JPH04325088A - Protein derivative and production thereof - Google Patents

Abstract

PURPOSE:To obtain a protein derivative having an extremely prolonged life in blood in comparison with protein alone while nearly maintaining physiological activity of protein as it is by reacting a protein with a specific long-chain carboxylic acid imide ester in an aqueous solution at a specific pH. CONSTITUTION:A protein (e.g. superoxide dismutase) is reacted with a long-chain carboxylic acid imide ester [e.g. N-(13-carboxytridecanoyloxy)succinic acid imide] shown by formula I [W is bifunctional long-chain hydrocarbon which may be interrupted with one or more of O, S and NR (R is lower alkyl) in an aqueous solution at pH6-10 to give the objective protein derivative shown by the formula [protein] [Z]. ([protein] is protein containing X residue obtained by eliminating one H from amino group instead of amino group; [Z] is group shown by formula II; X is average value of number of amide bonds between [Z] and [protein] and is 1-8 number).

Description

[Detailed description of the invention]

0001

[Industrial Application Field] The present invention provides an enzyme or protein having physiological activity (hereinafter, enzymes and proteins are collectively referred to as proteins) whose physiological activity is largely retained while being significantly prolonged compared to that of the protein. The present invention relates to a novel protein derivative that has a long blood half-life, is non-antigenic, and can be administered in vivo, and a method for producing the same.

0002

BACKGROUND OF THE INVENTION Many attempts have been made to improve proteins using various modifiers. Polyethylene glycol (hereinafter abbreviated as PEG) can be cited as a modifier that has been most actively studied. PEG is used for example as anticancer agents such as asparaginase, arginase, and interleukin-2 (hereinafter referred to as IL).
-2), modification of urokinase as a thrombolytic agent, streptokinase, tissue plasminogen activator (hereinafter referred to as TPA), etc., treatment of enzyme deficiency. β-glucosidase, β-glucuronidase, α-galactosidase as an agent,
Modification of adenosine deaminase, etc., modification of uricase as a therapeutic agent for gout, modification of superoxide dismutase (hereinafter abbreviated as SOD) as an anti-inflammatory agent or therapeutic agent for ischemic diseases, etc. It is used to modify insulin as a therapeutic agent for diabetes, and to modify bilirubin oxidase as a therapeutic agent for hyperbilirubinemia. Furthermore, recently, human granulocyte colony stimulating factor (hereinafter referred to as G-CSF), which is one of the hematopoietic promoting factors, has been modified with PEG to extend its lifespan in the blood and to treat hematopoietic disorders. Attempts have also been made to use it in
-316400 Publication and International Publication WO90/06
(See Publication No. 952). In addition, as modifiers other than PEG, natural polymer substances such as serum albumin and dextran,
Examples include polyaspartic acid, partially half-esterified styrene-maleic anhydride copolymer (hereinafter abbreviated as SMA), and reactive derivatives of long-chain fatty acids (``protein hybrid'', Chapter 1, Chapter 2
Chapters, Chapters 3 and 6, Kyoritsu Publishing Co., Ltd., 1987
Published on April 1, 2016; “Protein Hybrid”
Chapters 3, 4 and 6, Kyoritsu Publishing Co., Ltd., 19
Published May 20, 1988; “New Knowledge of SOD”, 107
Page, Japan Axel Springer Publishing Co., Ltd., 1
(Reference: December 20, 1990).

0003

[Problem to be solved by the invention] SOD modified with serum albumin has antigenicity [Agents and Actions]
s), Vol. 10, p. 231 (1980)]. Also,
Although the structure of modifiers such as dextran, PEG, polyaspartic acid, and SMA can be specified using polymer chemistry, their molecular weights have a distribution, so the molecular weight of proteins modified with these agents is not constant, and pharmaceutical This poses a practical problem considering that it is desirable for the active ingredient to have a single chemical structure.

[0004] One object of the present invention is to modify proteins with a modifier having a single chemical structure, thereby retaining most of the physiological activity of the protein and significantly reducing blood flow compared to proteins. extended midlifespan, non-antigenic,
Another object of the present invention is to provide a novel protein derivative that can be administered to living organisms.

Another object of the present invention is to provide a method for producing the above protein derivative.

[0006]

[Means for Solving the Problems] According to the present invention, one of the above objects is achieved by the following general formula:

[Protein][Z]x

(wherein, [protein] represents a protein having x residues obtained by removing one hydrogen atom from an amino group instead of an amino group, and [Z] represents the following general formula

[0009]

[C3]

(In the formula, W may be interrupted by one or more oxygen atoms, sulfur atoms, or a group represented by -N(R)- (in the formula, R represents a lower alkyl group). represents a residue obtained by removing the hydroxyl group from one carboxyl group of a long-chain dicarboxylic acid (representing a long-chain hydrocarbon group) (hereinafter, this will be abbreviated as a long-chain carboxylic acid residue), and x is [ Z]
means the average number of amide bonds with [protein], 1
8).

[0011] According to the present invention, another object of the above is to combine a protein with the following general formula (I).

0012

[C4]

(In the formula, W may be interrupted by one or more oxygen atoms, sulfur atoms, or a group represented by -N(R)- (in the formula, R represents a lower alkyl group). long-chain carboxylic acid imide ester (hereinafter referred to as long-chain carboxylic imide ester (representing a long-chain hydrocarbon group))
This is achieved by providing a method for producing a protein derivative, which is characterized by reacting (abbreviated as I)) in an aqueous solution at pH 6 to 10.

Protein and long chain carboxylic acid imide ester (
The reaction with I) differs slightly depending on the protein, but usually the protein is dissolved in an aqueous solution of a salt such as sodium carbonate, sodium bicarbonate, sodium acetate, or sodium phosphate, and the resulting solution is injected with a powdered long-chain carboxylic acid. Acid imide ester (
I) or a long chain carboxylic acid imide ester (I) dissolved in an organic solvent such as dimethyl sulfoxide. During the reaction, the pH of the solution is 6 ~
It is necessary to maintain it within the range of 10. When the pH is lower than 6, the solubility of the long-chain carboxylic acid imide ester (I) decreases, making it difficult for the reaction to proceed. Furthermore, if the pH is higher than 10, the protein is often inactivated, making it impossible to efficiently obtain the protein derivative of the present invention. The reaction temperature is below the protein denaturation temperature, usually about 3 to 50°C.
Temperatures in the range of about 3 to 40 degrees Celsius are preferred, and temperatures in the range of about 3 to 40 degrees Celsius are more preferred. In addition, the reaction time is the reaction temperature,
Although it varies depending on the method of addition of the long-chain carboxylic acid imide ester (I), it is usually in the range of about 10 minutes to 30 days. The amount of long-chain carboxylic acid imide ester (I) used is in the range of about 1 to 100 moles per mole of protein. When using SOD as a protein,
The amount of long-chain carboxylic acid imide ester (I) used is SO
Preferably, it ranges from about 2 to 50 moles per mole of D1. The number of molecules of long-chain carboxylic acid residues to be bound to the protein can be adjusted by the amount used.

The reaction solution thus obtained contains the protein derivative, unreacted protein, long-chain carboxylic acid imide ester (I), etc., but the reaction solution is filtered, and the filtrate is gel-filtered. The resulting eluate containing the protein derivative is subjected to hydrophobic chromatography, ion exchange chromatography, etc. as necessary, concentrated by ultrafiltration, and lyophilized to obtain the protein derivative. Solid matter can be obtained.

Through the above reaction, the amino group of the protein reacts with the imidoester moiety of the long-chain carboxylic acid imide ester (I), producing a protein derivative.

In the protein derivative of the present invention, examples of the divalent long chain hydrocarbon group represented by W include the following groups.

(CH2)10, (CH2)11, (CH2)
2) 12, (CH2) 13, (CH2) 14, (CH2
)15, (CH2)16, (CH2)17, (CH2)
18, (CH2)19, (CH2)20, CH2CH=
CH(CH2)7, (CH2)2CH=CH(CH2)
7, (CH2)3CH=CH(CH2)7, (CH2)
4CH=CH(CH2)7, (CH2)5CH=CH(
CH2)7, (CH2)6CH=CH(CH2)7, (
CH2)7CH=CH(CH2)7, (CH2)8CH
=CH(CH2)7,(CH2)9CH=CH(CH2
)7, (CH2)10CH=CH(CH2)7, (CH
2) 11CH=CH(CH2)7, (CH2)8CH=
CHCH2, (CH2)8CH=CH(CH2)2, (
CH2)8CH=CH(CH2)3, (CH2)8CH
=CH(CH2)4,(CH2)8CH=CH(CH2
)5, (CH2)8CH=CH(CH2)6, (CH2
)8CH=CH(CH2)7, (CH2)8CH=CH
(CH2)8, (CH2)8CH=CH(CH2)9,
(CH2)8CH=CH(CH2)10, CH2CH=
CHCH2CH=CH(CH2)7, (CH2)2CH
=CHCH2CH=CH(CH2)7, (CH2)3C
H=CHCH2CH=CH(CH2)7, (CH2)4
CH=CHCH2CH=CH(CH2)7, (CH2)
5CH=CHCH2CH=CH(CH2)7, (CH2
)6CH=CHCH2CH=CH(CH2)7, (CH
2) 7CH=CHCH2CH=CH(CH2)7, (C
H2)8CH=CHCH2CH=CH(CH2)7, (
CH2)2-O-(CH2)7, (CH2)2-O-(
CH2)8, (CH2)2-O-(CH2)9, (CH
2) 2-O-(CH2)10, (CH2)2-O-(C
H2)11, (CH2)2-O-(CH2)12, (C
H2)2-O-(CH2)13, (CH2)2-O-(
CH2)14, (CH2)2-O-(CH2)15, (
CH2)2-O-(CH2)16, (CH2)2-O-
(CH2)17, (CH2)4-O-(CH2)5, (
CH2)4-O-(CH2)6, (CH2)4-O-(
CH2)7, (CH2)4-O-(CH2)8, (CH
2) 4-O-(CH2)9, (CH2)4-O-(CH
2) 10, (CH2)4-O-(CH2)11, (CH
2) 4-O-(CH2)12, (CH2)4-O-(C
H2)13, (CH2)4-O-(CH2)14, (C
H2)4-O-(CH2)15, (CH2)6-O-(
CH2)3, (CH2)6-O-(CH2)4, (CH
2) 6-O-(CH2)5, (CH2)6-O-(CH
2) 6, (CH2)6-O-(CH2)7, (CH2)
6-O-(CH2)8, (CH2)6-O-(CH2)
9, (CH2)6-O-(CH2)10, (CH2)6
-O-(CH2)11, (CH2)6-O-(CH2)
12, (CH2)6-O-(CH2)13, (CH2)
8-O-CH2, (CH2)8-O-(CH2)2, (
CH2)8-O-(CH2)3, (CH2)8-O-(
CH2)4, (CH2)8-O-(CH2)5, (CH
2) 8-O-(CH2)6, (CH2)8-O-(CH
2) 7, (CH2)8-O-(CH2)8, (CH2)
8-O-(CH2)9, (CH2)8-O-(CH2)
10, (CH2)8-O-(CH2)11, (CH2)
10-O-CH2, (CH2)10-O-(CH2)2
, (CH2)10-O-(CH2)3, (CH2)10
-O-(CH2)4, (CH2)10-O-(CH2)
5, (CH2)10-O-(CH2)6, (CH2)1
0-O-(CH2)7, (CH2)10-O-(CH2
)8, (CH2)10-O-(CH2)9, (CH2)
12-O-CH2, (CH2)12-O-(CH2)2
, (CH2)12-O-(CH2)3, (CH2)12
-O-(CH2)4, (CH2)12-O-(CH2)
5, (CH2)12-O-(CH2)6, (CH2)1
2-O-(CH2)7, CH2-O-(CH2)5CH
=CH(CH2)7, (CH2)2-O-(CH2)5
CH=CH(CH2)7, (CH2)3-O-(CH2
)5CH=CH(CH2)7, (CH2)4-O-(C
H2)5CH=CH(CH2)7, (CH2)5-O-
(CH2)5CH=CH(CH2)7, (CH2)2-
S-(CH2)7, (CH2)2-S-(CH2)8,
(CH2)2-S-(CH2)9, (CH2)2-S-
(CH2)10, (CH2)2-S-(CH2)11,
(CH2)2-S-(CH2)12, (CH2)2-S
-(CH2)13, (CH2)2-S-(CH2)14
, (CH2)2-S-(CH2)15, (CH2)2-
S-(CH2)16, (CH2)2-S-(CH2)1
7, (CH2)4-S-(CH2)5, (CH2)4-
S-(CH2)6, (CH2)4-S-(CH2)7,
(CH2)4-S-(CH2)8, (CH2)4-S-
(CH2)9, (CH2)4-S-(CH2)10, (
CH2)4-S-(CH2)11, (CH2)4-S-
(CH2)12, (CH2)4-S-(CH2)13,
(CH2)4-S-(CH2)14, (CH2)4-S
-(CH2)15, (CH2)6-S-(CH2)3,
(CH2)6-S-(CH2)4, (CH2)6-S-
(CH2)5, (CH2)6-S-(CH2)6, (C
H2)6-S-(CH2)7, (CH2)6-S-(C
H2)8, (CH2)6-S-(CH2)9, (CH2
)6-S-(CH2)10, (CH2)6-S-(CH
2) 11, (CH2)6-S-(CH2)12, (CH
2) 6-S-(CH2)13, (CH2)8-S-CH
2, (CH2)8-S-(CH2)2, (CH2)8-
S-(CH2)3, (CH2)8-S-(CH2)4,
(CH2)8-S-(CH2)5, (CH2)8-S-
(CH2)6, (CH2)8-S-(CH2)7, (C
H2)8-S-(CH2)8, (CH2)8-S-(C
H2)9, (CH2)8-S-(CH2)10, (CH
2) 8-S-(CH2)11, (CH2)10-S-C
H2, (CH2)10-S-(CH2)2, (CH2)
10-S-(CH2)3, (CH2)10-S-(CH
2) 4, (CH2)10-S-(CH2)5, (CH2
)10-S-(CH2)6, (CH2)10-S-(C
H2)7, (CH2)10-S-(CH2)8, (CH
2) 10-S-(CH2)9, (CH2)12-S-C
H2, (CH2)12-S-(CH2)2, (CH2)
12-S-(CH2)3, (CH2)12-S-(CH
2) 4, (CH2)12-S-(CH2)5, (CH2
)12-S-(CH2)6, (CH2)12-S-(C
H2)7, (CH2)2-N(CH3)-(CH2)7
, (CH2)2-N(CH3)-(CH2)8, (CH
2) 2-N(CH3)-(CH2)9, (CH2)2-
N(CH3)-(CH2)10, (CH2)2-N(C
H3)-(CH2)11, (CH2)2-N(CH3)
-(CH2)12, (CH2)2-N(CH3)-(C
H2) 13, (CH2)2-N(CH3)-(CH2)
14, (CH2)2-N(CH3)-(CH2)15,
(CH2)2-N(CH3)-(CH2)16, (CH
2) 2-N(CH3)-(CH2)17, (CH2)4
-N(CH3)-(CH2)5, (CH2)4-N(C
H3)-(CH2)6, (CH2)4-N(CH3)-
(CH2)7, (CH2)4-N(CH3)-(CH2
)8, (CH2)4-N(CH3)-(CH2)9, (
CH2)4-N(CH3)-(CH2)10, (CH2
)4-N(CH3)-(CH2)11, (CH2)4-
N(CH3)-(CH2)12, (CH2)4-N(C
H3)-(CH2)13, (CH2)4-N(CH3)
-(CH2)14, (CH2)4-N(CH3)-(C
H2) 15, (CH2)6-N(CH3)-(CH2)
3, (CH2)6-N(CH3)-(CH2)4, (C
H2)6-N(CH3)-(CH2)5, (CH2)6
-N(CH3)-(CH2)6, (CH2)6-N(C
H3)-(CH2)7, (CH2)6-N(CH3)-
(CH2)8, (CH2)6-N(CH3)-(CH2
)9, (CH2)6-N(CH3)-(CH2)10,
(CH2)6-N(CH3)-(CH2)11, (CH
2) 6-N(CH3)-(CH2)12, (CH2)6
-N(CH3)-(CH2)13, (CH2)2-N(
C2H5)-(CH2)7, (CH2)2-N(C2H
5)-(CH2)8, (CH2)2-N(C2H5)-
(CH2)9, (CH2)2-N(C2H5)-(CH
2) 10, (CH2)2-N(C2H5)-(CH2)
11, (CH2)2-N(C2H5)-(CH2)12
, (CH2)2-N(C2H5)-(CH2)13, (
CH2)2-N(C2H5)-(CH2)14, (CH
2) 2-N(C2H5)-(CH2)15, (CH2)
2-N(C2H5)-(CH2)16, (CH2)2-
N(C2H5)-(CH2)17, (CH2)4-N(
C2H5)-(CH2)5, (CH2)4-N(C2H
5)-(CH2)6, (CH2)4-N(C2H5)-
(CH2)7, (CH2)4-N(C2H5)-(CH
2) 8, (CH2)4-N(C2H5)-(CH2)9
, (CH2)4-N(C2H5)-(CH2)10, (
CH2)4-N(C2H5)-(CH2)11, (CH
2) 4-N(C2H5)-(CH2)12, (CH2)
4-N(C2H5)-(CH2)13, (CH2)4-
N(C2H5)-(CH2)14, (CH2)4-N(
C2H5)-(CH2)15, (CH2)6-N(C2
H5)-(CH2)3, (CH2)6-N(C2H5)
-(CH2)4, (CH2)6-N(C2H5)-(C
H2)5, (CH2)6-N(C2H5)-(CH2)
6, (CH2)6-N(C2H5)-(CH2)7, (
CH2)6-N(C2H5)-(CH2)8, (CH2
)6-N(C2H5)-(CH2)9, (CH2)6-
N(C2H5)-(CH2)10, (CH2)6-N(
C2H5)-(CH2)11, (CH2)6-N(C2
H5)-(CH2)12, (CH2)6-N(C2H5
)-(CH2)13, (CH2)2-O-(CH2)2
-O-(CH2)4, (CH2)2-O-(CH2)2
-O-(CH2)5, (CH2)2-O-(CH2)2
-O-(CH2)6, (CH2)2-O-(CH2)2
-O-(CH2)7, (CH2)2-O-(CH2)2
-O-(CH2)8, (CH2)2-O-(CH2)2
-O-(CH2)9, (CH2)2-O-(CH2)2
-O-(CH2)10, (CH2)2-O-(CH2)
2-O-(CH2)11, (CH2)2-O-(CH2
)2-O-(CH2)12, (CH2)2-O-(CH
2) 2-O-(CH2)13, (CH2)2-O-(C
H2)2-O-(CH2)14, (CH2)4-O-(
CH2)2-O-(CH2)2, (CH2)4-O-(
CH2)2-O-(CH2)3, (CH2)4-O-(
CH2)2-O-(CH2)4, (CH2)4-O-(
CH2)2-O-(CH2)5, (CH2)4-O-(
CH2)2-O-(CH2)6, (CH2)4-O-(
CH2)2-O-(CH2)7, (CH2)4-O-(
CH2)2-O-(CH2)8, (CH2)4-O-(
CH2)2-O-(CH2)9, (CH2)4-O-(
CH2)2-O-(CH2)10, (CH2)4-O-
(CH2)2-O-(CH2)11, (CH2)4-O
-(CH2)2-O-(CH2)12, (CH2)2-
S-S-(CH2)7, (CH2)2-S-S-(CH
2) 9, (CH2)2-S-S-(CH2)11, (C
H2) 2-S-S-(CH2)13, (CH2)2-S
-S-(CH2)15, (CH2)4-S-S-(CH
2) 5, (CH2)4-S-S-(CH2)7, (CH
2) 4-S-S-(CH2)9, (CH2)4-S-S
-(CH2)11, (CH2)4-S-S-(CH2)
13, (CH2)6-S-S-(CH2)3, (CH2
)6-S-S-(CH2)5, (CH2)6-S-S-
(CH2)7, (CH2)6-S-S-(CH2)9,
(CH2)6-S-S-(CH2)11, (CH2)8
-S-S-CH2, (CH2)8-S-S-(CH2)
3, (CH2)8-S-S-(CH2)5, (CH2)
8-S-S-(CH2)7, (CH2)8-S-S-(
CH2)9

The protein derivatives obtained by the above reaction are mixtures obtained by reacting proteins with long chain carboxylic acid imide esters (I) of various numbers of molecules, and each protein derivative has a bond per molecule. The number of long-chain carboxylic acid residues present is not the same. therefore,
In the above general formula representing the protein derivative of the present invention, x means the average number of long-chain carboxylic acid residues bonded to one protein molecule. However, if protein derivatives having the same number of long-chain carboxylic acid residues that bind to proteins are desired, the protein derivatives obtained by the above method may be further subjected to operations such as gel filtration and ion exchange chromatography. By this, a desired protein derivative can be obtained. In addition, in the above reaction and post-reaction treatment,
The carboxyl group of a protein derivative may form an alkali metal salt or an ammonium salt, and protein derivatives having a carboxyl group that forms such a salt are also included in the protein derivative of the present invention.

The protein derivative of the present invention has a number of long-chain carboxylic acid residues in the range of 1 to 8 bound to one protein molecule, and has a significantly extended half-life in blood compared to unmodified protein. It has a period. Among the protein derivatives of the present invention, the neocarzinostatin (hereinafter abbreviated as NCS) derivative has alanine at the 1st position of NCS and 20
Preferably, both amino groups of the lysine at position are modified with long-chain carboxylic acid imide ester (I).

[0021] Examples of proteins that can be used as raw materials include the following.

Asparaginase, arginase, interleukin-1, IL-2, interleukin-3, interleukin-4, interleukin-5, interleukin
Leukine-6, interleukin-7, interleukin-8, urokinase, prourokinase, streptokinase, TPA, β-glucosidase, β-glucuronidase, α-galactosidase, adenosine Deaminase, uricase, SOD, insulin, bilirubin oxidase, G-CSF, granulocyte-macrophagic colony-stimulating factor, macrophagic colony-stimulating factor, N
CS, catalase, elastase, erythropoietin,
Interferon-α, interferon-β, interferon-γ, tumor necrosis factor-α, tumor necrosis factor-β
, nerve growth factor, epidermal growth factor, ovalbumin, platelet-derived growth factor, thrombomodulin, α1-antitrypsin, bone morphogenetic protein, cartilage-derived factor, fibroblast growth factor, growth hormone, transforming growth factor-β (TGF-
β), blood coagulation factor IX, protein C, protein S, insulin-like growth factor, calcitonin, somatostatin, tissue metalloprotease inhibitor (TIM
P), atrial natriuretic hormone, CD-4 protein, cystatin, calpastatin, ulinastatin, parathyroid hormone

The long chain carboxylic acid imide ester (I) has the following general formula (II):

HO2C-W-CO2H
(II)

One equivalent of N-
It can be produced by dehydration condensation in the presence of hydroxysuccinimide and dicyclohexylcarbodiimide (hereinafter abbreviated as DCC).

Furthermore, long-chain carboxylic acid imide ester (I
) is prepared by the following general formula (III) by dehydrating and condensing long-chain dicarboxylic acid (II) with 1 equivalent of benzyl alcohol in the presence of DCC.

[0027]

[C5]

Long-chain dicarboxylic acid monobenzyl ester (hereinafter referred to as long-chain dicarboxylic acid monobenzyl ester (II), where W is as defined above)
After obtaining the long-chain dicarboxylic acid monobenzyl ester (III) according to a conventional method, N-
By reacting with hydroxysuccinimide, the following general formula (IV)

[0029]

[C6]

A long chain dicarboxylic acid monobenzyl monosuccinimide ester (hereinafter abbreviated as long chain dicarboxylic acid diester (IV)) represented by the formula (wherein W is as defined above), and then It can also be produced by removing the benzyl ester moiety of the long-chain diester dicarboxylic acid (IV) by a conventional hydrolysis method.

The long-chain carboxylic acid imide ester (I) used as a raw material in the present invention has a fatty acid moiety. Therefore,
Proteins modified with such long-chain carboxylic acid imide esters (I) have reversible binding properties with serum proteins and biological membranes, thereby extending their blood half-life.
In addition, the transferability to organs is improved.

In the long-chain carboxylic acid imide ester (I), the number of atoms in the main chain of the long-chain hydrocarbon group represented by W is 8 to
It is preferable that it is 28, and it is more preferable that it is 10-20. When modifying SOD, it is particularly preferable that the number of atoms in the main chain of the long-chain hydrocarbon group represented by W in the long-chain carboxylic acid imide ester (I) is 10 to 15. Modified proteins obtained by reacting long-chain carboxylic acid imide esters (I) with less than 8 main chain atoms with proteins are not preferred because they have poor binding ability to serum proteins. Long-chain carboxylic acid imidoester (I) having more than 28 main chain atoms has a pH of 6 to
The solubility of 10 in an aqueous solution becomes poor, making it difficult to bind such a long-chain carboxylic acid imide ester (I) to a protein.

The protein derivative of the present invention effectively exhibits the pharmacological actions known to be possessed by the protein. For example, SOD derivatives have excellent anti-ulcer effects, as is clear from the results of Test Example 2 described below. Furthermore, SOD derivatives also have pharmacological actions such as anti-inflammatory action, anti-arrhythmia action, anti-ischemic injury action, and anti-cerebral edema action. Furthermore, NCS derivatives have excellent anticancer effects.

[0034] It has been confirmed that the protein derivative has low toxicity in toxicity tests.

From the above results, protein derivatives can be used as therapeutic and preventive agents for various diseases depending on the pharmacological action that the protein is known to have.

SOD derivatives are effective against various diseases involving active oxygen radicals, and are particularly used as anti-inflammatory agents, anti-ulcer agents, therapeutic agents for ischemic diseases, cerebral edema therapeutic agents, and paraquat therapeutic agents. be able to. SOD derivatives are also useful as medicines for reducing the side effects of anticancer drugs caused by active oxygen radicals. Furthermore, SOD derivatives are useful as therapeutic agents for skin diseases such as burns, trauma, and various dermatitis. In addition to the medicinal effects mentioned above, SOD derivatives have medicinal effects that SOD is originally known to have [Modern Igaku, Vol. 39, No. 2, 339 (1984); Medicine and Pharmacy, Vol. 14, No. 1, 55 (1985); Experimental Medicine, 4, No. 1 (1986), special feature/In vivo free radicals and diseases; and Fragrance Journal, No. 79, 89 (1986)], and also shows medicinal efficacy against diseases involving active oxygen radicals, for which SOD has no medicinal efficacy.

[0037] NCS derivatives are also useful as anticancer agents.

[0038] The dosage of the protein derivative varies depending on the disease, severity of the patient, drug tolerance, etc. For example, S
The dosage of OD derivatives is usually 0.00 mg per day for adults.
In the range of 1 to 500 mg, preferably 0.5
The amount ranges from 100 mg to 100 mg. The dosage of NCS derivatives varies depending on the administration method, the malignancy of the cancer, the type of cancer, the patient's medical condition and general condition, the progress of the cancer, etc.
It also varies depending on whether the purpose is to prevent lymph node metastasis during surgery, etc., or for treatment purposes, but the usual amount is 0 per day for adults.
．． 01 to 100 mg, preferably 0.1
Amounts ranging from ~10 mg. These doses are suitably administered at once or in divided doses. For administration, any form suitable for the administration route can be taken. To administer NCS derivatives to humans,
Administration methods include local tissue administration at the primary site of cancer, site of cancer removal after surgery, intradermal, subcutaneous, intramuscular, intravenous, intraarterial, oral, etc., and local application, spray, suppository, and intravesical administration. External administration methods such as injection are preferred.

The protein derivatives can be prepared for administration using any conventional method of formulation. A pharmaceutical composition containing at least one protein derivative is prepared by conventional means using any pharmaceutical carrier, excipients, and other medically acceptable additives.

[0040] When the pharmaceutical composition is an oral preparation, the preparation is preferably provided in a form suitable for absorption from the gastrointestinal tract. Tablets and capsules for oral administration are in unit dosage form and include binders such as syrup, acacia, gelatin, sorbitol, tragacanth, polyvinylpyrrolidone, etc.; excipients such as lactose, corn starch, calcium phosphate, sorbitol, glycine etc.; lubricants, such as magnesium stearate, talc, polyethylene glycol, silica, etc.; disintegrants, such as sodium lauryl sulfate, etc.; customary excipients may be included. The tablets may be coated by methods well known in the art. Oral liquid preparations may be aqueous or oily suspensions, solutions, syrups, elixirs, etc., or they may be dry products that are redissolved in water or other suitable vehicle before use. good. Such liquid preparations may contain commonly used excipients such as suspending agents such as sorbitol syrup, methyl cellulose, etc.
sugar, glucose/sugar syrup, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, aluminum stearate gel, hydrogenated edible fat, etc.; emulsifiers,
e.g. lecithin, sorbitan monooleate, gum arabic, etc.; water-insoluble vehicles, e.g. almond oil, fractionated coconut oil, oily esters, propylene glycol, ethanol, etc.; preservatives, e.g. p-hydroxybenzoic acid. It may also contain methyl, propyl p-hydroxybenzoate, sorbic acid, and the like.

[0041] When preparing an injection, the protein derivative is dissolved in a solvent such as physiological saline or glucose solution for injection, and the amount of the protein derivative is 2 to 20 mg/solvent.
Adjust the concentration to 10 ml and inject subcutaneously, intramuscularly, or intravenously using conventional methods. At the time of preparation, pH adjusters, buffers, stabilizers, preservatives, solubilizers, etc. may be added to the aqueous solution if necessary.

[0042] The above pharmaceutical composition may contain the protein derivative at a concentration of generally about 0.01 to 50% by weight, preferably about 0.1 to 20% by weight, depending on its form.

[0043]

[Examples] The present invention will be specifically explained below with reference to Examples. Note that the present invention is not limited to these Examples. 1H-NMR was measured using tetramethylsilane as an internal standard, and IR was measured by the KBr tablet method.

Reference Example 1 N-(13-carboxytridecanoyloxy)
Synthesis of succinimide 1,14-tetradecanedioic acid (1.0 g, 3.
87 mmol) in 15 ml of anhydrous tetrahydrofuran
A solution of N-hydroxysuccinimide (445 mg, 3.87 mmol) in 5 ml of anhydrous tetrahydrofuran and N,N were dissolved in the resulting solution.
- Dimethylaminopyridine hydrochloride (3.1 mg,
After stirring for 30 minutes,
A solution of DCC (799 mg, 3.87 mmol) in 5 ml of anhydrous tetrahydrofuran was added and stirred overnight. After filtering the reaction solution and concentrating the filtrate under reduced pressure,
The residue was subjected to silica gel column chromatography [Developing solution:
A mixture of benzene and chloroform (volume ratio: 1:3)
)] was separated and purified, and showed the following physical property values.N-(13
- Carboxytridecanoyloxy)succinimide (510 mg, 37%) was obtained. m. p. 116 to 118 °C FD-MS (m/z): [M+H] + 3561H-N
MR (CDCl3, 270 MHz): δ 1.1
8 ~ 1.47 (m, 16H), 1.63
(m, 2H), 1.74 (m, 4H), 2
．． 35 (t, 2H), 2.57 (t, 2H
), 2.84 (s, 4H), 7.85 ~ 1
0.50 (br,1H)IR (cm-1): 2
920, 2850, 1825, 1790, 17
40, 1725, 1710, 1210, 107
0

Reference Example 2 Synthesis of N-(15-carboxypentadecanoyloxy)succinimide 1,16-hexadecanedioic acid (1.0 g, 3.
49 mmol) in 30 ml of anhydrous tetrahydrofuran
A solution of N-hydroxysuccinimide (402 mg, 3.49 mmol) in 10 ml of anhydrous tetrahydrofuran and N,N were dissolved in the resulting solution.
- Dimethylaminopyridine hydrochloride (2.8 mg,
After stirring for 30 minutes, a solution of DCC (720 mg, 3.49 mmol) in 10 ml of anhydrous tetrahydrofuran was added and stirred overnight. After filtering the reaction solution and concentrating the filtrate under reduced pressure, the residue was subjected to silica gel column chromatography [Developing solution: a mixture of benzene and chloroform (volume ratio: 1
3)] and showed the following physical property values.
(15-carboxypentadecanoyloxy)succinimide (429 mg, 32%) was obtained. m. p. 118.5 ~ 121 °C FD-MS (m
/z): [M+H]+ 3841H-NMR (CDCl
3,270 MHz): δ 1.18 to 1.4
5 (m, 20H), 1.62 (m, 2H
), 1.74 (m, 4H), 2.34 (t
, 2H), 2.60 (t, 2H), 2.83
(s,4H)IR (cm-1): 2920,
2850, 1825, 1790, 1740, 1
725, 1710, 1210, 1070

Reference Example 3 Synthesis of N-(17-carboxyheptadecanoyloxy)succinimide 1,18-octadecanedioic acid (1.0 g, 3.
18 mmol) in 30 ml of anhydrous tetrahydrofuran
A solution of N-hydroxysuccinimide (366 mg, 3.18 mmol) in 10 ml of anhydrous tetrahydrofuran and N,N were dissolved in the resulting solution.
- Dimethylaminopyridine hydrochloride (2.5 mg,
After stirring for 30 minutes, a solution of DCC (656 mg, 3.18 mmol) in 10 ml of anhydrous tetrahydrofuran was added and stirred overnight. After filtering the reaction solution and concentrating the filtrate under reduced pressure, it was subjected to silica gel column chromatography [Developing solution:
Mixture of benzene and chloroform (volume ratio: 1:3
．． 5)], and showed the following physical property values.
17-carboxyheptadecanoyloxy)succinimide (480 mg, 37%) was obtained. m. p. 120 ~ 122.5 °C FD-MS (m
/z): [M+H]+ 4121H-NMR (CDCl
3,270 MHz): δ 1.13 to 1.4
7 (m, 24H), 1.63 (m, 2H
), 1.75 (m, 4H), 2.34 (t
, 2H), 2.60 (t, 2H), 2.84
(s, 4H), 5.0~7.0 (br, 1
H) IR (cm-1): 2920, 2850,
1825, 1790, 1740, 1725, 1
710, 1210, 1070

Reference Example 4 N-(19-carboxynonadecanoyloxy)
Synthesis of succinimide 1,20-eicosandioic acid (1.0 g, 3.1
8 mmol) in 50 ml of anhydrous tetrahydrofuran
A solution of N-hydroxysuccinimide (336 mg, 2.92 mmol) in 10 ml of anhydrous tetrahydrofuran and N,
N-dimethylaminopyridine hydrochloride (2.3 mg
, 0.015 mmol) and stirred for 30 minutes, and then added DCC (602 mg, 2.92 mmol).
) in 10 ml of anhydrous tetrahydrofuran was added and stirred overnight. After filtering the reaction solution and concentrating the filtrate under reduced pressure, it was separated and purified using silica gel column chromatography [developing solution: a mixture of benzene and chloroform (volume ratio: 1:3.5)], and the following physical properties were obtained. Show N
-(19-carboxynonadecanoyloxy)succinimide (420 mg, 33%) was obtained. m. p. 121.5 to 124 °C FD-MS (m
/z): [M+H]+ 4401H-NMR (CDCl
3,270 MHz): δ 1.14 to 1.4
5 (m, 28H), 1.63 (m, 2H
), 1.74 (m, 4H), 2.35 (t
, 2H), 2.60 (t, 2H), 2.84
(s,4H)IR (cm-1): 2920,
2850, 1825, 1790, 1740, 1
725, 1710, 1210, 1070

Reference Example 5 Synthesis of N-(21-carboxyheneicosanoyloxy)succinimide 1,20-docosanedioic acid (1.0 g, 2.70
mmol) in 70 ml of anhydrous tetrahydrofuran, and to the resulting solution a solution of N-hydroxysuccinimide (311 mg, 2.70 mmol) in 10 ml of anhydrous tetrahydrofuran and N,N.
- Dimethylaminopyridine hydrochloride (2.1 mg,
After stirring for 30 minutes, a solution of DCC (602 mg, 2.70 mmol) in 10 ml of anhydrous tetrahydrofuran was added and stirred overnight. After filtering the reaction solution and concentrating the filtrate under reduced pressure, it was subjected to silica gel column chromatography [Developing solution: a mixture of benzene and chloroform (volume ratio: 1:3.
5)] was separated and purified and showed the following physical properties.
1-carboxyheneicosanoyloxy)succinimide (440 mg, 35%) was obtained. m. p. 122 ~ 124.5 °C FD-MS (m
/z): [M+H]+ 4681H-NMR (CDCl
3,270 MHz): δ 1.12 to 1.4
3 (m, 16H), 1.63 (m, 2H
), 1.74 (m, 4H), 2.34 (t
, 2H), 2.60 (t, 2H), 2.84
(s,4H)IR (cm-1): 2920,
2850, 1825, 1790, 1740, 1
725, 1710, 1210, 1070

Reference Example 6 Synthesis of 1,14-tetradecanedioic acid monobenzyl ester 1,14-tetradecanedioic acid (5.0 g,
19.4 mmol) in tetrahydrofuran 80 ml
benzyl alcohol (2.
1 g, 19.4 mmol) in 10 ml of tetrahydrofuran and N,N-dimethylaminopyridine hydrochloride (15 mg, 0.1 mmol) were added, and after stirring for 30 minutes, DCC (4.0 g,
19.4 mmol) of tetrahydrofuran 10
ml solution was added and stirred at room temperature for 20 hours. After filtering the reaction solution and concentrating the filtrate under reduced pressure, it was separated and purified using silica gel column chromatography [developing solution: a mixture of hexane and diethyl ether (volume ratio: 2:1)], and the following physical properties were obtained. 1,14-tetradecanedioic acid monobenzyl ester (2.42 g, 38
%) was obtained. m. p. 73.5-74°C 1H-NMR (CDCl3, 270 MHz):
δ 1.17 to 1.40 (m, 16H), 1
．． 50 ~ 1.70 (m, 4H), 2.23
~ 2.39 (m, 4H), 5.11 (s,
2H), 7.32 (m, 5H), 7.40
~9.35 (br, 1H)

Reference Example 7 Synthesis of N-(13-benzyloxycarbonyltridecanoyloxy)succinimide 1,14-tetradecanedioic acid monobenzyl ester (2.4 g, 6.89 mmol) was added to 30 ml of tetrahydrofuran. Dissolve and add N to the resulting solution
- Hydroxysuccinimide (793 mg, 6.
89 mmol) of tetrahydrofuran 15 ml
solution and N,N-dimethylaminopyridine hydrochloride (3.3 mg, 0.02 mmol),
After stirring at room temperature for 30 minutes, DCC (1.42 g
, 6.89 mmol) of tetrahydrofuran 15
ml solution was added and reacted at room temperature for 15 hours. After filtering the reaction solution and concentrating the filtrate under reduced pressure, it was separated and purified using silica gel column chromatography [developing solution: a mixture of hexane and ethyl acetate (volume ratio: 2:1)], and the following physical properties were obtained. N-(13-benzyloxycarbonyltridecanoyloxy)succinimide (2.31 g, 75%) was obtained. m. p. 6
1.5 to 62.5°C 1H-NMR (CDCl3,
270 MHz): δ 1.05 to 1.46
(m, 16H), 1.63 (m, 2H),
1.72 (m, 2H), 2.33 (t, 2
H), 2.58 (t, 2H), 2.79 (
s, 4H), 5.11 (s, 2H), 7.
33 (m, 5H)

Reference Example 8 N-(13-carboxytridecanoyloxy)
Synthesis of succinimide N-(13-benzyloxycarbonyltridecanoyloxy)succinimide (2.28 g, 5.
12 mmol) and 15 ml of tetrahydrofuran
228 mg of 10% palladium on carbon was added to the resulting solution, and the mixture was stirred under a hydrogen atmosphere for 15 hours. After filtering the reaction solution and concentrating the filtrate under reduced pressure, the residue was recrystallized (from ethanol) to give N-(13-
Carboxytridecanoyloxy)succinimide (
1.71 g, 94%) was obtained. m. p. 116 to 118 °C FD-MS (m/z): [M+H] + 3561H-N
MR (CDCl3, 270 MHz): δ 1.1
8 ~ 1.47 (m, 16H), 1.63
(m, 2H), 1.74 (m, 4H), 2
．． 35 (t, 2H), 2.57 (t, 2H
), 2.84 (s, 4H), 7.85 ~ 1
0.50 (br,1H)IR (cm-1): 2
920, 2850, 1825, 1790, 17
40, 1725, 1710, 1210, 107
0

Example 1 N-(13-carboxytridecanoyloxy)
Synthesis of SOD derivative by reaction of succinimide and SOD Human red blood cell type SOD aqueous solution (71.2 mg/ml
2.6 ml of 0.5 M sodium bicarbonate aqueous solution (pH 8.0) was added to 1.4 ml of N-(13-carboxytridecanoyloxy)succinimide obtained in Reference Example 8. A solution obtained by dissolving 1.0 mg of dimethyl sulfoxide in 0.2 ml of dimethyl sulfoxide was gradually added under stirring. After stirring overnight at room temperature and filtering, the reaction solution was purified with Sephadex G-25 (Se
phadex G-25: trade name, manufactured by Pharmacia)
The polymer fraction was subjected to gel filtration using as a carrier [eluent: 10 mM ammonium bicarbonate aqueous solution], and the polymer fraction was collected. This fraction was directly used as DEAE-Sepharose (
DEAE-Sepharose Fast Flow:
A mixture of 10mM Tris-HCl buffer (pH 8) and 0.15M sodium chloride aqueous solution, 10mM Tris-HCl buffer (pH
8) and 0.20M aqueous sodium chloride solution, 1
0mM Tris-HCl buffer (pH 8) and 0.25
Elute sequentially with a mixture of M sodium chloride aqueous solution, and separate each fraction (hereinafter, these fractions will be referred to as fraction A, fraction A,
Fraction B and Fraction C) were collected. These fractions were treated with Sephadex G-25 (Sephadex G-25).
After desalting by gel filtration using ex G-25 (trade name, manufactured by Pharmacia) as a carrier [eluent: 10 mM ammonium bicarbonate aqueous solution], the high molecular fractions were lyophilized to separate the respective fractions. 33 mg of SOD derivative (hereinafter referred to as SOD derivative A) from minute A;
SOD derivative from fraction B (hereinafter referred to as SOD derivative B)
18 mg of SOD derivative (hereinafter abbreviated as SOD derivative C) from fraction C.
I got mg. When the amino groups of each of SOD derivatives A, B and C were quantified by the trinitrobenzenesulfonic acid (hereinafter abbreviated as TNBS) method, it was found that SO
It was confirmed that 3.6, 4.4, and 5.4 amino groups of the raw material SOD were modified in D derivatives A, B, and C, respectively.

[0053] SOD used and obtained SOD derivative C
The respective electropherograms are schematically shown in FIGS. 1(a) and (b). Further, the infrared absorption spectrum of SOD derivative C is shown in FIG.

Example 2 Synthesis of SOD derivative by reaction of N-(17-carboxyheptadecanoyloxy)succinimide and SOD Human red blood cell type SOD aqueous solution (88.9 mg/
ml) to 1.12 ml with 1.88 ml of water and 0.5M aqueous sodium bicarbonate solution (pH 8.0)0
．． 8 ml was added, and then N − obtained in Reference Example 3 was added.
(17-carboxyheptadecanoyloxy)succinimide 3.9 mg dimethyl sulfoxide 0
．． A solution obtained by dissolving 2 ml was gradually added under stirring. After stirring overnight at room temperature and filtering, the reaction solution was poured into Sephadex G-25.
: Product name, manufactured by Pharmacia) as a carrier [Eluate: 10mM ammonium bicarbonate aqueous solution]
The high molecular weight fraction was collected. Use this fraction as is D
EAE-Sepharo
Ion exchange chromatography using SE Fast Flow (trade name, manufactured by Pharmacia) as a carrier [
Eluate: a mixture of 10mM Tris-HCl buffer (pH 8) and 0.20M sodium chloride aqueous solution],
A fraction containing the SOD derivative was collected. This fraction was subjected to gel filtration using Sephadex G-25 (trade name, manufactured by Pharmacia) as a carrier [eluent: 10mM ammonium bicarbonate aqueous solution], and after desalting, the polymer fraction was separated. was freeze-dried to obtain 18 mg of SOD derivative. When the amino groups of the obtained SOD derivative were quantified by the TNBS method, it was found that the raw material SOD
It was confirmed that 2.0 of the amino groups were modified.

The electropherograms of the SOD used and the SOD derivative obtained are schematically shown in FIGS. 3(a) and 3(a).
Shown in b). Moreover, the infrared absorption spectrum of the obtained SOD derivative is shown in FIG.

Example 3 N-(19-carboxynonadecanoyloxy)
Synthesis of SOD derivative by reaction of succinimide and SOD In Example 2 N-(17-carboxyheptadecanoyloxy)succinimide 3.9 m
In place of g, N −(19 −
Carboxynonadecanoyloxy)succinimide 4
．． Perform the same procedure except that 1 mg was used, and
14 mg of D derivative was obtained. When the amino groups of the obtained SOD derivative were quantified by the TNBS method, it was found that the raw material SO
It was confirmed that 2.0 of the amino groups in D were modified.

The electropherograms of the SOD used and the SOD derivative obtained are schematically shown in FIGS. 5(a) and 5(a).
Shown in b). Moreover, the infrared absorption spectrum of the obtained SOD derivative is shown in FIG.

Example 4 Synthesis of NCS derivative by reaction of N-(15-carboxypentadecanoyloxy)succinimide with NCS 50 mg of NCS was dissolved in 18 ml of 0.5M sodium bicarbonate aqueous solution under ice cooling. N-(15-carboxypentadecanoyloxy)succinimide 7 obtained in Reference Example 2 was added to the solution obtained.
A solution obtained by dissolving 9.8 mg in 2 ml of dimethyl sulfoxide was gradually added dropwise while stirring. After stirring for 2 weeks at 4°C in the dark and filtering, the reaction solution was purified with Sephadex G-25 (Sephadex G-25).
G-25 (trade name, manufactured by Pharmacia) was subjected to gel filtration [eluent: 10 mM aqueous ammonium bicarbonate solution] using G-25 (trade name, manufactured by Pharmacia) as a carrier, and a high molecular fraction was collected. This fraction was directly used as DEAE-Sepharose (DEAE-Sepharose).
Ion exchange chromatography using pharose Fast Flow (trade name, manufactured by Pharmacia) as a carrier [eluent: 10mM Tris-HCl buffer (p
8) and a 0.20 M aqueous sodium chloride solution], and a fraction containing the NCS derivative was collected. This fraction was treated with Sephadex G-25 (Sephadex G-25).
After desalting by gel filtration using G-25 (trade name, manufactured by Pharmacia) as a carrier [eluent: 10 mM ammonium bicarbonate aqueous solution], the polymer fraction was freeze-dried to obtain 7 mg of the NCS derivative. I got it. Obtained NC
When the amino groups of the S derivative were quantified by the TNBS method, no free amino groups were detected.

The electropherograms of the used NCS and the obtained NCS derivative are schematically shown in FIGS. 7(a) and ().
Shown in b). Moreover, the infrared absorption spectrum of the obtained NCS derivative is shown in FIG.

Example 5 Synthesis of NCS derivative by reaction of N-(17-carboxyheptadecanoyloxy)succinimide with NCS In Example 4 N-(15-carboxypentadecanoyloxy)succinimide 79. 8
mg of N-(17
- The same operation was performed except that 85.6 mg of carboxyheptadecanoyloxy)succinimide was used to obtain 5 mg of the NCS derivative. Obtained NCS
When the amino group of the derivative was quantified by the TNBS method,
No free amino groups were detected.

The electropherograms of the used NCS and the obtained NCS derivative are schematically shown in FIGS. 9(a) and 9(a).
Shown in b). Moreover, the infrared absorption spectrum of the obtained NCS derivative is shown in FIG.

Test Example 1 Blood Concentration of SOD Derivatives A rat (male Wistar strain, 7 weeks old, weight approximately 200 g) was cannulated from the femoral vein under anesthesia with pentobarbital, and a heparin solution (1000 U/cm) was injected into the femoral vein.
ml) was injected. A solution obtained by dissolving SOD or SOD derivative in physiological saline (10 mg/
0.2 ml of each rat was injected into the femoral vein. Blood was collected in 0.2 ml portions over time.
Blood concentrations were determined by measuring SOD activity in plasma. FIG. 11 shows the changes over time in the blood concentration of SOD and the blood concentration of SOD derivatives.

Test Example 2 Effect of SOD derivatives on acute gastric mucosal lesions (gastric ulcers) in rats After fasting SD male rats (weight 200 g) overnight, they were placed in a stress cage with 3 rats per group and restrained. The rats were immersed from the chest down in water at 22°C to apply stress to the rats. After 6 hours, the rats were removed from the water, bled to death, and their stomachs were removed. Tissues were fixed by injecting 1% formalin into the gastric lumen. After fixation, the length of the linear ulcer on the mucosal surface was measured, and the sum of the lengths was taken as the ulcer index.

[0064] Furthermore, the control group received 0.5 ml.
2 mg/rat of the SOD derivative C obtained in Example 1 was intravenously administered to the test group as a 0.2 ml physiological saline solution 5 minutes before water immersion restraint. The results are shown in the table below.

[0065]

[Table 1]

As is clear from Table 1, the S
A significant anti-ulcer effect of the OD derivative was observed.

[0067]

[Effects of the Invention] According to the present invention, the biological activity of proteins is largely retained, the lifespan in the blood is significantly extended compared to proteins, there is no antigenicity, and administration to living bodies is possible. A novel protein derivative is provided. The protein derivative has a reversible interaction with serum proteins and can be easily transferred to the lesion. Further, according to the present invention, there is provided a method for producing the above-mentioned protein derivative by reacting an amino group of a protein with a long-chain carboxylic acid imide ester (I) having a single chemical structure.

[Brief explanation of drawings]

FIG. 1 is a schematic diagram showing the results of electrophoresis, (a) is a schematic diagram of the electropherogram of SOD used in Example 1, and (b) is a schematic diagram of the SOD derivative C obtained in Example 1. A schematic diagram of the electropherogram of .

FIG. 2 is a diagram showing an infrared absorption spectrum of SOD derivative C obtained in Example 1.

FIG. 3 is a schematic diagram showing the results of electrophoresis, in which (a) shows a schematic diagram of the electropherogram of SOD used in Example 2, and (b) shows a schematic diagram of the electropherogram of SOD used in Example 2. A schematic diagram of an electropherogram is shown.

FIG. 4 is a diagram showing an infrared absorption spectrum of the SOD derivative obtained in Example 2.

FIG. 5 is a schematic diagram showing the results of electrophoresis, in which (a) shows a schematic diagram of the electropherogram of SOD used in Example 3, and (b) shows a schematic diagram of the electropherogram of SOD used in Example 3. A schematic diagram of an electropherogram is shown.

FIG. 6 is a diagram showing an infrared absorption spectrum of the SOD derivative obtained in Example 3.

FIG. 7 is a schematic diagram showing the results of electrophoresis, in which (a) shows a schematic diagram of the electropherogram of NCS used in Example 4, and (b) shows a schematic diagram of the electropherogram of NCS derivative obtained in Example 4. A schematic diagram of an electropherogram is shown.

FIG. 8 is a diagram showing an infrared absorption spectrum of the NCS derivative obtained in Example 4.

FIG. 9 is a schematic diagram showing the results of electrophoresis, in which (a) shows a schematic diagram of the electropherogram of NCS used in Example 5, and (b) shows a schematic diagram of the electropherogram of the NCS derivative obtained in Example 5. A schematic diagram of an electropherogram is shown.

FIG. 10 is a diagram showing an infrared absorption spectrum of the NCS derivative obtained in Example 5.

FIG. 11 is a diagram showing changes in blood concentration over time measured in Test Example 1, where (1), (2), (3) and (4) are SOD and the SOD derivative obtained in Example 1, respectively. A, S
1 shows changes over time in blood concentrations of OD derivative B and SOD derivative C.

Claims (2)

[Claims] [Claim 1] The following general formula [protein] [Z]
represents a protein having x residues excluding hydrogen atoms,
[Z] is the following general formula [Formula 1] (wherein, W is one or more oxygen atoms, sulfur atoms, or -
One carboxyl of a long-chain dicarboxylic acid represented by N(R)- (represents a divalent long-chain hydrocarbon group optionally interrupted by a group represented by the formula, R represents a lower alkyl group) (represents a residue obtained by removing a hydroxyl group from a group, x means the average number of amide bonds between [Z] and [protein], and represents a number in the range of 1 to 8). [Claim 2] Protein and the following general formula [Formula 2] (wherein, W is one or more oxygen atoms, sulfur atoms, or -
A long-chain carboxylic acid imide ester represented by N(R)- (represents a divalent long-chain hydrocarbon group optionally interrupted by a group represented by the formula, R represents a lower alkyl group). p
2. The method for producing a protein derivative according to claim 1, wherein the reaction is carried out in an aqueous solution of H6 to H10.