibodies against nicotinic acetylcholine receptors are removed from the blood by adsorption, and the treated blood passing through the column is then circulated into the patient's blood vessels.

(2) Blood taken from the patient's blood vessel is first separated into blood cell components and plasma components, and the separated plasma components are sent to a column filled with an adsorbent, where antibodies against nicotinic acetylcholine receptors are adsorbed from the plasma components. The treated plasma components removed by and passed through the column are mixed with the separated blood cell components, and the resulting mixture is then circulated into the patient's blood vessels.

[Example] The present invention will be explained below with reference to Examples, but the present invention is not limited by the Examples.

Example 1 Formula H-Lys-Lys-Gly-Phe-Ly
It was synthesized by a solid phase synthesis method using Model 430A manufactured by s-His-Phe-Biosystems. Styrene-vinylbenzene copolymer having 4 to [N-(1-butoxycarbonyl)glycyloxymethyl]phenylacetamidomethyl groups at a ratio of 0.78 mmol/g (resin) [Styrene and divinylbenzene composition] 99:1] [Applied Biosystems (U.S.)
Using 0.64 g of PAM Glycine and t-Boc-Gly (manufactured by Lied Biosystems), the corresponding L- Aspartic acid, L-cysteine, glycine, L-
Histidine, leucine, lysine, L-proline, L-luonine, enylalanine, L-tyrosine and valine were sequentially linked. In the condensation reaction, each of the above amino acids is converted into tLN-(t-butoxycarbonyl)-0'-bensica1carbonyl') -N''-1-
sil-histidine anhydride, N-(t-butoxycarbonyl)-L-ro/lysine anhydride, N″-(t-butoxycarbonyl)I@-benzyloxycarbonyl-lysine anhydride, N-(t-butoxycarbonyl)-L
-7'loline anhydride, N-(t-butoxycarbonyl)
-□3-Benzyl-L-) leonine anhydride, N'-(1
-butoxycarbonyl)-shi-phenylalanine anhydride, N-(t-butoxycarbonyl)-0'-benzyl-
-Tyrosine anhydride and -N-(-butoxycarbonyl)-L-valine anhydride were used, and the amount used was about twice the molar amount relative to the substrate. The condensation reaction was carried out at room temperature, and the reaction time varied depending on the type of amino acid to be condensed, but was within the range of 18 to 30 minutes. Also N”-(
Since the conversion rate is low in the condensation reaction using L-butoxycarbonyl)-N'm'-tosyl-L-histidine anhydride, after completing the series of operations shown in Table 1, step 4 in Table 1 is further carried out. The condensation reaction was carried out again by repeating steps 6 to 6.

After completing the reaction operations for all amino acids, the resulting resin was filtered with diethyl ether and diluted with diethyl ether on a glass filter.
Wash sequentially with dichloromethane and methanol,
Next, 2.1 g of dry resin was obtained by vacuum drying. Polytrifluoromonochrome aoethylene reaction vessel (HF-reactor type I manufactured by Peptide Institute Co., Ltd.)
Hydrogen fluoride, anisole and ethylmethyl sulfide were removed from the dry phase mixture under reduced pressure, and the residue was filled with diethyl ether on a glass filter.
Washed for minutes. The obtained residue was extracted with a 2N acetic acid aqueous solution, and the extract was freeze-dried to obtain 0.5 mg of a crude peptide.

The obtained crude product was subjected to preparative reverse phase high performance liquid chromatography [Column: octadecylated silica gel (particle size: 5 μm).
) Packed column (inner diameter: loms, length = 30 ha)
(Develosi manufactured by Kemuco Co., Ltd.)
l) OD S 1oanφX 30Gms')
; By purification with a mixed solvent of acetonitrile and water containing 0.05% by volume of trifluoroacetic acid as a mobile phase (the concentration of acetonitrile was gradually changed from 20% to 35% by volume in 20 minutes)] , 50 t@g 14 of the purified peptide of interest was prepared.

The obtained purified product was subjected to analytical reverse phase high performance liquid chromatography [Column: octadecylated silica gel (particle size: 5 μm).
) Packed column (inner diameter: 4m+e, length = 150mm)
(Tosoh Corporation!l!TSKgelODS-: Flow rate: 1 m12/min; Detection method Absorbance at dual wavelength 2i0 nm) showed a single sharp peak at 18.1 min. The molecular weight of the purified product determined by mass spectrometry (atomic bombardment) was 2737 (theoretical value: 27:(7,15).In addition, the product obtained by hydrolyzing the purified product with hydrochloric acid was The results of amino acid composition analysis were as follows (numbers in parentheses indicate theoretical values): Lysine: 5.22 (5), Glycine: 1.95 (2), Phenylalanine = 2, 0f (
2), Histidine: 0.97(1), Valine: 0
．． 93(1), Tyrosine: 3.07(3), Threonine: 2.05(2).

Cystine: 0.87(1), Proline: 2.13(
2), Aspartic acid: 2.10 (2), Leucine: 1.01 (1).

Examples 2 to 16 The peptides shown in Table 2 were obtained by solid phase synthesis and purification of peptides in the same manner as in Example 1. However, in Examples 2 and 10, as the solid phase resin, 4-[N-(t-butoxycarbonyl)glycyloxymethyl]phenylacetamidomethyl group was added to 0.
78 mmol/g country, PAM manufactured by Applied Biosystems (App!ied[3iHystelIs)
Glycine +t -Boc -G
Using lyl, Example 3, Example 5, Example 4-1 Example 8,
and in Example 12, styrene having 4-[N-(t-butoxycarbonyl)-o'-benzyl-α-L-asbartyloxymethylcophenylacetamidomethyl group at a ratio of 0.78 mmol/g (resin). - Granular resin consisting of divinylbenzene copolymer [component ratio (molar ratio) of styrene and divinylbenzene: 99:1] [Applied Biosystems, USA]
PAM aspartic acid (Aspa
rtic acid), t-BoaL-As
p (OBZI) ], Example 4 and Example 6
Then, the 4-[N-(t-butoxycarbonyl)-05-benzyl-α-L-glutamyloxymethyl]phenylacetamidomethyl group was 0.78 mmol/g (resin).
Styrene-divinylbenzene copolymer having a proportion of [
Composition ratio (mole ratio) of styrene and divinylbenzene, 99
granular resin [Applied Biosystems (U.S.A.)
Consisting of a styrene-divinylbenzene copolymer having L-lysyloxymethyl]phenylacetamidomethyl groups at a ratio of 0.78 mmol/g (resin) [composition ratio (mole ratio) of styrene and divinylbenzene, 99:l] Granular resin [U.S. Applied Biosystems (＾p
PAM Lysine (PliedBiosystems)
Lysine), t-Boc-L-Lys
(C12-Z)] and Examples 13 to 16
From 6 styrene-divinylbenzene copolymers having α-amino-p-methylbenzyl groups at a ratio of 0.78 mmol/g (resin) [composition ratio (mole ratio) of styrene and divinylbenzene: 99:1] granular resin [Applied Biosystems (U.S.)
ysLea+s) company! f! p-Methyl BHA resin (p
-Methyl BHA Re5in)] was used. In addition, in the condensation reaction, monoglutamic acid, 12-aminododecanoic acid, and 18-aminooctadecanoic acid were converted to N-(t-butoxycarbonyl)OS-benzyl-glutamic anhydride, 12-(t-butoxycarbonylamino)dodecane, respectively. Table 3 shows the molecular weight determined by acid-free FAB mass spectrometry and the amino acid composition analysis of the product obtained by hydrolysis with hydrochloric acid.

No. table Numbers in parentheses indicate theoretical values.

No. table (continuation) Numbers in parentheses indicate theoretical values.

Table (continued) Table (continued) Numbers in parentheses indicate theoretical values.

Table 4 (Continued) Peptides shown in Table 4 were obtained by performing phase combination and purification. However, as the solid phase resin, Example 17,
In Example 18 and Example 26, 4″r-(N-(t-
Consisting of a styrene-divinylbenzene copolymer having (butoxycarbonyl)glycyloxymethylcophenylacetamidomethyl groups at a ratio of 078 mmol/g (resin) [composition ratio (mole ratio) of styrene and divinylbenzene = 99:1] Granular resin [P manufactured by Applied Biosystems, USA]
AM Glycine, t-B
oa -Glyl was used, and in Examples 19, 21, 24, and 28, 4-[N-(t-butoxycarbonyl-04=benzyl-α-L-asbartyloquinemethyl]phenylacetamidomethyl Copolymerization K
A granular resin consisting of a styrene-divinylbenzene copolymer E having a ratio of 0.78 mmol/g (resin), a composition ratio (mole ratio) of styrene and divinylbenzene of 99 to l] was manufactured by Applied Biosystems (Appli) in the United States.
ed Biosystems) PAM Asparagi 1 phenylacetamidomethyl group 0.78 mmol/
A granular resin [U.S. Applied Co., Ltd.
Biosystems (AppliedBtosys+te)
ms) PAM glutamic acid (G1utaa+i
c acid), t-Boa-L-Glu
(OBzl)
-lysyloxymethyl]phenylacetamidomethyl group at a ratio of 0.78 mmol/g (resin) [mol ratio of styrene and divinylbenzene = 99:1]. Resin [Applied Biosystems (U.S.)
d Biosysta+*s) PAM lysine (Ly
sine), t-Boa-L-Lyg (C1
2-Z) ], and in Examples 24 to 32, α-
Amino-p-methylbenzine group 0.711 mmol/
Styrene-divinylbenzene p-Methyl BHA resin (p-Methyl
BHARe5in)] was used. In addition, in the condensation reaction, L-glutamic acid, 12-aminododecanoic acid, and 18-aminooctadecanoic acid are each converted to N-(t
-butoxycarbonyl)O5-benzyl-glutamic anhydride, 12-(t-butoxycarbonylamino)dodecanoic anhydride and 18-(t-butoxycarbonylamino)octadecanoic anhydride.

When the purified peptides obtained were subjected to analytical reverse phase high performance liquid chromatography, a single peak was observed in each case. Table 5 shows the molecular weights of these purified products determined by FAB mass spectroscopy and the amino acid composition analysis values of the products obtained by hydrolysis with hydrochloric acid.

No. table No. table (continuation) Numbers in parentheses indicate theoretical values.

Table (continued) Table (continued) Numbers in parentheses indicate theoretical values.

No. Table (continued) Numbers in parentheses indicate theoretical values.

Examples 33 and 34 By performing solid phase synthesis and purification of peptides in a manner similar to that in Example 1, compounds of the formula H-G[Y-Phe-
Lys -His -Phe -Val-Tyr -T
Peptide represented by yr -Thr-Oll (Example 3
3) and formula 8 formula% were obtained. However, as a solid phase resin, 4-[N(t-
butoxycarbonyl)-0'-benzine-α-L-aspartyloxymethyl]phenylacetamidomethyl group at 0.78 t remol/g (m fat) PAM Aspartic acid, tB -B
oc-L-Asp(0Bzl)] was used.

1 The obtained purified peptide was subjected to analytical reverse phase high performance liquid chromatography, and the amounts and amino acid composition analysis values of the products obtained by hydrolysis using hydrochloric acid are shown in Table 6. show.

0.5 g of N-hydroxysuccinimide and 1.0 g of dicyclohexylcarbodiimide were added to the resulting suspension. og was added and the mixture was shaken and stirred at room temperature overnight. The resulting mixture was washed with 0.02 mol/Q phosphate buffer (pH: 7.4) and filtered with suction. The obtained particles were mixed with 2 of the peptide obtained in Example 1.
0.02 mol/Q phosphate buffer (pH: 7.4) containing 0.011E, and the mixture was
Stirred overnight at a temperature of .degree. The resulting mixture was passed under suction. The solution was subjected to analytical reverse phase high performance liquid chromatography, but no residual unreacted peptide was observed (immobilization rate of peptide on carrier: approximately 100%). In this way, about lag of cellulose particles on which 20 mg of the peptide obtained in Example 1 was immobilized were obtained.

(b) 1 g each of the cellulose particles on which the peptides obtained as described above are immobilized were added to a 0.02 mol/Q phosphate buffer containing 0.15 mol/Q sodium chloride (pH 7.4) Thermo-cleonics (1:1 ecjro-nucleonitics) suspended in 5- and heated at 121'C under pressure in an autoclave sterilizer for 20 min at 1 degree.
cs) CP G-10-1000 log, γ
The reaction was carried out in a toluene solution containing 5-aminopropyltriethoxysilane under heating and reflux for 24 hours. The resulting mixture is washed with dioxane obtained by distillation in the presence of metallic sodium,
Suction failed. Dioxane 100- obtained by distilling the obtained particles in the presence of metallic sodium
3 g of succinic acid was added to this suspension and the mixture was stirred at room temperature overnight. The resulting mixture is
Wash with dioxane obtained by distillation in the presence of metallic sodium and suction. The particles obtained are suspended in dioxane 50a+Q obtained by distillation in the presence of metallic sodium, and this suspension is
-0.5 g of hydroquine succinimide and 10 g of dicyclohexylcarbodiimide were added and the mixture was stirred at room temperature overnight.

The resulting mixture was diluted with 0.02 mol/Q phosphate buffer (
pH: 7.4) and filtered with suction. About 10 g of lath particles containing peptide 20B obtained in Example 3 were obtained from the obtained particles (peptide immobilization rate: about 100%).
.

(b) Same procedure as in Reference Example 2(b) except that Ig of the porous glass particles on which the peptide was immobilized obtained as described above was used instead of 1 g of polyvinyl alcohol particles on which the peptide was immobilized. According to the method, an adsorbent was obtained.

Reference Examples 4 to 16 (a) Reference examples except for using 20 mg of the peptide shown in Table 7! (a), Reference Example 2 (a) or Reference Example 3
Particulate carriers on which peptides were immobilized were obtained by the same method as in either (a). Table 7 shows the particulate carrier used and the immobilization rate of the peptide on the carrier.

(b) Reference except that 1 g of the particulate carrier on which the peptide obtained as above was immobilized was used instead of 1 g of polyvinyl alcohol particles on which the peptide obtained in Reference Example 2(a) was immobilized. The adsorbents were each obtained by a method similar to that in Example 2(b).

Example 5 Example 5 Example 6 Example 7 Example 8 Example 9 Example 1O Example What was obtained in Example 11 What was obtained in Example 12 What was obtained in Example 13 What was obtained in Example 14 What was obtained in Example 15 Cellulose particles about 97 Cellulose particles about 100 Polyvinyl alcohol particles approx. 94 Polyvinyl alcohol particles approx. 96 Cellulose particles approx. 94 Cellulose particles approx. 99 Cellulose particles approx. 99 Cellulose particles approx. 98 Polyvinyl alcohol particles approx. 92 Polyvinyl alcohol particles approx. 100 Porous glass particles Approximately 96 CM-Cellulofine CH Polyvinyl alcohol particles; CM-Toyopearl 650C manufactured by Tosoh Corporation Porous glass particles: CP manufactured by Electro-nucleonics, USA
G-10-1000 Reference Example 17 (a) Reference Example 1 Example 1 was prepared in the same manner as in (a) except that 20 mg of the peptide obtained in Example 33 was used instead of the peptide 2Q+ig obtained in Example 1. Approximately log cellulose particles were obtained on which 15.0 mg of the peptide obtained in 33 was immobilized (peptide immobilization rate: approximately 75%).

(b) By the same method as in Reference Example 2(b) except that 1 g of the cellulose particles on which the peptide was immobilized obtained as described above was used instead of the polyvinyl alcohol particles tg on which the peptide was immobilized. , an adsorbent was obtained.

Reference Example 18 (a) Example 3 was prepared in the same manner as in Reference Example 2(a) except that 20 mg of the peptide obtained in Example 34 was used instead of 20 mg of the peptide obtained in Example 2.
15.2B of the peptide obtained in step 4 was immobilized.
,! ! Approximately log polyvinyl alcohol particles were obtained (immobilization rate of Bebuti 1: approximately 76%).

(b) 1 g of polyvinyl alcohol particles obtained by the above immobilization operation was obtained in Reference Example 2 (a); Polyvinyl alcohol particles 1 on which peptide was immobilized
An adsorbent was obtained in the same manner as in Reference Example 2(b) except that g was used instead of g.

Reference Examples 19-34 Reference Example 1 except that 30 mg of the peptide shown in Table 8 was used.
(a), Reference Example 2(a), or Reference Example 3(a), a particulate carrier on which a peptide was immobilized was obtained, respectively. Table 8 shows the particulate carrier used and the immobilization rate of the peptide on the carrier.

(b) Reference except that 1 g of the particulate carrier on which the peptide obtained as above was immobilized was used instead of 1 g of polyvinyl alcohol particles on which the peptide obtained in Reference Example 2(a) was immobilized. The adsorbents were each obtained by a method similar to that in Example 2(b).

No. m-23 obtained in Example 21 24 Obtained in Example 22 25 Obtained in Example 23 26 Obtained in Example 24 27 Obtained in Example 25 28 Obtained in Example 26 29 Obtained in Example 27 30 Obtained in Example 28 31 Obtained in Example 29 32 Obtained in Example 30 33 The results obtained in Example 31 table cellulose particles cellulose particles polyvinyl alcohol particles polyvinyl alcohol particles cellulose particles cellulose particles cellulose particles cellulose particles polyvinyl alcohol particles polyvinyl alcohol particles porous glass particles Approximately 98 Approximately 100 Approximately 95 Approximately 95 Approximately 95 Approximately 98 Approximately 100 Approximately 100 Approximately 90 About io.

Approximately 95 CM-Cellulofine CH Polyvinyl alcohol particles: CM-Toyovar 650C manufactured by Tosoh Corporation Porous glass particles/CP manufactured by Electro-nucleonics, USA
G-10-1000 Test Example 1 Adsorbent 50B obtained in Reference Example 1 was added to 0.5-ml serum of a myasthenia gravis patient and suspended at a temperature of 37°C for 3 hours. The resulting suspension was centrifuged and the supernatant was quenched. The concentration of human antibody against nicotinic acetylcholine receptor in the obtained supernatant was determined by the Con A method [Protein Nucleic Acid Enzyme, Vol. 26, pp. 1578-'1591 (1981).
etc.]. In other words, the test solution contains nicotinic acetylcholine receptors and radiolabeled α-
By successively contacting with bungarotoxin, the resulting treated solution was passed through a column packed with Sepharose ((S″6ph3rose)) on which concanavalin, A (Con A) was immobilized, and the radioactivity of the column was measured. The amount of human antibody that inhibits the binding between α-bungarotoxin and nicotinic acetylcholine receptor contained in the test solution was quantified as the toxin binding inhibition activity (rate of decrease in column radioactivity). The results are shown in Table 9.For comparison, the results obtained when using the cellulose particles (without heat treatment) on which the peptide obtained in Reference Example 1(a) was immobilized, as well as the Cellulose particles on which glycine is immobilized, obtained by the same method as in Reference Example 1(a) except that glycine is used instead of the peptide obtained in Example!, and cellulose particles on which this glycine is immobilized. Reference Example + Results obtained when using an adsorbent heat-treated at 121°C in the same manner as in (b) except that it is used instead of cellulose particles on which peptides are immobilized are also shown. It is shown in Table 9.

In Test Example 1, serum was suspended in the same manner except that the adsorbents obtained in Reference Examples 2 to 18 were used instead of the adsorbent obtained in Reference Example 1. Human antibodies against nicotinic acetylcholine receptors were determined. The results obtained are shown in Table 10. In addition, the same glycine-immobilized cellulose particles used for comparison in Test Example 1 were used at 12 t'c.
The results obtained when using the adsorbent obtained by heat treatment are shown in Table 1θ.

1 Peptide obtained in Example 1 8028 1 Peptide obtained in Example 1 too 24,
Peptide obtained in Example 1 121 2
04-Glycine without heat treatment Test Example 2 Reference Example Reference Example Reference Example Reference Example Reference Example Reference Example Reference Example Reference Example Reference Example 1 Reference Example 1 Reference Example 1 Reference Example 1 Reference Example 1 Reference Example 1 Reference Example 1 Reference Example 1 Obtained in Table 2 3 4 5 6 7 8 9 0 1 2 1/3 4 5 6 7 Test Example 3 Test Example 1 obtained in Reference Example 1 Serum was suspended in the same manner except that the adsorbents obtained in Reference Examples 19 to 34 were used instead of the obtained adsorbent), and the results were shown in Table 1. The concentration of human antibody was determined. The results obtained are shown in Table 11. Table 11 shows the results obtained when using an adsorbent obtained by heat-treating the same glycine-immobilized cellulose particles used for comparison in Test Example 1 at 121'C. show.

Reference Example 21 Reference Example 22 Reference Example 23 Reference Example 24 Reference Example 25 Reference Example 26 Reference Example 27 Reference Example 28 Reference Example 29 Reference Example 30 Reference Example 31 Reference Example 32 Reference Example 33 Novel peptides useful for efficiently manufacturing adsorbents are provided.

Patent applicant Juzo Iizuka, Director of the Agency of Industrial Science and Technology [Effects of the invention]

Claims (1)

[Claims] General formula H-X-Gly-A-Lys-His-B-Val-T
yr-Tyr-Thr-Cys-Cys-Pro-As
p-Thr-Pro-Tyr-Leu-Asp-Y-Z [wherein A and B each represent Phe or Tyr, X and Y each represent a single bond, or Asp, Glu, Lys and the formula ▲ There are mathematical formulas, chemical formulas, tables, etc.▼ (In the formula, n represents an integer from 1 to 17.)
represents a peptide residue formed by a peptide bond of 2 to 10 amino acid residues selected from the group consisting of divalent groups represented by or at least one type of amino acid residue selected from the group, and Z is Represents a hydroxyl group or an amino group, and each C in Cys-Cys of the above amino acid sequence
The mercapto groups of ys may be bonded to each other to form a disulfide bond. ] Peptide indicated by.