JPH09124505A - Therapeutic agent/preventive for corneal problem - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject medicine suppressing ectocornea disorder caused by ultraviolet light, ecotocornea disorder and occurrence of cornea cloud, comprising a lecithinized superoxide dismutase as an active ingredient. SOLUTION: This therapeutic agent/preventive for corneal problem comprises a lecithinized superoxide dismutase [preferably an SOD of the formula (SOD is a superoxide dismutase; X is a residue after removal of a hydrogen atom of a hydroxyl group at the 2-position from a lysolecithin containing a hydroxyl group at the 2-position of glycerol; (m) is >=1; (n) is >=2] bonded through a chemical cross-linking to lysolecithin. An SOD obtained by chemically modifying a cysteine as an amino acid at the 111 position of SOD derived from human, containing copper and zinc at the active center is preferably used as the SOD. The objective medicine preferably contains 0.01-0.1wt.% lecithinized SOD in an eye drop. The eye drop is applied once to several times usually and one or three drops are applied per time.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a therapeutic / preventive agent for corneal disorders containing lecithinized superoxide dismutase as an active ingredient.

[0002]

2. Description of the Related Art Superoxide dismutase (hereinafter, referred to as superoxide dismutase)
Sometimes abbreviated as SOD) is an animal, plant, widely distributed in the living body such as microorganisms, superoxide anion radical is the active oxygen rich in reactivity (hereinafter, simply O ₂ ^-
It is also abbreviated)) is known as an enzyme that decomposes. 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)). Generally, when administered intravenously, SOD has a low cell affinity and its half-life in blood is only 4 to 6 minutes, and SOD is rapidly excreted in urine. In order to extend the blood half-life of this SOD, SOD was added to Ficoll, polyethylene glycol,
Attempts have been made to modify them with rat albumin, dextran, etc. to form macromolecules. However, SOD modified with ficoll or polyethylene glycol
It has been reported that the original enzyme activity of SOD is significantly reduced and the cell affinity is still low. It has been reported that SOD modified with rat albumin has antigenicity in the human body. further,
The SOD modified with dextran is excellent in that the anti-inflammatory action originally possessed by SOD is enhanced, but it has been reported that this also has antigenicity in the human body.

In recent years, a substance in which a biologically active protein such as SOD is chemically modified with lecithin (phosphatidylcholine: hereinafter sometimes abbreviated as PC) has been reported. It has been reported that this PC-binding modified bioactive protein has markedly increased cell affinity and is significantly different in biodistribution from conventional non-modified bioactive proteins. Further, it is said that enhancement of pharmacological activity of bioactive proteins, reduction of side effects, and promotion of absorption can be expected. In addition, PC SOD (hereinafter, P
It is also reported that C-SOD is sometimes abbreviated as "C-SOD" (Japanese Patent Laid-Open No. 3-163100, Japanese Patent Laid-Open No. 3-170438, US Pat. 109, 118). Furthermore, it is said to be useful as an anti-inflammatory agent without side effects such as antigenicity. Japanese Unexamined Patent Publication (Kokai) No. 6-54681 discloses PC-S for respiratory resistance by Forssman antiserum.
The effect of OD is described. In addition, PC-SO
The effect of D on ulcerative gastrointestinal disorder has also been reported. Furthermore, the effect of PC-SOD on motor neuron diseases such as amyotrophic lateral sclerosis has also been reported. However, the use of lecithinized superoxide dismutase for ophthalmic use, in particular, the therapeutic or preventive effect on corneal disorders has not been studied so far.

[0004]

SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to provide a therapeutic / preventive agent for corneal disorders, which comprises lecithinized superoxide dismutase as an active ingredient for preventing or treating corneal disorders.

[0005]

[Means for Solving the Problems] In view of the above circumstances, the present inventors have used rat as a model animal to induce corneal disorders, and then administered an eye drop containing lecithinized superoxide dismutase to the eye drop for a period of time. Observation of corneal epithelial lesion area and corneal opacification revealed that ophthalmic solution containing lecithinized superoxide dismutase suppresses UV-induced corneal epithelial injury and suppresses corneal opacification that occurs during healing of epithelial injury. It was The present invention has been completed through further research based on such findings.

That is, the gist of the present invention is (1) a therapeutic / preventive agent for corneal disorders containing lecithinized superoxide dismutase as an active ingredient, (2) corneal disorders occurring in corneal epithelium. Corneal disorder therapeutic / preventive agent, (3) corneal disorder is an ultraviolet-induced corneal epithelial disorder, the corneal disorder therapeutic / preventive agent according to (1), (4) the dosage form being eye drops (1) to (3) A therapeutic / preventive agent for corneal disorders according to any one of (5), A therapeutic / prophylactic agent for corneal disorders according to (4), wherein the lecithinized superoxide dismutase content is 0.001 to 1% by weight. )
The therapeutic / preventive agent for corneal disorders according to the above (1) or (2), wherein the lecithinized superoxide dismutase is a superoxide dismutase bound to lecithin through a chemical crosslink, and (7) a lecithin bound through a chemical crosslink. The superoxide dismutase is represented by the following general formula (I) SOD- [C (O)-(CH ₂ ) _n- C (O) -X] _m (I) (In the formula, SOD represents superoxide dismutase, and X is Represents a 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 is the average number of bonds to one SOD molecule, represents an integer of 1 or more, and n is 2 or more. Represents the integer). The therapeutic / preventive agent for corneal disorders according to (6), which is represented by: (8) The therapeutic / preventive agent for corneal disorders according to (7), wherein in the general formula (I), SOD is of human origin. In the general formula (I), the SOD is a human-derived Cu / Zn superoxide dismutase, wherein the amino acid at position 111 of the amino acid sequence is S- (2-hydroxyethylthio) cysteine, (7). Corneal disorder therapeutic / preventive agent, (10) In the general formula (I), n is 2 or more and 1
The therapeutic / preventive agent for corneal disorders according to any one of (7) to (9), which is any integer of 0 or less, (11) the above formula (10), wherein n is 3 Corneal disorder therapeutic / preventive agent, (12) in the general formula (I),
The therapeutic / preventive agent for corneal disorders according to any one of (7) to (11) above, wherein m is a number of 1 or more and 16 or less, and (13)
In the general formula (I), m is 4, relates to the therapeutic / preventive agent for corneal disorders according to the above (12).

[0007]

Embodiments of the present invention will be described below. (1) The therapeutic / prophylactic agent for corneal disorders of the present invention is a therapeutic / prophylactic agent for corneal disorders, which comprises lecithinized superoxide dismutase, particularly superoxide dismutase bound to lecithin through chemical crosslinking, as an active ingredient. In the present specification, "lecithin" includes, in addition to ordinary lecithin which means phosphatidylcholine, "lysolecithin" obtained by removing one molecule of fatty acid bonded to the 1-position or 2-position of glycerol. Further, the “superoxide dismutase (SOD)” in the present specification is not particularly limited in its origin and the like as long as it can exhibit its original function of decomposing active oxygen O ₂ ⁻ in the living body.
It is possible to widely use SODs possessed by various animals and plants or microorganisms. However, the present invention is an invention for pharmaceutical use, and considering that it is preferable to reduce antigenicity in vivo as much as possible, depending on the animal species to which the therapeutic / preventive agent for corneal disorders of the present invention is administered. Therefore, it is preferable to select an appropriate SOD as appropriate. For example, when the human is considered to need the therapeutic / prophylactic agent for corneal disorders of the present invention most, human-derived SOD is used in the present invention in order to reduce the antigenicity in the human body as much as possible. It is preferable. Among them, human-derived Cu / ZnSOD (human-derived SOD containing copper and zinc in the active center; hereinafter sometimes abbreviated as human Cu / ZnSOD) has a large intracellular expression level and is also genetically engineered. The production technique by the technical method was established at the time of completion of the present invention, and it is possible to prepare a large amount at the present time, which is particularly preferable.

The human Cu / ZnSOD has a natural human Cu / ZnSOD produced from human tissue; a human Cu / ZnSOD produced by a genetic engineering technique; an amino acid sequence substantially the same as the natural human Cu / ZnSOD. Recombinant human Cu / ZnSOD and the like are available, and any human Cu / ZnSOD can be used as a material for superoxide dismutase (PC-SOD) bound to lecithin through the chemical crosslinking used in the present invention.
The amino acid at position 111 of natural human Cu / ZnSOD is cysteine, but human C obtained by converting the position 111 to serine by a protein engineering method such as site-directed mutagenesis.
u / ZnSOD (JP-A-62-130684) and human Cu / ZnS chemically modified with 111 cysteine.
OD (JP-A-6-199895) has also been reported, and any of these PC-SODs made from human Cu / ZnSOD can be used as an active ingredient of the therapeutic / preventive agent for corneal disorders of the present invention. . And these human Cu /
Among ZnSOD, 111-position cysteine, which is uniform in charge and molecular weight and has stable SOD activity, is chemically modified. For example, this cysteine is S- (2-
Human Cu / Z with (hydroxyethylthio) cysteine
It is preferable to use nSOD as a material for the PC-SOD. In this specification, human Cu / ZnSOD is obtained by partially converting amino acids by site-directed mutagenesis or the like, or is obtained by chemically modifying some amino acids of human Cu / ZnSOD. Including those, it is simply called human Cu / ZnSOD.

The PC-SOD used in the present invention is usually formed from a lecithin derivative obtained by previously binding a chemical cross-linking agent to the residue of lecithin to superoxide dismutase (SOD) by the chemical cross-linking agent. It can be obtained by binding one or more lecithins via chemical cross-linking. The lecithin that binds to SOD is not particularly limited here, but PC-SOD to which the lysolecithin is bound is preferably used in the present invention. Suitable PC-SOD in the present invention has the formula (I) _{SOD-[C (O) - (CH 2} ) n -C (O) -X ] _m (I) (wherein, SOD is superoxide dismutase X represents a 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 represents an integer of 1 or more, and n represents an integer of 2 or more). It is represented by. Here, the “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” represented by X is represented by the following formula (II) —O—CH (CH ₂ OR) [CH ₂ OP (O) (O ⁻ ) (OCH ₂ CH ₂ N ⁺ (CH ₃ ) ₃ )] (II) (wherein R is a fatty acid residue (acyl group)). In particular, a saturated or unsaturated fatty acid residue having 10 to 28 carbon atoms is preferable as R above, and more preferable is a myristoyl group, a palmitoyl group, a stearoyl group, an icosanoyl group, a docosanoyl group, or a saturated fatty acid having 14 to 22 carbon atoms. A residue, particularly preferred is a palmitoyl group which is a saturated fatty acid residue having 16 carbon atoms. Further, in the above formula (I), the residue —C (O) — (CH ₂ ) _n —C (O) — represents a chemically crosslinked residue. Residues of the chemical crosslinking of the formula HO-C (O) - ( CH 2) n -C (O) linear dicarboxylic acid represented by -OH, anhydrides of the dicarboxylic acids, the dicarboxylic acid Hydroxyl groups at both ends of the chemical cross-linking agent molecule such as ester, halide of this dicarboxylic acid or other reactive derivative of this dicarboxylic acid (in the case of the above reactive derivative of dicarboxylic acid, it corresponds to the hydroxyl groups at both ends of this dicarboxylic acid. It is the residue excluding the part).

Since this residue can crosslink and bond SOD and lecithin, they are collectively referred to as "chemical crosslinks" in the present specification. The chemical crosslink in the above formula (I) is bonded to the above-mentioned lysolecithin residue (II) by an ester bond at one end thereof. Further, it is considered that the other end of this chemical crosslink is directly bonded to the amino group of SOD by an amide bond or the like. Note that in this chemical crosslinking, group - (CH _{2) n} - is a bivalent group obtained by removing one by one hydrogen atom from a carbon atom at both ends of a linear alkane,
That is, it is an alkylene group. n is an integer of 2 or more, preferably n is an integer of 2 to 10, and particularly preferably 3. Further, m in the formula (I) represents the average number of lecithins bound to the SOD1 molecule through the above-mentioned chemical cross-linking. This m is an integer of 1 or more, and m
Is preferably 1 to 16, and is particularly preferably 4. The binding of the lecithin derivative to SOD can be specifically performed based on, for example, the method described in JP-A-6-54681, whereby PC-
SOD can be manufactured. Details of the manufacturing process of this PC-SOD 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
Regarding nSOD, a pharmaceutically acceptable salt among salts with an alkali, such as an alkali metal salt, an alkaline earth metal salt and an ammonium salt, can be used in the present invention. The thus-produced PC-SOD can be blended as an active ingredient in the dosage form described below to give a desired therapeutic / preventive agent for corneal disorders.

(2) The therapeutic / prophylactic agent for corneal disorders of the present invention can be prepared as follows. As for the administration of lecithinized superoxide dismutase, local administration is preferable, and eye drop administration is particularly preferably used. As a dosage form for eye drop administration, an eye drop solution is preferable. As a general method for preparing eye drops, a lyophilized preparation of lecithinized superoxide dismutase may be prepared by dissolving it in sterile purified water. If necessary, an isotonic agent, a pH adjusting agent, a buffering agent may be used. ,
Additives commonly used in eye drops such as preservatives can be added as appropriate. Additives for the preparation of eye drops are not particularly limited, the enzyme activity of lecithinized superoxide dismutase is not inhibited, does not impair the stability, and is generally used as an additive for eye drops. Whatever is present can be used as a substrate.

However, since the stability of lecithinized superoxide dismutase is impaired in the presence of salts, it is preferable to minimize the use of salts. Examples of additives include glycerin and mannitol as isotonic agents, hydrochloric acid and sodium hydroxide as pH adjusting agents, phosphate buffers as buffering agents, and paraoxybenzoic acid esters and chlorides as preservatives. Examples thereof include benzalkonium.

The content of lecithinized superoxide dismutase used in eye drops is usually 0.001 to 1.
It is 0% by weight, preferably 0.005 to 0.5% by weight, more preferably 0.01 to 0.1% by weight. In the examples, the effect of the eye drop of the present invention was examined using a rat corneal injury model, but when it is used for human treatment / prevention, a high concentration of about 10 times the concentration in normal rats is required. Become.

When the therapeutic / preventive agent for corneal disorders of the present invention is clinically applied as an eye drop, it is usually 1 depending on symptoms.
Apply 1 to 3 drops once to several times a day. Regarding the safety of lecithinized superoxide dismutase,
As described in detail later, an acute toxicity test was conducted in rats and cynomolgus monkeys. No deaths were observed, and no changes were observed in general condition, body weight, or autopsy findings. The lethal dose of PC-SOD is estimated to be 100 mg / Kg or more.

The therapeutic / prophylactic effect of lecithinized superoxide dismutase on corneal damage is clarified by the effect of reducing corneal damage using a rat corneal damage model. Using a model in which corneal epithelial damage was induced by ultraviolet irradiation as a rat corneal damage model, it was clarified by examining the therapeutic / preventive effects of corneal damage by instilling eye drops containing PC-SOD at various concentrations. It The effect is determined by measuring the area of corneal epithelial damage and measuring the degree of corneal opacity. As a comparative control, non-lecithinized superoxide dismutase (U-SO
A similar experiment is performed using D). As a result, as shown in the examples, PC-SOD shows a markedly superior effect to U-SOD, and the effect is concentration-dependent. This comparative study of PC-SOD and U-SOD clearly supports the effect of lecithinization of SOD.

As is clear from the above facts, PC-SOD has the effect of remarkably suppressing corneal epithelial damage and promoting its recovery, and is useful for the treatment and prevention of corneal damage.

[0018]

EXAMPLES Examples of the present invention will be specifically described below as production examples and test examples. However, these should not limit the technical scope of the present invention.

[Production Example] (1) Human Cu / ZnSOD derivative [human-derived superoxide dismutase in which the 111th amino acid of SOD is S- (2-hydroxyethylthio) cysteine]
Production of this human Cu / ZnSOD derivative
It can be obtained by the method described in JP-A-199895. The specific procedure is shown below. Human SOD
Well-known Escherichia coli 545πHR (pHT351) prepared by introducing the gene was cultured, and the obtained cultured cells were crushed to obtain a crude extract of human Cu / ZnSOD. Ammonium sulfate precipitation, Q-Sepharose FF was added to this crude extract.
A column (manufactured by Pharmacia) was sequentially subjected to ammonium sulfate precipitation for purification, and the filtrate filtered through a 0.2 μm filter was freeze-dried to obtain a colorless solid (recombinant human apo SOD). The recombinant human apo SOD was dissolved in 10 mM triethanolamine buffer (pH 7.0), bis (2-hydroxyethyl) disulfide was added, and the pH was adjusted again to 7.0, followed by stirring at 4 ° C. for 24 hours. NaCl to this,
2M acetate buffer (pH 5.0) and 0.1M CuCl
₂ aqueous solution was added, and the mixture was stirred at 4 ° C. for 13 hours. After this stirring, the pH was adjusted to 7.0 and ammonium sulfate precipitation (4 ° C, 100%
The resulting precipitate (saturated) was collected. The precipitate was desalted by dialysis with 10 mM triethanolamine buffer (pH 7.0). The desalted fraction was passed through a Q-Sepharose FF column, the concentration of NaCl was increased stepwise in the order of 5 mM, 10 mM, 20 mM, and 50 mM to develop, and the SOD fraction was collected. Next, this SOD fraction was subjected to ammonium sulfate precipitation (4 ° C., 100% saturation), and the resulting precipitate was collected. The precipitate was used as an aqueous solution, dialyzed against a 0.5 M NaCl aqueous solution, and then purified water, and lyophilized after completion of these dialysis to obtain a human SOD derivative (hereinafter, sometimes simply referred to as SOD). The amino acid at position 111 of this human SOD derivative is S- (2-
It became hydroxyethylthio) cysteine.

(2) Production of PC-SOD: the above human SO
Using the D derivative, PC-SOD was produced by the method described in JP-A-6-54681. Synthesis of Active Ester of 2- (4-Hydroxycarbonylbutyroyl) lysolecithin (-A) Synthesis of (-A) 2- (4-Hydroxycarbonylbutyroyl) lysolecithin Chloroform-pyridine of lysolecithin having a hydroxyl group at the 2-position of glycerol (80 ml / 20 ml) suspension, 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. The target compound was fractionated by TLC, the solvent was concentrated under reduced pressure, and the residue was purified by a column packed with ODS (octadecylsilane) to obtain the title compound.

(-B) Synthesis of active ester form of 2- (4-hydroxycarbonylbutyroyl) lysolecithin The carboxylic acid obtained in -A is dissolved in dichloromethane and cooled at 0 ° C, and N-hydroxysuccinimide is added thereto. And tetrazole are added in sequence, then DCC (1,3-
A solution of dicyclohexylcarbodiimide) in dichloromethane was slowly added dropwise, and the mixture was stirred at room temperature for 15 hours. After stirring, the insoluble matter was filtered through Celite to obtain a dichloromethane solution of the active ester compound. Synthesis of PC-SOD in which an average of four lecithin derivatives are bound per SOD molecule 50 mM borate buffer (pH 8.
SOD prepared according to the above process dissolved in 5)
And a 0.8-fold molar amount of the active ester form of 2- (4-hydroxycarbonylbutyroyl) lysolecithin with respect to all amino groups of this SOD were reacted by the following method.

Dichloromethane of the active ester solution was distilled off and the residue was 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 this 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
Elution at 8.5), PC-SOD elution 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)).
)), The residual amino groups of SOD were analyzed by the TNBS method (trinitrobenzene sulfonate, Goodwin, JF, etc., Clin. Che.
m., Vol. 16, p. 24 (1970)).
The number of lecithin derivatives bonded per D1 molecule was determined to be 4.0 on average. The property of this PC-SOD is a blue-green to green transparent aqueous solution with a pH of 7-
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.

[Test Example] PC manufactured by the above manufacturing example
-SOD (concentration 30 mg / ml; specific activity 3.07 × 10 ³ U
/ Mg protein) was subjected to a toxicity test and a drug efficacy test. P
C-SOD was added to a 5% mannitol aqueous solution to give a final concentration of 5 mg / ml.
A solution (hereinafter, sometimes abbreviated as PC-SOD solution) was used so that The property of this PC-SOD solution is a blue-green to green transparent aqueous solution.
H 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 100 mg / Kg of PC-SOD solution was administered via tail vein to 5 male and 5 female SD rats. 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 rat was observed, and no change was observed in any of the general condition, body weight, and autopsy findings. Therefore, the lethal dose of PC-SOD by intravenous administration was estimated to be 100 mg / Kg or more. . Acute toxicity test using monkeys The PC-SOD solution was administered to two female cynomolgus monkeys at 100 mg / Kg via a vein in the extremities. 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 dead cynomolgus monkeys were observed, and no change was observed in general condition, body weight, or autopsy findings. Therefore, the lethal dose of PC-SOD by intravenous administration was estimated to be 100 mg / Kg or more. .

(2) Drug Efficacy Test Effect of PC-SOD on UV-induced corneal epithelial disorder Superoxide dismutase (SOD) is effective by specifically eliminating the superoxide anion (O ₂ ⁻ ) which is one of the active oxygen. It is an in-vivo substance that exhibits an oxidative action and plays an important role in the biological defense mechanism. However, SOD also does not have sufficient tissue affinity or cell affinity when it is administered from the outside, as is the case with ordinary foreign proteins.
Therefore, using a model in which rats were exposed to ultraviolet rays to induce corneal damage, lecithinized S was added to enhance tissue affinity.
The effect of the eyedrops of the present invention containing OD (PC-SOD) as a main component was examined, and as a comparative control, the eyedrops of SOD (U-SOD) that was not lecithinized were also examined. As a control, an ophthalmic solution containing only the base (2.55 g of glycerin dissolved in 100 ml of sterile purified water) was used.

Experimental Materials Preparation of Eye Drops Freeze-dried powder of PC-SOD synthesized in Production Example 0.01
2.55g of glycerin in 100ml of sterile purified water
Was added to adjust the pH to 7.0 with a trace amount of 1N-hydrochloric acid and 1N-sodium hydroxide aqueous solution, and 0.01% PC-SO was added.
D eye drops were prepared. By changing the amount of PC-SOD, 0.001% and 0.003% eye drops were prepared in the same manner.
In addition, 0.1% U-SOD ophthalmic solution used in the comparative experiment was similarly prepared.

As experimental animals, 6-week-old S. D. Twelve rats (purchased from Japan SLC, Inc.) were used, and the above eye drops were 0.01% PC-SOD, 0.1.
An eye drop consisting only of% U-SOD and the base was used.

Experimental Method Twelve rats were divided into 3 groups, and 0.1 ml per 100 g of body weight of Nembutal ^R injection solution (Dainippon Pharmaceutical Co., Ltd.) was intraperitoneally administered for anesthesia. After sandwiching the upper and lower eyelids of both eyes of the rat with a clip for document binding and fully opening the eyeball, a 312 nm ultraviolet lamp (Cosmo Bio, Cat No. CSL6
The rats were placed under B) and irradiated with 1 kJ / m ² of ultraviolet rays per rat. Immediately after irradiation, 0.01 for each group
% PC-SOD, 0.1% U-SOD and base 1
Both eyes were instilled 6 times a day at 1.5 hour intervals. Immediately after ultraviolet irradiation, 1% of 1% fluorescein-Na was added every 12 hours.
After instilling L, thoroughly washing with physiological saline and staining the lesion, observation was performed with a slit lamp, and an anterior segment of the eye was photographed with a medical Nikkor equipped with a cobalt filter.
For the corneal epithelium lesion area, the fluorescein-stained area on the obtained photograph was used as an image analysis system (COSMOZONE 1
SA, Nikon). In addition, corneal opacity that occurs during the healing process of epithelial disorders was also observed, and the scores were evaluated according to the table below.

Statistical Analysis Significance of the stained area of fluorescein was examined by a two-sided test of Dunnett's multiple comparison test using a parametric method. The significance of the corneal opacity score was examined by a two-sided test of the Sheffe type multiple comparison test using a nonparametric method. The significance level was 5% or less in all cases. Corneal opacity score No opacity is observed ..... 0 A degree of opacity is slightly observed. The boundary between the turbid and non-turbid areas is unclear. 1 Semi-transparent areas can be easily identified, and the boundary between the turbid and non-turbid areas is clear. Something ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ 2 Obvious opaque region spreads over the entire cornea ・ ・ ・ 3

Results and Conclusions (1) Effect of PC-SOD on corneal epithelial disorder 0.01-% PC-SOD, 0.1% U-SOD and the time course of fluorescein-stained area in the base ophthalmic group are shown in FIG. . In the base eye drop group, corneal epithelial damage began to be recognized 24 hours after the irradiation with ultraviolet rays, and reached a peak 36 hours after the irradiation. After 48 hours of irradiation, the area of the epithelial lesion gradually decreased, and after 84 hours of irradiation, almost all cases were cured. In the 0.01% PC-SOD eye drop group, corneal epithelial damage began to be recognized 12 hours after irradiation, and irradiation 4
Although it reached a peak after 8 hours, the degree of epithelial damage was significantly suppressed as compared with the base group. After that, the area of injury decreased, and all the cases were cured 72 hours after irradiation. Also, 0.1
The% U-SOD eye drop group showed almost the same course as the 0.01% PC-SOD eye drop group, and the anterior healing was achieved 120 hours after irradiation.

(2) Effect of PC-SOD on corneal opacity Corneal opacity was not observed in any of the groups until 48 hours after irradiation with ultraviolet rays. The corneal opacity score from 36 hours to 120 hours after ultraviolet irradiation is shown in FIG. In the base instillation group, corneal opacity began to be recognized 60 hours after the irradiation with ultraviolet rays, reached a peak 84 hours after the irradiation, and then gradually disappeared. In the 0.01% PC-SOD eye drop group, turbidity began to be recognized 60 hours after irradiation similarly to the base group, but the opacity score showed remarkable suppression as compared with the base eye drop group, and 72 hours of irradiation. After the peak, it gradually disappeared and 96 hours after irradiation, the corneal opacity was significantly suppressed as compared with the base group. In addition, in the 0.1% U-SOD group, irradiation 4
After 8 hours, turbidity appeared, and similar to the 0.01% PC-SOD group, it showed a tendency of suppression as compared with the base ophthalmic solution group.
The peak appeared 2 hours later, and then the turbidity gradually disappeared. From the above results, PC-SOD showed a remarkable suppression of UV-induced corneal epithelial damage at a concentration of 0.01%, and significantly suppressed corneal opacity that occurs during the healing process of epithelial damage. The suppression effect was about the same as or higher than that of 0.1% U-SOD, which was 10 times higher concentration.

Regarding the effect of low-concentration PC-SOD on UV-induced corneal epithelial damage, PC-SOD significantly suppressed the UV-induced corneal epithelial damage at a concentration of 0.01%, and the corneal opacity generated during the healing process of epithelial damage was significant. Suppressed to. Therefore, using the same model, the PC-SOD eye drop group (0.00
The effect of 1%, 0.003% and base) was investigated. The experimental materials, eye drops, experimental animals, and experimental methods were the same as those described above.

Results and Conclusions (1) Effect of PC-SOD on corneal epithelial disorder 0.001% PC-SOD, 0.003% PC-SO
FIG. 3 shows the time-dependent changes in the fluorescein-stained area in D and the base ophthalmic solution group. Ultraviolet irradiation 1 in base ophthalmic group
The corneal epithelial disorder started to be recognized after 2 hours, and reached a peak 36 hours after irradiation. After that, the area of the lesion gradually disappeared, and almost 72 cases were healed 72 hours after irradiation. In the PC-SOD eye drop group, epithelial damage began to be recognized 24 hours after irradiation, reached a peak 36 hours after irradiation, and thereafter the damage gradually disappeared. The onset of corneal epithelial disorder and the course of healing were not so different between the base group and the PC-SOD eye drop group, but the degree of the disorder at the peak was PC.
-SOD showed remarkable inhibition in the eye drop group, and its action was 0.0
03% was stronger.

(2) Effect of PC-SOD on Corneal Opacity The results of evaluation of corneal opacity scores from 36 hours to 144 hours after UV irradiation are shown in FIG. Corneal opacity was not observed in any of the groups until 36 hours after UV irradiation. In the base instillation group, corneal opacity began to be recognized 48 hours after the irradiation with ultraviolet rays, reached a peak 72 hours after the irradiation, and then gradually disappeared. 0.001% PC-SOD
In the eye drop group, cloudiness occurred 60 hours after irradiation, and
After a lapse of time, it peaked and then gradually disappeared. 0.00
With 3% PC-SOD, corneal opacity began to occur 60 hours after irradiation, and showed a peak 84 hours after irradiation, but the degree of opacity was significantly suppressed in any observation period as compared with the base ophthalmic group. Indicated.

From the above results, PC-SOD shows a tendency to suppress the UV-induced corneal epithelial damage, and its action is 0.
003% was stronger. In addition, 0.003% PC-SO was used to prevent corneal opacity during healing of epithelial disorders.
It can be seen that D shows a more remarkable suppressing effect than 0.001% PC-SOD.

[0036]

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide an eye drop having excellent preventive and therapeutic effects on ultraviolet ray-induced corneal disorders.

[Brief description of the drawings]

FIG. 1 is a graph showing the effect of PC-SOD-containing eye drops on UV-induced corneal epithelial lesions measured by the fluorescein staining method.

FIG. 2 is a diagram showing the effect of PC-SOD-containing eye drops on ultraviolet ray-induced corneal epithelial damage as seen from corneal opacity.

FIG. 3 is a graph showing the effect of low-concentration PC-SOD-containing eye drops on UV-induced corneal epithelial damage measured by the fluorescein staining method.

FIG. 4 is a graph showing the effect of low-concentration PC-SOD-containing eye drops on UV-induced corneal epithelial damage as seen from corneal opacity.

Claims (13)

[Claims] 1. A therapeutic / preventive agent for corneal disorders, which comprises lecithinized superoxide dismutase as an active ingredient. 2. The therapeutic / preventive agent for corneal disorders according to claim 1, wherein the corneal disorders occur in the corneal epithelium. 3. The therapeutic / preventive agent for corneal disorders according to claim 1, wherein the corneal disorder is an ultraviolet-induced corneal epithelial disorder. 4. The method according to claim 1, wherein the dosage form is ophthalmic solution.
The therapeutic / prophylactic agent for corneal disorders according to any one of items. 5. The therapeutic / preventive agent for corneal disorders according to claim 4, wherein the lecithinized superoxide dismutase content is 0.001 to 1% by weight. 6. The therapeutic / prophylactic agent for corneal disorders according to any one of claims 1 to 5, wherein the lecithinized superoxide dismutase is a superoxide dismutase bound to lecithin through a chemical crosslink. 7. A superoxide dismutase bound to lecithin through a chemical crosslink is represented by the following general formula (I) SOD- [C (O)-(CH ₂ ) _n- C (O) -X] _m (I). (In the formula, SOD represents a superoxide dismutase, X represents a 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, and m is the average number of bonds to one SOD molecule. And represents an integer of 1 or more, and n represents an integer of 2 or more). The therapeutic / preventive agent for corneal disorders according to claim 6, which is represented by 8. The therapeutic / preventive agent for corneal disorders according to claim 7, wherein in the general formula (I), the SOD is of human origin. 9. In the general formula (I), SOD is human-derived Cu /, in which the amino acid at position 111 of the amino acid sequence is S- (2-hydroxyethylthio) cysteine.
The therapeutic / preventive agent for corneal disorders according to claim 7, which is Zn superoxide dismutase. 10. In the general formula (I), n is 2 or more and 1
The therapeutic / prophylactic agent for corneal disorders according to any one of claims 7 to 9, which is an integer of 0 or less. 11. The therapeutic / preventive agent for corneal disorders according to claim 10, wherein n is 3 in the general formula (I). 12. In the general formula (I), m is 1 or more and 1
The therapeutic / prophylactic agent for corneal disorders according to any one of claims 7 to 11, which is a number of 6 or less. 13. The therapeutic / preventive agent for corneal disorders according to claim 12, wherein in the general formula (I), m is 4.