JPH09117279A - Lecithinized superoxide dismutase and medicine comprising the same as active ingredient - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a lecithinized superoxide dismutase having enhanced pharmacological activity and a medicine comprising the same as an active ingredient. SOLUTION: This lecithinized superoxide dismutase is obtained by bonding lysolecithin by chemical cross-linking to a human type superoxide dismutase which coordinates copper and/or zinc and into which a 2-hydroxyethylthio group is introduced to the mercapto group of cystine at the 111 position. The objective medicine comprises the lecithinized superoxide dismutase as an active ingredient.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a lecithinized superoxide dismutase, and a medicine containing the same as an active ingredient.

[0002]

2. Description of the Related Art Although attempts to enhance the effects of drugs and reduce side effects have been made for a long time, one of the drug delivery systems (D
DS). What is DDS?
It is an attempt to shift the drug as selectively as possible for a necessary time, thereby enhancing the effect of the drug and significantly reducing systemic side effects.

There are various carriers used for DDS, and examples thereof include liposomes and lipid microspheres. The liposome is a liposome in which naturally occurring lipids, such as lecithin and cholesterol, are dissolved in an organic solvent, diffused in water by ultrasonication, and the drug is encapsulated therein. On the other hand, lipid microspheres are soybean oil suspended in water together with lecithin, and lecithin is present on the surface thereof, and a drug is encapsulated inside.

In both cases, the drug is encapsulated mainly by physical bonding inside. Liposomes have poor stability, and lipid microspheres require that the drug to be encapsulated be lipophilic, and moreover specialized manufacturing equipment must be used.

On the other hand, superoxide dismutase (hereinafter, abbreviated as SOD) is a superoxide anion which is widely distributed in the living body of animals, plants, microorganisms and the like, and has active oxygen with a high degree of free (reactive) reactivity. It is known as an enzyme that decomposes radicals. In terms of drugs, prophylactic and therapeutic agents for various diseases caused by superoxide (for example, inflammation, osteoarthritis, rheumatoid arthritis, ultraviolet irradiation-induced disorders, premature oxygen retinopathy, cataracts, anticancer agents such as adriamycin) Expected as side effects, obstacles associated with resumption of blood flow to the ischemic area, use during myocardial infarction or organ transplant, etc. (For anti-inflammatory agents, see Pharmacia, Vol. 17, p. 411 (1981)) ).

[0006]

[Problems to be Solved by the Invention] When SOD is intravenously administered, it has a low cell affinity and a half-life in blood of only 4 to 6 minutes, and SOD is rapidly excreted in urine. . To increase the blood half-life of SOD,
Attempts have been made to enlarge the protein by modifying it with ficoll, polyethylene glycol, rat albumin, and dextran.

However, it has been reported that SOD modified with ficoll or polyethylene glycol has antigenicity. When modified with dextran, S
Although the anti-inflammatory effect of OD is enhanced, the effect of suppressing immunogenicity is not observed.

[0008] Various conventionally known modified SODs have problems in practical use due to the above-mentioned reasons already reported, or a decrease in tissue permeability due to macromolecularization. Therefore, under the present circumstances, none of the modified SODs has been clinically applied.

On the other hand, the inventors of the present invention examined the conversion of SOD to DDS, and as a result, found a lecithinized superoxide dismutase in which lecithin was bound to SOD by chemical crosslinking (see JP-A-3-163100). . In addition, as this SOD, a human-derived SOD in which copper and / or zinc is coordinated and the 111-position is serine
Was also found to be suitable (JP-A-6-54681).
Reference).

[0010]

The present inventors have examined lecithinized superoxide dismutase, which is completely different from these conventional ones and has an excellent effect, and as a result, the following specific superoxide dismutase was identified. The lecithinized superoxide dismutase used was found.

The present invention is this specific lecithinized superoxide dismutase, and a drug containing it as an active ingredient. A lecithinized superoxide dismutase represented by the following formula [1]. A- [C (O)-(CH ₂ ) _n C (O) -B] _m ... [1] However, A: 2 in the mercapto group of cysteine at position 111, to which copper and / or zinc is coordinated. -Hydroxyethylthio group-introduced residue of human-type superoxide dismutase, B: residue of lysolecithin having a hydroxyl group at the 2-position of glycerol, excluding the hydrogen atom of the hydroxyl group at the 2-position, m: 1 or more Integer, n: an integer of 2 or more.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION The lecithinized superoxide dismutase of the present invention has a remarkably different biodistribution and cell affinity from conventional SOD, and has a very uniform residual activity of 90% or more. Therefore, it can be expected to enhance the pharmacological activity of SOD, reduce side effects, and promote absorption.

The lecithinized superoxide dismutase of the present invention is usually a lecithin derivative in which a cross-linking agent is chemically bonded to a residue of lysolecithin, and a copper and / or zinc coordinated cysteine at position 111 Human-type superoxide dismutase having a 2-hydroxyethylthio group introduced into a mercapto group (hereinafter, Cu-Zn-type Cys-
111-ME-h-SOD)).

B is a residue of lysolecithin having a hydroxyl group at the 2-position of glycerol represented by the following formula [2], excluding the hydrogen atom of the hydroxyl group at the 2-position. _{-O-CH (CH 2 OR)} [CH 2 OP (O) (O -) (OCH 2 CH 2 N + (CH 3) 3] in ... [2] above formulas, R fatty acid residue (acyl group ), And
Particularly, a saturated to unsaturated fatty acid residue having 8 to 30 carbon atoms is preferable. Particularly preferred R is a myristoyl group, a palmitoyl group, a stearoyl group, or another saturated fatty acid residue having 14 to 22 carbon atoms. When the Cu-Zn type Cys-111-ME-h-SOD has two or more lysolecithin residues, the Rs at the lysolecithin residues may be different.

In the above formula [1], -C (O)-(CH ₂ ) _n C (O)-represents the residue of the chemical cross-linking agent. Residues of the chemical crosslinking agent, HO-C (O) - (CH 2) n C (O) linear dicarboxylic acid represented by -OH, and their anhydrides, their esters, other that the dicarboxylic It is a residue obtained by removing both hydroxyl groups of a chemical cross-linking agent consisting of a reactive derivative of an acid. Hereinafter, this residue is referred to as a chemical crosslink.

This chemical crosslink is linked to the lysolecithin residue by an ester bond. The other end of the chemical bridge is considered to be directly bonded to the amino group of Cu-Zn type Cys-111-ME-SOD by an amide bond or the like. In this formula, n is an integer of 2 or more, - (CH _{2) n} - represents a straight-chain alkylene group, especially n is preferably a linear alkylene group having 2 to 10.

Lecithinated Cu-Zn represented by the above formula [1]
The type Cys-111-ME-h-SOD is produced, for example, by the Cu-Zn type recombinant h-SOD and the lysolecithin derivative of the formula [3]. _{ZC (O) - (CH 2} ) in _{n C (O) -B ··· [} 3] the above formulas, B, n are the same as in the formula [1]. In formula [3], Z represents a hydroxyl group or a group obtained by removing a carbonyl group from a group forming an active ester. For example, p-nitrophenol, 3,5-trichlorophenol, pentafluorophenol, 2,4-dinitrophenol, N-hydroxysuccinimide, N-hydroxypiperidine, N-hydroxy-5-norbornene-2,3-dicarboxylic acid imide , A group obtained by removing a hydrogen atom from a hydroxyl group of a hydroxyl group-containing compound such as 8-hydroxyquinoline or 2-hydroxypyridine. As a method for synthesizing an active ester, a known method can be used (Izumiya et al., “Basics and Experiments of Peptide Synthesis” (198
5) Published by Maruzen Co., Ltd.).

A lysolecithin derivative represented by the formula [3]
Examples of the binding method with Cu-Zn type Cys-111-ME-h-SOD include the following. When Z is a hydroxyl group in the formula [3], it is carried out by the carbodiimide method. Examples of the carbodiimides include diethylcarbodiimide, diisopropylcarbodiimide, dicyclohexylcarbodiimide, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and the like. For example, an aqueous solution of the compound [3] of 1 to 10 wt% is adjusted to pH 4 to 6 with hydrochloric acid, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide is added at room temperature or 0 ° C., and the pH is adjusted to 4 again. Prepare ~ 6. SOD is added and the pH is kept at 4 to 6 at room temperature or 0 ° C for 1 hour, and then stirred for 5 to 20 hours.
Obtain s-111-ME-h-SOD.

In the formula [3], when Z represents a group excluding a carbonyl group from a group forming an active ester, Cu-
It can be directly bound to Zn-type Cys-111-ME-h-SOD.
The reaction is carried out by mixing the lecithin derivative and the Cu-Zn type Cys-111-ME-h-SOD in an aqueous solution of a salt such as sodium borate, sodium phosphate, potassium phosphate or sodium bicarbonate.

If necessary, N, N-dimethylformamide, N-methylpyrrolidone, N, N-dimethylacetamide,
An organic solvent such as sulfolane, dimethyl sulfoxide, acetone, 1,4-dioxane and methanol can be added in advance. The reaction temperature is preferably -20 to 50 ° C, 0 to 20 ° C
Is more preferred. The reaction time varies depending on the reaction temperature and the mixing method, but is usually 2 to 24 hours.

The lecithin derivative represented by the formula [3] is charged in an amount of 0.1 with respect to the amino group of Cu-Zn type Cys-111-ME-h-SOD.
A suitable amount is 2 to 8 mol. Cu-
The number of molecules of the lecithin derivative (formula [3]) bound to the Zn-type Cys-111-ME-h-SOD can be adjusted.

The reaction solution thus obtained contained lecithinized Cu-Zn type Cys-111-ME-h-SOD and unreacted Cu-Zn type Cys--1.
Although 11-ME-rh-SOD and unreacted lecithin derivative coexist, the reaction mixture was subjected to gel filtration and ion exchange column chromatography to obtain the desired lecithinized Cu-
Zn type Cys-111-ME-h-SOD can be obtained. Also, the lecithinized Cu-Zn type Cys-111-ME-hS obtained in this way
OD is usually a mixture of Cu-Zn type Cys-111-ME-h-SOD obtained by binding various numbers of lecithin derivatives.

Cu-Zn type Cys-111-ME as an active ingredient compound
-If lecithinized Cu-Zn type Cys-111-ME-h-SOD such that the number of lecithin derivatives bound to h-SOD becomes uniform, lecithinized Cu obtained by the above method is desired. -
The Zn-type Cys-111-ME-h-SOD was further subjected to operations such as gel filtration and ion-exchange column chromatography to give the desired number of lecithin derivatives bound lecithinized Cu-Zn type.
It is possible to obtain Cys-111-ME-h-SOD. Cu-Zn type Cy
The number of lecithin bonds per molecule of s-111-ME-h-SOD (m in formula [1]) is not particularly limited, but is preferably 1 to 16, and particularly preferably 1 to 10.

The compound of the formula [3] can be produced by reacting an acid anhydride represented by the formula [4] with lysolecithin represented by H-B or by the formula [5]. It is obtained by a method of reacting the dicarboxylic acid half ester anhydride with lysolecithin represented by H-B. [-C (O) - (CH 2) n C (O) -] = O ··· [4] [Z'-OC (O) - (CH 2) n C (O) -] 2 = O · .. [5] Here, B and n are the same as in the case of the formula [1]. Z'represents a protecting group for a carboxyl group, for example, an alkyl group, a methoxymethyl group, a benzyl group, a phenacyl group, a t-butyldimethylsilyl group, a triethylsilyl group, a trimethylsilyl group and the like.

The reaction for producing the compound of the formula [3] using these acid anhydrides and half ester anhydrides is usually carried out in a solvent, optionally in the presence of an organic base. As the reaction solvent, for example, a halogenated hydrocarbon such as chloroform is used, and as the organic base, for example,
Pyridine, piperidine, triethylamine, 4-dimethylaminopyridine, 4-piperidinopyridine and the like are used. The reaction temperature is preferably 20 to 80 ° C, more preferably 40 to 60 ° C. The reaction time is usually 2 to 24 hours.

The production method of the formula [5] is obtained by mixing the carboxylic acid half ester with carbodiimide in a solvent such as benzene, toluene, chloroform, dichloromethane and tetrahydrofuran. As the carbodiimide, for example, diethylcarbodiimide, diisopropylcarbodiimide, dicyclohexylcarbodiimide, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and the like are used. The reaction temperature may be in the range of −20 ° C. to solvent reflux temperature, but preferably 0 ° C. to room temperature.

Cu-Zn type Cys-111-ME-h-SOD used in the present invention
Can be obtained, for example, by reacting human superoxide dismutase with bis (2-hydroxyethyl) disulfide (see JP-A-6-199895). Human-type superoxide dismutase has an amino acid sequence substantially the same as that of natural human SOD, and can be obtained, for example, by the method described in JP-A-61-111690, or as a commercially available product. It is also available.

Examples of the form of the preparation of the present invention include injections, rectal absorbents, nasal absorbents and the like. Injections include, for example, buffering agents, isotonic agents, pH adjusting agents,
It is prepared by dissolving in a stabilizer and distilled water for injection dissolved in an appropriate amount and sterilized through a sterilization filter and then dispensed into ampoules or vials and freeze-dried.

The dose of the lecithinized superoxide dismutase of the present invention to human is not particularly limited, but about 0.0001 to 100 mg / person is suitable, and about 0.001 to 10 mg / person is particularly preferable. Note that 1
mg corresponds to 3000 units (U). The present invention will be specifically described below, but the present invention is not limited to these examples.

[0030]

【Example】

(Synthesis example 1) Synthesis of 9-benzyloxycarbonyl-1-nonanoic acid anhydride 9-benzyloxycarbonyl-1-nonanoic acid 15 g (51 mmol)
Is dissolved in 50 ml of benzene and cooled to 0 ° C, and DCC (1,3-
Dicyclohexylcarbodiimide) (5.8 g, 28 mmol) was added, and the mixture was stirred at room temperature for 15 hours. The insoluble material was filtered through Celite and concentrated under reduced pressure to give the title compound.

Synthesis Example 2 Synthesis of 2- (9-benzyloxycarbonylnonanoyl) lysolecithin Lysolecithin 3 g in which the 2-position of glycerol is a hydroxyl group
2.59 g of DMP (N, N-dimethylaminopyridine) in a suspension of (5.9 mmol) of chloroform-pyridine (80 ml / 20 ml).
(17.7 mmol) and 10.0 g (17.7 mmol) of 9-benzyloxycarbonyl-1-nonanoic acid anhydride were added, and the mixture was stirred at 60 ° C. for 15 hours. After that, the reaction solution was concentrated under reduced pressure, chloroform: methanol: water = 4: 5: 1 (10 ml) was added to the residue to dissolve it, and an ion exchange column (Dowex 50W-
X8).

The target compound was fractionated by TLC, the solvent was concentrated under reduced pressure, and the residue was purified by silica gel column to obtain 3.91 g (5.0 mmol, 85%) of the title compound. ¹ H-NMR (CDCl ₃ ) 0.84 (t, 3H), 1.20 (brs), 1.50-1.70 (brs, 6H), 2.20-2.40 (b
rs, 6H), 3.38 (s, 9H), 3.80-4.00 (m, 4H), 4.20-4.40 (m, 4H),
5.10 (s, 2H), 5.20 (m, 1H), 7.30 (m, 5H).

(Synthesis Example 3) Synthesis of 2- (9-hydroxycarbonylnonanoyl) lysolecithin 2.91 g (5.00 mmol) of 2- (9-benzyloxycarbonylnonanoyl) lysolecithin obtained in Synthesis Example 2 was added to methanol-water. Dissolve in (225ml / 25ml) and palladium hydroxide 3.
0 g was added. After purging with hydrogen, the mixture was stirred for 15 hours at 1 atm and room temperature. After filtration through Celite and concentration under reduced pressure, the residue was purified by a silica gel column to give the title compound 2.37 (3.41 mmol, 61
%).

Synthesis Example 4 Synthesis of Active Ester of 2- (9-Hydroxycarbonylnonanoyl) lysolecithin 2.0 g (2.98 mmol) of the carboxylic acid obtained in Synthesis Example 3 was dissolved in 50 ml of dichloromethane and heated to 0 ° C. Cooled, N-hydroxysuccinimide 343 mg (2.98 mmol), tetrazole 20
9 mg (2.98 mmol) were added in this order. Next, DDC 769 mg (3.
73 mmol) was dissolved in 8 ml of dichloromethane. This solution was slowly added dropwise, and the mixture was stirred at room temperature for 15 hours. The insoluble matter was filtered through Celite to obtain a dichloromethane solution of the active ester form.

(Synthesis Example 5) Synthesis of 11-benzyloxycarbonyl-1-undecanoic acid anhydride It was synthesized from 11-benzyloxycarbonyl-1-undecanoic acid in the same manner as in Synthesis Example 1.

(Synthesis example 6) Synthesis of 2- (11-benzyloxycarbonylundecanoyl) lysolecithin Synthesis was carried out in the same manner as in synthesis example 2 from the acid anhydride obtained in synthesis example 5.

(Synthesis Example 7) Synthesis of 2- (11-hydroxycarbonylundecanoyl) lysolecithin Synthesis was carried out in the same manner as in Synthesis Example 3 from the benzyl ester compound obtained in Synthesis Example 6.

Synthesis Example 8 Synthesis of Active Ester of 2- (11-Hydroxycarbonylundecanoyl) lysolecithin Similar to Synthesis Example 4, it was synthesized from the carboxylic acid obtained in Synthesis Example 7.

(Synthesis Example 9) Synthesis of 6-benzyloxycarbonyl-1-hexanoic anhydride This was synthesized from 6-benzyloxycarbonyl-1-hexanoic acid in the same manner as in Synthesis Example 1.

(Synthesis Example 10) Synthesis of 2- (6-benzyloxycarbonylhexanoyl) lysolecithin Synthesis was carried out in the same manner as in Synthesis Example 2 from the acid anhydride obtained in Synthesis Example 9.

(Synthesis Example 11) Synthesis of 2- (6-hydroxycarbonylhexanoyl) lysolecithin Synthesis was carried out in the same manner as in Synthesis Example 3 from the benzyl ester compound obtained in Synthesis Example 10.

Synthesis Example 12 Synthesis of Active Ester of 2- (6-Hydroxycarbonylhexanoyl) lysolecithin Similar to Synthesis Example 4, it was synthesized from the carboxylic acid obtained in Synthesis Example 11.

(Synthesis Example 13) Synthesis of 2- (4-hydroxycarbonylbutyroyl) lysolecithin Synthesis was carried out in the same manner as in Synthesis Example 3 from glutaric anhydride. Purification was performed by a column packed with ODS (octadecylsilane). ¹ H-NMR (CDCl ₃ ) 0.84 (t, 3H), 1.20 (brs), 1.52-1.60 (brs, 2H), 1.80-1.95
(m, 2H), 2.20-2.40 (m, 6H), 3.35 (s, 9H), 3.780 (m, 4H), 3.90
-4.35 (m, 4H), 5.20 (m, 1H).

(Synthesis Example 14) Synthesis of active ester form of 2- (4-hydroxycarbonylbutyroyl) lysolecithin Synthesis was carried out in the same manner as in Synthesis Example 4 from the carboxylic acid obtained in Synthesis Example 13.

[Example] Lecithinated Cu-Zn type Cys-111-ME-h-SOD was prepared by using the compound prepared in the above-mentioned synthesis example.
It was manufactured using the method of -C. Method A: Distill off dichloromethane of the active ester solution,
Cu-Zn type Cys-111- dissolved in 50 mM borate buffer (pH 8.5)
ME-h-SOD solution is added, and the mixture is reacted at 0 ° C. for 1 hour and further at room temperature for 2 hours. The reaction solution is filtered, and Sephacryl S-300
A gel filtration column using (Pharmacia) as a carrier is applied and eluted with the same buffer as the reaction buffer. Then, the lecithinized Cu-Zn type Cys-111-ME-h-SOD elution fractions are collected and concentrated by ultrafiltration.

Method B: Dichloromethane of the active ester solution was distilled off and dissolved in DMF. This is added to 50 mM borate buffer (pH 8.5), insoluble matter is filtered, dissolved in the same buffer, and added dropwise to a Cu-Zn type Cys-111-ME-h-SOD solution cooled to 0 ° C. . At this time, Cu-Zn type Cys-111-ME-h-SOD solution was added to D
Add 50% MF. After stirring for 15 hours at 0 ° C., purify as in Method A.

Method C: To a Cu-Zn type Cys-111-ME-h-SOD solution dissolved in 50 mM borate buffer (pH 8.5), 20% DMF was added and cooled to 0 ° C. A DMF solution of the active ester prepared in the same manner as above is slowly added dropwise. After stirring for 15 hours at 0 ° C., purify as in Method A.

(Example 1) Cu-Zn type Cys-111-ME-h-SOD Lecithinated Cu-Zn type Cys-111-ME-hS having an average of two lecithin derivatives bonded per molecule.
Synthesis of OD Cu-Zn type Cys-11 dissolved in 50 mM borate buffer (pH 8.5)
According to the method A, 1-ME-h-SOD was reacted with 0.4-fold molar amount of the active ester synthesized in Synthesis Example 4 relative to all amino groups of Cu-Zn type Cys-111-ME-h-SOD. . The reaction solution was purified by gel filtration and ion exchange chromatography. Lowry method for protein concentration (Lowry, OH et al., (1951)
J. Biol. Chem., 193, 265), Cu-Zn type Cys-111-ME-h-SO.
Residual amino groups on D were analyzed by the TNBS method (trinitrobenzenesulfonic acid sodium salt, Goodwin, JF et al., (1970) Cl
in. Chem., 16, 24) by performing Cu-Zn type Cys-111-
When the number of bound lecithin derivatives per molecule of ME-h-SOD was determined, it was 2.0 on average.

(Example 2) Cu-Zn type Cys-111-ME-h-SOD Lecithinated Cu-Zn type Cys-111-ME-hS having an average of four lecithin derivatives bonded per molecule.
Synthesis of OD Cu-Zn type Cys-11 dissolved in 50 mM borate buffer (pH 8.5)
According to the method B, 1-ME-h-SOD was reacted with 0.8-fold molar amount of the active ester synthesized in Synthesis Example 14 relative to all amino groups of Cu-Zn type Cys-111-ME-h-SOD. . Purification was carried out in the same manner as in Example 1, and the number of bonded lecithin derivatives per molecule of Cu-Zn type Cys-111-ME-h-SOD was determined, and the average was 4.0.

(Example 3) Cu-Zn type Cys-111-ME-h-SOD Lecithinated Cu-Zn type Cys-111-ME-hS having an average of eight lecithin derivatives bonded per molecule.
Synthesis of OD Cu-Zn type Cys-11 dissolved in 50 mM borate buffer (pH 8.5)
According to Method C, 1-ME-h-SOD was reacted with the active ester synthesized in Synthesis Example 8 in a 2.0-fold molar amount with respect to all amino groups of Cu-Zn type Cys-111-ME-h-SOD. . Purification was carried out in the same manner as in Example 1, and the number of bonded lecithin derivatives per molecule of Cu-Zn type Cys-111-ME-h-SOD was determined, and the average was 8.0.

(Example 4) Lecithinated Cu-Zn type in a mouse ischemic paw edema model
Inhibitory effect of Cys-111-ME-h-SOD ICR mice (male, 6 weeks old) were purchased from Charles River Japan Co., Ltd. and used in the experiment. Lecithinized Cu-Zn type Cys-111-as a test drug from the tail vein of ICR mice
ME-h-SOD or Cu-Zn type Cys-111-ME-h-SOD was administered, and the right ankle was wrapped 5 times with a commercially available rubber band (1x1 mm, diameter 42 mm). After ischemia for 20 minutes as it was, the rubber band was removed with scissors and re-refluxed. After 30 minutes, the thickness of the right foot was measured using a gauge. At this time, the thickness of the left foot was measured as a control.

As the test drug, lecithinated Cu-Zn type Cy
s-111-ME-h-SOD (synthesized in Example 2, 30000 U / kg or 60
000U / kg) or Cu-Zn type Cys-111-ME-h-SOD
(60000 U / kg was used) was used. Ma as statistical processing
nn-Whitney U test is used to perform a significant difference test, and P <0.
05 was judged to have a significant difference. Table 1 shows the test results.

[0053]

[Table 1]

From these results, the lecithinated Cu-of Example 2 was
Zn-type Cys-111-ME-h-SOD 30000U / kg and 60000U / kg administration groups were P <0.05 and P <0.01, respectively, compared with the control group.
It was judged that there was a difference, and it was effective. Also, Cu-
The lecithinized Cu-Zn type Cys-111-ME-h-of Example 2 was found to have an effect even when compared with the Zn type Cys-111-ME-h-SOD 60000U / kg administration group. SOD is Cu-Zn type Cys-111-
It was judged to be effective because it significantly reduced free radicals compared to ME-h-SOD.

Further, as a test drug, human-derived superoxide dismutase (hereinafter referred to as Cu-Zn type Ser-111-rh-SOD) in which the 111-position coordinated with copper and zinc is represented by serine
Table 2 shows the results of the comparison with the case of using the lecithinized product (referred to as) (synthesized in the same manner as in Example 2). As a result, it was found that the lecithinized Cu-Zn type Cys-111-ME-h-SOD had a higher inhibitory effect than the lecithinized Cu-Zn type Ser-111-h-SOD.

[0056]

[Table 2]

As described above, the lecithinized Cu-of Example 2 was used.
Zn-type Cys-111-ME-h-SOD showed an inhibitory effect on ischemic foot edema in mice. In addition, lecithinated Cu-Zn type Cys-111-
As a result of intravenous administration of ME-h-SOD to mice at 60000 U / kg, no death was observed in any of them.

[0058]

INDUSTRIAL APPLICABILITY The lecithinized superoxide dismutase of the present invention is bound to lecithin through chemical cross-linking with superoxide dismutase. Compared with conventional modified products, it can be expected that the biodistribution and the cell affinity will be remarkably different, and it has the effect of enhancing the pharmacological activity.

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Claims (4)

[Claims] 1. A lecithinized superoxide dismutase represented by the following formula [1]. A- [C (O)-(CH ₂ ) _n C (O) -B] _m ... [1] However, A: 2 in the mercapto group of the cysteine at position 111, to which copper and / or zinc is coordinated. -Hydroxyethylthio group-introduced residue of human-type superoxide dismutase, B: residue of lysolecithin having a hydroxyl group at the 2-position of glycerol, excluding the hydrogen atom of the hydroxyl group at the 2-position, m: 1 or more Integer, n: an integer of 2 or more. 2. The lecithinized superoxide dismutase according to claim 1, wherein n is an integer of 2-10. 3. The lecithinized superoxide dismutase according to claim 1 or 2, wherein m is on average 1-16. 4. A medicine comprising the lecithinized superoxide dismutase of claim 1, 2 or 3 as an active ingredient.