JPH10338645A - Agent for improving functional disorder accompanied by cerebrovascular disease - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject improving agent by including a specific superoxide dismutase(SOD). SOLUTION: This improving agent contains a SOD bound to lecithin through a chemical crosslink and expressed by the formula [X is a residue obtained by removing H from the 2-hydroxyl group of lysolecithin having the hydroxyl group at the 2-position of glycerol: (m) is 1 or larger which is an average bond number per SOD molecule; (n) is an integer of >=2] [preferably a human- originated SOD which contains copper and zinc at the active center and whose amino acid at the 111-position of the amino acid sequence is S-(2- hydroxyethylthio)cysteine], and is useful for improving allolalia caused by cerebral hemorrhage and/or cerebral infarction, dyskinesia caused by paralysis, and/or hypanakinesis accompanied by central nervous system disorder.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a medicament containing lecithinated superoxide dismutase as an active ingredient. More specifically, the present invention relates to an agent for improving dysfunction associated with cerebrovascular disorders, an agent for improving motor dysfunction associated with disorders of the central nervous system, and an agent for improving speech disorders, which contains lecithinated superoxide dismutase as an active ingredient.

[0002]

2. Description of the Related Art Superoxide dismutase (hereinafter, referred to as superoxide dismutase)
SOD is sometimes widely distributed in living organisms such as animals, plants, and microorganisms, and is a reactive oxygen species that is highly reactive among the active oxygen groups (hereinafter sometimes abbreviated as ROS). oxide anion radicals ^- known as enzymes that degrade (hereinafter, simply O ₂ may be abbreviated as). From the pharmacological point of view, this SOD is used for the treatment of various inflammations caused by radicals and the removal of radicals generated after the use of anti-rheumatic drugs, the use of anti-thrombotic agents used in myocardial infarction and organ transplantation. It is expected to be applied. Recently, application of this SOD to gastric mucosal injury has also been studied, and its therapeutic effect is expected (Lipid Peroxide Research, Vol. 16, p. 74 (1992)).

In general, when administered intravenously, SOD has a low cell affinity and has a blood half-life of only 4 to 6 minutes, and SOD is rapidly excreted in urine. This S
In order to extend the blood half-life of OD, attempts have been made to modify SOD with ficoll, polyethylene glycol, rat albumin, dextran, or the like to form a macromolecule.

[0004] However, it has been reported that SOD modified with ficoll or polyethylene glycol greatly reduces the intrinsic enzyme activity of SOD and still has low cell affinity. In addition, it has been reported that rat albumin-modified SOD exhibits antigenicity in the human body. Furthermore, SOD modified with dextran is excellent in that the anti-inflammatory action inherent to SOD is enhanced, but it is also reported that antigenicity in the human body is also recognized.

[0005] In recent years, there has been reported a substance obtained by chemically modifying a biologically active protein such as SOD with lecithin (phosphatidylcholine: hereinafter sometimes abbreviated as PC). It has been reported that this PC-binding modified bioactive protein has a markedly increased cell affinity and distribution in a living organism is significantly different from conventional unmodified bioactive proteins. It is also said that enhancement of the pharmacological activity of the biologically active protein, reduction of side effects, and promotion of absorption can be expected.

[0006] Also, PC-based SOD (hereinafter referred to as PC-SOD)
(Sometimes abbreviated as "abbreviated") has been reported to have an effect of promoting the healing of inflammation caused by burns (Japanese Patent Application Laid-Open No. Hei 3-16).
3100, JP-A-3-170438, U.S. Pat. No. 5,109,118). Furthermore, it is said that it is useful as an anti-inflammatory agent without side effects such as antigenicity. JP-A-6-54681 describes the effect of PC-SOD on respiratory resistance by Forssman antiserum. The effect of PC-SOD on ulcerative gastrointestinal disorders has also been reported. Furthermore, PC-SO for motor neuron diseases such as amyotrophic lateral sclerosis
D (JP-A-8-208511) and PC-SOD for acute renal failure (JP-A-9-52843)
JP-A-9-59178), the effect of PC-SOD on virus (JP-A-9-59178) and P-SOD on lupus nephritis
The effect of C-SOD (JP-A-9-110717) has been reported.

[0007]

As described above, further applications of PC-SOD, which can play various roles in vivo, to pharmaceutical applications are expected in the industry.

[0008] The problem to be solved by the present invention is to establish a new pharmaceutical use of the above-mentioned PC-SOD and to provide this pharmaceutical.

[0009]

Means for Solving the Problems The present inventor has sought for the applicability of PC-SOD for various medical uses in order to meet the above-mentioned problems.

[0010] As a result, PC-SOD is extremely excellent in improving the dysfunction associated with cerebrovascular disorders.
-SOD was found to be extremely useful as an active ingredient of an agent for improving dysfunction associated with cerebrovascular disorders, and completed the present invention.

[0011] That is, the present inventors provide an agent for improving dysfunction associated with cerebrovascular disorder, comprising as an active ingredient SOD to which lecithin is chemically bonded. Preferably, the lecithin chemically bound SOD is a lecithin bound SOD via chemical crosslinking. SOD bound to lecithin via chemical crosslinking is preferably of the following general formula (I)
It is represented by

[0012]

Embedded image SOD— [C (O) — (CH ₂ ) _n —C (O) —X] _m (I) (wherein SOD represents superoxide dismutase, and X represents a hydroxyl group at the 2-position of glycerol. Represents the residue of the lysolecithin having the hydrogen atom at the 2-position hydroxyl group removed, m represents the average number of bonds to one SOD molecule, represents 1 or more, and n represents an integer of 2 or more)

[0013] Preferably, the SOD is a human-derived SOD. More preferably, SOD has the amino acid sequence 1
Human / human Cu / ZnSOD in which the amino acid at position 11 is S- (2-hydroxyethylthio) cysteine.

N is preferably any integer from 2 to 10. More preferably, n is 3. Also,
m is preferably a number from 1 to 16 on average. More preferably, m is 4 on average.

[0015]

Embodiments of the present invention will be described below. The agent for improving dysfunction associated with cerebrovascular disorders of the present invention (hereinafter, also simply referred to as “the agent for improving the present invention”) is effective for SOD chemically bound to lecithin, particularly SOD bound to lecithin via chemical crosslinking. It is a dysfunction improving agent associated with cerebrovascular disorders as a component.

[0016] In the present specification, "lecithin" means not only ordinary lecithin meaning phosphatidylcholine, but also fatty acid 1 bound to position 1 or 2 of glycerol.
Includes "lysolecithin" from which molecules have been removed.

Furthermore, SOD herein, active oxygen O ₂ in vivo ^- as long as capable of exhibiting its inherent function of decomposition, its origin and the like are not particularly limited, various animals and plants, or It is possible to widely use the SOD of a microorganism. However, considering that it is preferable to reduce antigenicity in a living body as much as possible in pharmaceutical applications, it is preferable to appropriately select an appropriate SOD according to the animal species to which the improving agent of the present invention is administered. . For example, when targeting a human who is most likely to need the improving agent of the present invention, it is preferable to use a human-derived SOD in the present invention in order to reduce antigenicity in the human body as much as possible. And among them,
Human-derived Cu / ZnSOD (human-derived SOD containing copper and zinc in the active center; hereinafter may be abbreviated as human Cu / ZnSOD) has a large amount of expression in cells,
In addition, production technology using genetic engineering techniques has been established,
It is particularly preferred because it can be prepared in large quantities.

This human Cu / ZnSOD includes natural human Cu / ZnSOD produced from human tissue; human Cu / ZnSOD produced by genetic engineering techniques; and an amino acid sequence substantially identical to natural human Cu / ZnSOD. Recombinant human Cu / ZnSOD having
/ SOD in which some amino acids in the amino acid sequence of ZnSOD are chemically modified or altered, and any human Cu / ZnSOD of SOD (PC-SOD) chemically bound to lecithin used in the present invention is used. It can be a material.

For reference, this natural human Cu / Zn
The amino acid sequence of the SOD protein is shown in SEQ ID NO: 1. Actual human Cu / ZnSOD is a dimer of a protein having this amino acid sequence.

As shown in the amino acid sequence of this protein, the amino acid at position 111 of natural human Cu / ZnSOD is cysteine. This position was converted to serine by protein engineering techniques, for example, site-directed mutagenesis. Human Cu / Zn SOD (JP-A-62-130684)
Further, human Cu / ZnSOD chemically modified at the cysteine at position 111 (Japanese Patent Application Laid-Open No. Hei 6-199895) has also been reported.
Any of C-SOD can be used as an active ingredient of the improving agent of the present invention. And these human Cu / Zn
Among SODs, charge and molecular weight are uniform and S
Human Cu / ZnS in which the cysteine at position 111 is chemically modified, for example, this cysteine is S- (2-hydroxyethylthio) cysteine, which has stable OD activity.
Preferably, OD is used as a material for the PC-SOD.

In this specification, human Cu / ZnSOD obtained by partially converting amino acids by site-directed mutagenesis or the like, or human Cu / ZnSOD
Is simply referred to as human Cu / ZnSOD, including those obtained by chemically modifying some amino acids.

In the present specification, "chemically bonded" means that the compound is covalently bonded, and as long as the compound is covalently bonded, the presence or absence of a chemical crosslink described later is not determined. It doesn't matter. That is, PC may be directly covalently bonded to SOD, or PC may be covalently bonded to SOD via chemical crosslinking.

The production method of the PC-SOD used in the present invention is not particularly limited as long as the lecithin is a chemically bound SOD. Usually, a chemical cross-linking agent is previously bound to the lecithin residue. The lecithin derivative can be obtained by bonding one or more lecithin derivatives to the SOD via chemical crosslinking formed by the chemical crosslinking agent.

The SOD chemically bound to lecithin contained in the improving agent of the present invention is not particularly limited, but it is particularly preferable to use SOD bonded to lecithin via chemical crosslinking.

The lecithin chemically bonded to SOD is not particularly limited, but it is preferable to use PC-SOD to which lysolecithin is chemically bonded.

The preferred PC-SOD in the present invention is represented by the following formula (I).

[0027]

Embedded image SOD— [C (O) — (CH ₂ ) _n —C (O) —X] _m (I) (wherein SOD represents superoxide dismutase, and X represents a hydroxyl group at the 2-position of glycerol. Represents the residue of the lysolecithin having the hydrogen atom at the 2-position hydroxyl group removed, m represents the average number of bonds to one SOD molecule, represents 1 or more, and n represents an integer of 2 or more)

Here, "glycerol 2" represented by X
The residue of lysolecithin having a hydroxyl group at the 2-position, excluding the hydrogen atom of the 2-position hydroxyl group, is represented by the following formula (II).

[0029]

Embedded image -O-CH (CH ₂ OR) [CH ₂ OP (O) (O ⁻ ) (OCH ₂ CH ₂ N ⁺ (CH ₃ ) ₃ )] (II) (where R is a fatty acid residue (Acyl group)

Particularly, a saturated or unsaturated fatty acid residue having 10 to 28 carbon atoms is preferable as R, and more preferably a myristoyl group, a palmitoyl group, a stearoyl group, an icosanoyl group, a docosanoyl group, and other groups having 14 to 14 carbon atoms.
Among them, there are 22 saturated fatty acid residues, and particularly preferred is a palmitoyl group which is a saturated fatty acid residue having 16 carbon atoms.

In the above formula (I), the residue -C (O)-
(CH ₂ ) _n —C (O) — represents a residue of a chemical bridge. The residue of this chemical bridge has the formula HO—C (O) — (CH ₂ ) _n
A linear dicarboxylic acid represented by —C (O) —OH, an anhydride of the dicarboxylic acid, an ester of the dicarboxylic acid,
Hydroxyl groups at both ends of a chemical crosslinking agent molecule such as a halide of this dicarboxylic acid or another reactive derivative of this dicarboxylic acid (in the case of the reactive derivative of the dicarboxylic acid, a portion corresponding to the hydroxyl group at both ends of the dicarboxylic acid) ).

Since this residue can crosslink SOD and lecithin to form a chemical bond (covalent bond), this is generically referred to herein as "chemical crosslink". The chemical crosslink in the formula (I) is linked at one end to the lysolecithin residue of the formula (II) by an ester bond.

It is considered that the other end of the chemical cross-linking is directly bonded to the amino group of SOD by an amide bond or the like. In this chemical cross-linking, the group — (CH ₂ )
_n- is a divalent group formed by removing one hydrogen atom from each of carbon atoms at both ends of a linear alkane, that is, an alkylene group. n is an integer of 2 or more, preferably n is 2 to
It is an integer of 10, particularly preferably 3.

Further, m in the above formula (I) represents the average number of lecithin bound to one SOD molecule through the above chemical crosslinking. This m is a number of 1 or more, and m is 1
To 16 are preferable, and 4 is particularly preferable.

The binding of the above lecithin derivative to SOD is
Specifically, it can be performed based on, for example, the method described in Japanese Patent Application Laid-Open No. 6-54681, and thereby a PC-SOD can be manufactured. This PC-S
Details of the manufacturing process of the OD will be described in a manufacturing example described later.

The PC-SOD used in the present invention may be a pharmaceutically acceptable salt. The salt that can be used specifically depends on the isoelectric point of PC-SOD used. For example, when the isoelectric point of the PC-SOD molecule is acidic, a pharmaceutically acceptable salt with an alkali can be used. Conversely, when the isoelectric point of the PC-SOD molecule is alkaline, a pharmaceutically acceptable salt with an acid can be used. For example, the isoelectric point of human Cu / ZnSOD obtained by converting the amino acid sequence at position 111 to serine is acidic, and the isoelectric point of PC-SOD obtained by converting this into PC is also acidic. Therefore, this 111-position converted human Cu / Z
As for nSOD, pharmaceutically acceptable salts of salts with alkali, for example, alkali metal salts, alkaline earth metal salts, ammonium salts and the like can be used in the present invention.

The PC-SOD manufactured as described above is
The desired improving agent of the present invention can be formulated as an active ingredient in the dosage form described below. Although the improving agent of the present invention can be widely applied as a dysfunction improving agent associated with cerebrovascular disorders in mammals including humans, it is particularly preferable to use the ameliorating agent as a dysfunctional improving agent associated with cerebrovascular disorders in humans.

The term “cerebrovascular disorder” refers to “a state in which ischemia or hemorrhage is caused by abnormal cerebral blood vessels and the brain is functionally or organically affected”. Specifically, cerebral hemorrhage, hypertensive intracerebral hemorrhage, Examples include infarction, cerebral thrombosis, cerebral embolism, transient ischemic attack, hypertensive encephalopathy, and the like.

The ameliorating agent of the present invention can be widely applied to such dysfunctions associated with cerebrovascular disorders. Among these cerebrovascular disorders, dysfunction associated with cerebral hemorrhage and / or cerebral infarction is particularly effective as a target disease for the administration of the ameliorating agent of the present invention, and dysfunction associated with cerebral hemorrhage is a target disease for administration of the ameliorating agent of the present invention. Extremely effective.

The dysfunction is not limited as long as it is a dysfunction caused by a cerebrovascular disorder. Examples of the dysfunction include speech impairment, motor dysfunction due to paralysis, visual impairment, hearing impairment, olfaction impairment, taste impairment and the like. Sensory disorders, dementia, and the like. Among these dysfunctions, speech disorders due to cerebrovascular disorders and / or motor dysfunctions due to paralysis are extremely effective as target diseases to which the improving agent of the present invention is administered.

The specific names of cerebrovascular disorders and dysfunctions described above are merely examples, and the disorders to which the agent of the present invention can be applied are not limited to these exemplified disorders. PC-SOD, which is an active ingredient of the ameliorating agent of the present invention, improves cerebral microcirculation disorder due to cerebrovascular disorder, and further improves dysfunction by suppressing direct damage of O ₂ ⁻ to cerebral nerve cells. It is possible to do.

In the present invention, any dosage form can be appropriately selected as the dosage form of the ameliorating agent of the present invention, depending on the nature and progress of the target disease. That is, the improving agent of the present invention can be injected (intramuscularly, subcutaneously, intradermally, intravenously), orally,
It can be orally or parenterally selected and administered depending on the administration method such as inhalation. These drugs can be formulated as appropriate according to these administration methods. The most preferred administration method of the improving agent of the present invention is intravenous injection.

There are no particular restrictions on the dosage form that can be selected. For example, injections (solutions, suspensions, emulsions, solids for dissolution at the time of use, etc.), tablets, capsules, granules, powders, solutions, liposomes Agents, gels, external powders, sprays, inhalants, suppositories and the like can be widely selected. The most preferred dosage form of the injection of the present invention is an injection.

In preparing these preparations, ingredients used in the preparation of ordinary pharmaceuticals, such as excipients, binders, lubricants, coloring agents and disintegrants, can be added.
The amount of PC-SOD, which is an active ingredient in the improving agent of the present invention, and the dose of the improving agent of the present invention depend on the method of administration of the preparation, the dosage form, the purpose of use, the specific symptoms of the patient, the weight of the patient, and the like. Although it is a matter to be determined individually and is not particularly limited, the clinical dose of PC-SOD is about 100 U / Kg to about 10,000 U / Kg per day [1 U (unit) in the present specification. Can be obtained by a cytochrome c method (xanthine-xanthine oxidase-cytochrome c system; pH 7.8, 30 ° C .; J. Biol. Chem., Vol. 244, N).
o. 22, 6049-6055 (1969)) indicates the amount of PC-SOD enzyme that inhibits the reduction rate of cytochrome c by 50%. Further, the administration interval of the above preparation may be every other day or every day, and is not particularly limited. The administration can be performed about once a day, or can be divided into two to four times a day.

The present invention relates to a method in which lecithin is chemically bound to S
Also provided is an agent for improving motor dysfunction associated with central nervous system disorders, which comprises OD as an active ingredient. The central nervous system disorder here is not particularly limited as long as it is a disorder of the brain and spinal cord, and vascular disorders (for example, cerebral hemorrhage, hypertensive intracerebral hemorrhage, cerebral infarction, cerebral thrombosis, cerebral embolism, transient cerebral ischemic attack, Hypertensive encephalopathy, etc.), inflammation (eg, encephalitis, myelitis, etc.). Among these, vascular disorders (cerebrovascular disorders) are preferred, cerebral hemorrhage and / or cerebral infarction are more preferred, and cerebral hemorrhage is particularly preferred.

The motor dysfunction referred to herein is not particularly limited as long as it is a motor dysfunction, and examples thereof include motor dysfunction due to paralysis. The PC-SOD contained as an active ingredient of this drug, the administration method, dosage form, and dosage of this drug are as described in detail above.

Although this drug can be widely applied to mammals including humans, it is particularly preferable to use it for humans. The present invention also provides a speech disorder improving agent containing as an active ingredient SOD to which lecithin is chemically bonded.

PC contained as an active ingredient of this drug
-SOD, the administration method, dosage form, and dosage of this drug are as described in detail above. This drug can also be widely applied to mammals including humans, but is particularly preferably used for humans. This drug is considered to be applicable regardless of whether the speech disorder is caused by central nervous system or peripheral nervous system.

[0049]

EXAMPLES Examples of the present invention will be specifically described below as Production Examples, Test Examples and Formulation Examples. However, these should not limit the technical scope of the present invention.

[0050]

[Production example]

(1) Production of human Cu / Zn SOD derivative [human SOD in which the amino acid at position 111 of SOD is S- (2-hydroxyethylthio) cysteine]: This human Cu / Z
The nSOD derivative was produced by the method described in JP-A-6-199895.

That is, a known E. coli 545πHR (p
HT351) was cultured, and the obtained cultured cells were disrupted to obtain a crude extract of human Cu / ZnSOD. The crude extract was precipitated with ammonium sulfate, Q-Sepharose FF (Pharmacia)
Column, ammonium sulfate precipitation in order to purify, further 0.2μ
The filtrate filtered through a m filter was lyophilized to give a colorless solid (recombinant human apo SOD). This recombinant human apo SOD was added to a 10 mM triethanolamine buffer (pH 7.
0), added with bis (2-hydroxyethyl) disulfide, readjusted to pH 7.0, and stirred at 4 ° C for 24 hours. NaCl, 2M acetate buffer (pH
5.0) and 0.1 M CuCl ₂ aqueous solution, and added at 4 ° C.
For 13 hours. After the stirring, the pH was adjusted to 7.0, and the precipitate formed by ammonium sulfate precipitation (4 ° C., 100% saturation) was collected. The precipitate was dissolved in a 10 mM triethanolamine buffer (pH 7.0) and dialyzed against a 10 mM triethanolamine buffer (pH 7.0) to desalinate. The desalted fraction was passed through a Q-Sepharose FF column and developed by gradually increasing the NaCl concentration in the order of 5 mM, 10 mM, 20 mM, and 50 mM, and the SOD fraction was collected. Then, this SOD
The fraction was precipitated with ammonium sulfate (4 ° C., 100% saturation), and the resulting precipitate was collected. This precipitate is used as an aqueous solution,
and then lyophilized to obtain a human SOD derivative (hereinafter simply referred to as SO
D).

The amino acid at position 111 of this human SOD derivative is S- (2-hydroxyethylthio) cysteine.

(2) Production of PC-SOD: PC-SOD
Was produced by the method described in JP-A-6-54681. Specifically, it is as follows.

(A) Synthesis of active ester of 2- (4-hydroxycarbonylbutyroyl) lysolecithin Synthesis of 2- (4-hydroxycarbonylbutyroyl) lysolecithin Chloroform-pyridine of lysolecithin in which the 2-position of glycerol is a hydroxyl group 80ml / 20ml), add DM
AP (N, N-dimethylaminopyridine) and glutaric anhydride were added, and the mixture was stirred at 60 ° C. for 15 hours. Thereafter, the reaction solution was concentrated under reduced pressure, and chloroform: methanol:
The solvent was dissolved by adding a solvent of water = 4: 5: 1, and the solution was passed through an ion exchange column (Dowex 50W-X8, manufactured by Dow Chemical Company) equilibrated with the same solution. After fractionating the target compound by TLC and concentrating the fractionated solution under reduced pressure, the residue was purified by a column packed with ODS (octadecylsilane) to obtain the title compound.

Synthesis of Active Esters of 2- (4-Hydroxycarbonylbutyroyl) lysolecithin The carboxylic acid obtained above was dissolved in dichloromethane, cooled at 0 ° C., and N-hydroxysuccinimide and tetrazole were sequentially added thereto. , Then DCC (1,3-
A dichloromethane solution of dicyclohexylcarbodiimide) was slowly added dropwise, and the mixture was stirred at room temperature for 15 hours. After stirring, the insolubles were filtered through celite to obtain a dichloromethane solution of the active ester.

(B) Synthesis of PC-SOD having an average of four lecithin derivatives bound to one molecule of SOD SOD produced according to the above-mentioned step (1) dissolved in 50 mM borate buffer (pH 8.5) and this SOD Was reacted with the active ester of 2- (4-hydroxycarbonylbutyroyl) lysolecithin synthesized in the above (a) in a 0.8-fold molar amount with respect to all amino groups of the above by the following method.

The dichloromethane in the active ester solution was distilled off and dissolved in DMF (N, N-dimethylformamide). This was added to a 50 mM borate buffer (pH 8.5).
, And after filtration of insolubles, dissolve in the same buffer
Was added dropwise to the SOD solution to which 50% of DMF was added. After stirring at 0 ° C. for 15 hours, the reaction solution was filtered,
The mixture was applied to a gel filtration column using Sephacryl S-300 (manufactured by Pharmacia) as a carrier, and a 50 mM borate buffer (pH
8.5), and the PC-SOD eluted fractions were collected and purified by ion exchange chromatography. This was then concentrated by ultrafiltration, and the protein concentration was determined by the Lowry method (Lowry, Oh et al., J. Biol. Chem., 193, 265 (1951)).
Year)), the remaining amino group of SOD was determined by the TNBS method (trinitrobenzene sulfonate method, Goodwin, JF, etc., Clin. Ch.
em., vol. 16, p. 24 (1970)).
The number of lecithin derivatives bonded per D1 molecule was determined to be 4.0 on average. The properties of this PC-SOD are a colorless to pale blue-green transparent aqueous solution, and the pH is 7 to
It was 8. In addition, when the molecular weight was examined by SDS-polyacrylamide gel electrophoresis, about 1800 per monomer of PC-SOD subunit (PC-SOD is a homodimer of subunit as described above)
It was 0.

[0058]

[Test Example] PC-SOD (concentration: 30 mg / ml; specific activity: 3.07 × 10 ³ U / protein m)
g) was tested for toxicity and pharmacology. PC
For -SOD, a solution (hereinafter sometimes abbreviated as PC-SOD solution) dissolved in a 5% mannitol aqueous solution to a final concentration of 5 mg / ml was used. The properties of this PC-SOD solution are a colorless to pale blue-green transparent aqueous solution,
Was 6-8. The osmotic pressure ratio with respect to the physiological saline was about 1.

(1) Acute toxicity test Acute toxicity test using rats A PC-SOD solution was administered to 5 male and 5 female SD rats via the tail vein at 100 mg / Kg. For 14 days after the administration, observation of general condition and life and death and measurement of body weight were performed. The animals were necropsied on the 15th day, and the major organs were visually observed.

As a result, no dead rats were observed,
No change was observed in any of the general condition, body weight, and autopsy findings.
The lethal dose of D was estimated to be 100 mg / Kg or more.

Acute toxicity test using monkeys PC-SOD solution was administered to two female cynomolgus monkeys via the vein of the extremity at 100 mg / Kg. For 14 days after the administration, observation of general condition and life and death and measurement of body weight were performed.

As a result, no cynomolgus monkey died was observed, and no change was observed in any of the general condition and body weight. Therefore, the lethal dose of PC-SOD by intravenous administration was estimated to be 100 mg / Kg or more. .

(2) Efficacy Pharmacological Test In a phase II clinical study for examining the effect of PC-SOD on amyotrophic lateral sclerosis (ALS), the following results were obtained for an ALS patient (1) with cerebral hemorrhage. PC-SOD
Was administered intravenously.

[0064]

[Table 1] (Early November 1996, concurrent with cerebral hemorrhage) Feb. 3, 1997-Feb. 16, 1997 (2 weeks) PC-SOD administration 2 mg / day, Feb. 17, 1997-Mar. 2, 1997 (2 weeks) PC-SOD administration 4 mg / day March 3-early April 1997 PC-SOD administration 8 mg x 2 times / week

(A) PC for speech disorder due to cerebral hemorrhage
-Effect of SOD The speech ability of the ALS patient was continuously evaluated immediately after cerebral hemorrhage. The utterance ability was judged by a doctor according to the following evaluation criteria. 10-9: Can speak normally 8-7: Can speak almost normally 6-5: Can speak, but slightly inconvenient 4-3: Can speak, but inconvenient 2-1: Speak Can do, but is quite inconvenient 0: Can't speak at all

The results are shown in FIG. In FIG. 1A, cerebral hemorrhage occurs at the origin (96.11) on the horizontal axis.
As a result, the speech ability after cerebral hemorrhage was 3-4, but after PC-SOD administration, the speech ability gradually recovered, and finally the speech ability was restored to 8. From this, P
It was shown that C-DOS has an improving effect on dysfunction (speech disorder) associated with cerebrovascular disorder (cerebral hemorrhage).

(B) PC for hand paralysis due to cerebral hemorrhage
-Effect of SOD The left and right grip strength of the ALS patient was continuously evaluated after cerebral hemorrhage. The grip strength was measured by the following method. That is,
Using a standard mercury sphygmomanometer, the initial pressure is 20 mmHg, and the patient can hold the cuff as hard as possible without supporting the hand with anything.
You can hold it up to three times with one hand and take the highest value.

The results are shown in FIG. In FIG. 1B, cerebral hemorrhage occurs at the origin (96.11) on the horizontal axis.
Since the ALS patient has cerebral hemorrhage in the right brain, the grip strength of the left hand reflects the influence of both ALS and cerebral hemorrhage, and the grip strength of the right hand reflects only the influence of ALS. That is, the difference between the grip strength of the right hand and the grip strength of the left hand reflects the influence of cerebral hemorrhage.

As a result, the left hand grip strength (AL
Reflecting the effects of both S and cerebral hemorrhage) was 0 mmHg,
After PC-SOD administration, the grip strength gradually recovered and gradually approached the right hand grip strength (only reflecting the influence of ALS). From this, it was shown that PC-SOD has an improving effect on dysfunction (motor dysfunction due to hand paralysis; grip strength decrease) associated with cerebrovascular disorder (cerebral hemorrhage).

Incidentally, the right hand grip strength was also changed after PC-SOD administration.
Although it is gradually recovering, this is considered to be the effect of PC-SOD on ALS.

[0071]

[Formulation example]

(1) Injection PC-SOD (30 mg / m2) produced in the above Production Example
l) was dissolved in a 5% aqueous solution of mannitol to a final concentration of 5 mg / ml, which was aseptically filtered, dispensed into ampoules in 2 ml portions and sealed to produce injections.

(2) Tablets 100 mg of freeze-dried PC-SOD produced in the above-mentioned production example, lactose 670 mg, potato starch 150 m
g, 60 mg of crystalline cellulose and 50 mg of light anhydrous silicic acid were mixed, and a solution of 30 mg of hydroxypropyl cellulose in methanol (10% by weight of hydroxypropyl cellulose) was added and kneaded and granulated. Next, this was extruded through a screen having a diameter of 0.8 mm to obtain granules, and after drying, 15 mg of magnesium stearate was added and compression-molded to produce tablets of 200 mg.

(3) Capsules 100 mg of freeze-dried PC-SOD produced in the above production example, 150 mg of potato starch, 50 mg of light anhydrous silicic acid, 10 mg of magnesium stearate and lactose 7
65 mg was uniformly mixed, 200 mg of the mixture was dispensed and filled into hard capsules to produce capsules.

[0074]

The medicament according to the present invention has been directly shown in pharmacological tests in vivo to be effective in humans and not in model animals. In particular, for disorders of advanced functions such as speech, since the results using model animals do not always apply directly to humans, the results that the agent according to the present invention is effective for humans are as follows: The application of the drug according to the present invention to humans is extremely important and significant.

The dysfunctions associated with cerebrovascular disorders are manifested as a result of complex involvement of cerebral neuronal disorders and other factors caused by cerebrovascular disorders. Improvement alone cannot predict that all dysfunctions will be sufficiently improved. The ameliorating agent of the present invention very effectively ameliorates dysfunctions caused by the complex involvement of such various factors caused by cerebrovascular disorders, and its utility value is high.

According to the present invention, there is provided a remarkably effective remedy for dysfunction due to cerebrovascular disorder, motor dysfunction due to central nervous system disorder and speech disorder, particularly a remedy which can be sufficiently used in humans. .

[0077]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 153 Sequence type: Amino acid Topology: Linear Sequence type: Protein Sequence Ala Thr Lys Ala Val Cys Val Leu Lys Gly Asp Gly Pro Val Gln Gly 1 5 10 15 Ile Ile Asn Phe Glu Gln Lys Glu Ser Asn Gly Pro Val Lys Val Trp 20 25 30 Gly Ser Ile Lys Gly Leu Thr Glu Gly Leu His Gly Phe His Val His 35 40 45 Glu Phe Gly Asp Asn Thr Ala Gly Cys Thr Ser Ala Gly Pro His Phe 50 55 60 Asn Pro Leu Ser Arg Lys His Gly Gly Pro Lys Asp Glu Glu Arg His 65 70 75 80 Val Gly Asp Leu Gly Asn Val Thr Ala Asp Lys Asp Gly Val Ala Asp 85 90 95 Val Ser Ile Glu Asp Ser Val Ile Ser Leu Ser Gly Asp His Cys Ile 100 105 110 Ile Gly Arg Thr Leu Val Val His Glu Lys Ala Asp Asp Leu Gly Lys 115 120 125 Gly Gly Asn Glu Glu Ser Thr Lys Thr Gly Asn Ala Gly Ser Arg Leu 130 135 140 Ala Cys Gly Val Ile Gly Ile Ala Gln 145 150

[Brief description of the drawings]

FIG. 1 shows the effect of PC-SOD on ALS patients (complicated with cerebral hemorrhage).

Claims (13)

[Claims] 1. An agent for improving dysfunction associated with cerebrovascular disorders, comprising as an active ingredient superoxide dismutase to which lecithin is chemically bonded. 2. The superoxide dismutase chemically bound to lecithin is superoxide dismutase bound to lecithin via chemical crosslinking.
The agent for improving dysfunction associated with cerebrovascular disease according to the above. 3. The agent for improving dysfunction associated with cerebrovascular disorder according to claim 2, wherein the superoxide dismutase bonded to lecithin via chemical crosslinking is represented by the following general formula (I). Embedded image SOD— [C (O) — (CH ₂ ) _n —C (O) —X] _m (I) (wherein SOD represents superoxide dismutase, and X represents a hydroxyl group at the 2-position of glycerol. Represents the residue of the lysolecithin having the hydrogen atom at the 2-position hydroxyl group removed, m represents the average number of bonds to one SOD molecule, represents 1 or more, and n represents an integer of 2 or more) 4. The agent for improving dysfunction associated with cerebrovascular disorder according to claim 3, wherein in the general formula (I), the SOD is human-derived superoxide dismutase. 5. In the general formula (I), SOD is human-derived Cu /, wherein the amino acid at position 111 of the amino acid sequence is S- (2-hydroxyethylthio) cysteine.
The agent for improving dysfunction associated with cerebrovascular disorder according to claim 3, which is Zn superoxide dismutase. 6. In the general formula (I), n is 2 or more and 10 or more.
6. Any one of claims 3 to 5, which is one of the following integers:
The agent for improving dysfunction associated with cerebrovascular disorder according to the above item. 7. The agent for improving dysfunction associated with cerebrovascular disorder according to claim 6, wherein n is 3 in the general formula (I). 8. In the general formula (I), m is 1 on average.
The dysfunction-improving agent associated with cerebrovascular disorder according to any one of claims 3 to 7, which has a number of 16 or more. 9. In the general formula (I), m is 4 on average.
The agent for improving dysfunction associated with cerebrovascular disorder according to claim 8, which is 10. The ameliorating agent for dysfunction associated with cerebrovascular disorder according to any one of claims 1 to 9, wherein the cerebrovascular disorder is cerebral hemorrhage and / or cerebral infarction. 11. The agent for improving a dysfunction associated with a cerebrovascular disorder according to any one of claims 1 to 10, wherein the dysfunction is a motor dysfunction due to speech disorder and / or paralysis. 12. An agent for improving motor dysfunction associated with central nervous system disorders, comprising as an active ingredient superoxide dismutase to which lecithin is chemically bonded. 13. A speech disorder improving agent comprising, as an active ingredient, superoxide dismutase to which lecithin is chemically bonded.