JPH093094A - Serratio protease inhibitor - Google Patents

Abstract

PURPOSE: To obtain the subject inhibitor for preventing and treating an infectious disease of cornea caused by an enzyme produced by Serratia marcescens and capable of specifically inhibiting serratio protease by pharmaceutically manufacturing propioxanthine A as an active ingredient. CONSTITUTION: Propioxanthine A of the formula as an active ingredient obtained by culturing Kitasatosporia setae ISANK60684 (FERM P-7581) is dissolved in sterilized purified water containing an isotonizing agent, a buffer agent, a solubilizer, a stabilizer, an antiseptic, etc., and pharmaceutically manufactured to give the objective serratio protease inhibitor which has a specifically inhibiting action on serratio protease and is useful as a preventive and/or a therapeutic agent for an infectious diseases of cornea caused by an enzyme produced by Serratia marcescens.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a seratioprotease inhibitor and a preventive and / or therapeutic agent for corneal infection. More particularly, it plays an important role in the expression, such as corneal infections, Serratia marcescens (Serr
Atia marcescens ) has a molecular weight of 56,000 and is immunologically equivalent to Serratia piscatorum ( Serratia piscatorum ). The following formula

Embedded image The present invention relates to a seratiopeptidase inhibitor consisting of propioxatin A, and a preventive and / or therapeutic agent for corneal infections containing the inhibitor as an active ingredient.

[0002]

2. Description of the Related Art Extracellular proteases produced by microorganisms are known to be an important source of many bacterial infections. For example, destruction of immunoglobulins (IgG, IgA, etc.) that are important for protection against infection,
Inactivation of various proteases involved in the regulation of blood coagulation system, invasion into neuroblasts via a complex with α ₂ -macroglobulin, and cell destruction by protease reactivation have been reported (H. Maeda, et al., J. Biol. Chem., 2
62 , 10946-10950 (1987)). Most of the bacteria that produce the protease that is the cause are Gram-positive bacteria such as Gram-positive bacteria and Staphylococcus. Among these bacteria, Serratia genus and the like have been considered to be of little importance in etiology, but today it has been found that they are causative bacteria of some diseases as described above.

It has been clarified that the protease produced by Serratia marcescens among Serratia fungi decomposes and inactivates in vivo protease inhibitors and plays an important role in the expression of corneal infections and the like. . When this protease is administered to the eyes of mice, keratitis is induced in a short time (2 to 4 hours), and necrosis of corneal tissue occurs several hours later (5 to 8 hours), leading to blindness.

Serratia marcesense
It has been clarified that there are three types of proteases produced by scens ) having different molecular weights as shown in the table below (H. Maeda, et al., J. Biol. Chem.,
262 , P10946-10950 (1987); Hiroshi Maeda, Shinichi Miyagawa, The latest medicine, 43 , P790-794 (1988); Hiroshi Maeda, Biochemistry, Volume 63, P
14-21 (1991) ； K.Matsumoto, H.Maeda, K.Takata, R.Ok
umura, J. Bacteriol., 157 , P225-232 (1984); K. Naka
mura, et al., Necleic Acids Res., 14 , P5843-5855 (1
986)).

[0005]

[Table 1] Protease molecular weight produced by Serratia marcescens 56,000 60,000 73,000 Isoelectric point 5.3 4.4 5.3 7.3 Optimum pH 55-5 86-9 Inhibitor EDTA + -phosphoramidone- + DFP --- EDTA: Ethylenediaminetetraacetic acid DFP: Diisopropyl Fluorophosphoric acid

Among the above-mentioned three kinds of proteases, a molecular weight of 5 containing one zinc atom per 1 molecule of protease
It was revealed that 6,000 metal proteases (56K proteases) accounted for about 70% of the produced proteases in both production amount and activity, and were most strongly involved in the expression of pathogenicity. Therefore, the present inventors considered suppressing pathological expression such as corneal infection caused by 56K protease by an inhibitor of the protease. Although this protease is a metal protease containing a zinc atom, it is a known metal protease inhibitor (S-MPI
It has been clarified that it is not inhibited by (S-metal protease inhibitor), phosphoramidon, etc.), and a substance that specifically inhibits this protease is not known. Therefore, an object of the present invention is to provide a specific inhibitor of 56K protease, which is considered to be effective in the prevention and / or treatment of corneal infections and the like caused by the above 56K protease, and a cornea containing the inhibitor as an active ingredient. An object is to provide a preventive and / or therapeutic agent for infectious diseases.

[0007]

The inventors of the present invention have produced 5 of Serratia marcescens.
A substance that inhibits the 6K protease decided to search required for the microbial world, but, Serratia marcescens (Serratia
The protease itself produced by marcescens ) has a high pathogenicity and is difficult to carry out in an experimental operation. Therefore, a commercially available Serratia piscatorum , which is immunologically equivalent to 56K protease, produces the sera with the characteristics shown in Table 2. Microbes were screened using thioprotease.

[0008]

[Table 2] Serratia Piscatorum production to 56K proteases canceller thioprotease molecular weight 60,000 56,000 Isoelectric point 5.0-5.5 5.3 Optimum pH 9 5 inhibitor EDTA + + phosphoramidon - - S-MPI - ND PCMB - ND DFP - - EDTA: ethylenediaminetetraacetic acid S -MPI: S-metalloprotease inhibitor PCMB: p-chloromercuribenzoic acid DFP: diisopropylfluorophosphoric acid

As a result, it was confirmed that one strain isolated from soil in Kumamoto Prefecture could produce the above-mentioned protease inhibitor. As a result of analysis (see below), this inhibitor was found to be a known substance, propioxatin A. Propioxatin A is an inhibitor of enkephalinase B, and acts to delay the deactivation of enkephalin, which is a neurotransmitter showing an analgesic effect. This propioxatin A is a kind of actinomycete, Kitasatosporiaceae.
It has been reported that it is produced by Kitasatosporia setae SANK 60684, and its structure analysis and organic chemical synthesis method have also been performed (Inaoka, Y., et al., J. Ant.
ibiotics, 39 , 1368-1385 (1986)). However, the above-mentioned reference does not mention at all that propioxatin A specifically inhibits not only 56K protease produced by Serratia marcescens but also its immunological equivalent, seratioprotease. Absent. Propioxatin A specifically inhibits seratioprotease and its immunological equivalent, 56K
It was revealed for the first time that it would inhibit proteases and could suppress the development of pathogenicity such as corneal infections due to 56K protease.

That is, the present invention is: 1) The following formula

Embedded image And 2) a preventive and / or therapeutic agent for a corneal infection comprising the propioxatin A-inhibiting seratioprotease inhibitor, and 2) the propioxatin A described in 1) above as an active ingredient.

Hereinafter, the present invention will be described in detail. Serratioprotease is a kind of metal protease and is inhibited by metal chelating agents such as ethylenediaminetetraacetic acid (EDTA) and o-phenanthroline which are known as metal protease inhibitors. These metal chelating agents also strongly inhibit other metal proteases such as thermolysin. Other microbial metal protease inhibitors include phosphoramidon and talopeptin, which inhibit thermolysin but not seratioprotease.

On the other hand, the propioxatin A according to the present invention strongly inhibits seratioprotease but hardly inhibits thermolysin. Inhibition of thermolysin by propioxatin A is inhibited by the chelate forming ability of hydroxamic acid (-NH-OH) in the propioxatin A molecule, and the inhibition rate is about 10%. . Further, propioxatin A does not inhibit trypsin, α-chymotrypsin and subtilisin BPN ′ which are serine proteases, pepsin which is an acidic protease, and papain which is a thiol protease. That is, propioxatin A specifically inhibits the enzymatic action of seratioprotease.

Method for producing propioxatin A: Method using microorganisms Propioxatin A can be obtained by the present inventors using a strain isolated from soil in Kumamoto prefecture. That is, this strain may be inoculated into an appropriate medium and cultured according to a conventional method. Any medium can be used as the medium as long as the strain can grow and produce propioxatin A. For example, glucose, glycerol, lactose, starch, etc. are usually used as the carbon source, and meat extract, peptone, defatted soybean flour, yeast extract, malt extract, corn steep liquor, etc. are used as the nitrogen source, and phosphate is also used. Inorganic salts such as sodium salt, calcium salt and magnesium are added. The culture is carried out under aerobic conditions, for example, the aeration and stirring method or the shaking culture method. The culture temperature is preferably 25 to 35 ° C, and the pH is preferably 5 to 8. The culture time is about 34 to 42 hours, and the culture may be terminated when the inhibitory activity of propioxatin A reaches the maximum. The purification of propioxatin A may be carried out by subjecting the above culture solution to heat treatment and filtration to remove the bacterial cells, followed by ion exchange chromatography, gel filtration chromatography and the like.

The method of Inaoka et al. (J. Antibiotic
s, 39 , 1368-1385 (1986)), that is, the contract number FERM P-7581 to the Institute of Biotechnology, Institute of Industrial Science and Technology.
Kitasatosporia setae ( Kita
It can also be obtained by culturing as described above using satosporia setae ) SANK 60684.

Production Method of Propioxatin A-2: Synthetic Chemical Method Propioxatin A can also be synthesized by a known method according to the procedure of the following reaction scheme (J. Antibio).
tics, 39 , 1382-1385 (1986)).

[0016]

Embedded image

[Toxicity] The toxicity of propioxatin A according to the present invention is extremely low, and it can be judged that it is sufficiently safe for use as a medicine.

[Application to Pharmaceuticals] In order to use propioxatin A for the purpose of prevention or treatment of corneal infection, etc.,
Usually, it is used in the form of a solution, and can be formulated using a commonly used technique. For example, in the case of eye drops,
Isotonic agents such as sodium chloride and concentrated glycerin in sterile purified water, buffering agents such as sodium phosphate, polysorbate 80
And the like, stabilizers such as sodium edetate and preservatives such as benzalkonium chloride can be appropriately selected and prepared as necessary. When the inhibitor of the present invention is administered in the form of eye drops, it is usually used as a 0.0001% to 0.5% solution, although it varies depending on the symptoms, age, therapeutic effect, treatment time and the like. When administered parenterally as an injection, at least one inactive aqueous diluent (distilled water for injection, physiological saline, etc.) or an inactive nonaqueous diluent (propylene glycol, propioxatin A, It is used as a mixture with polyethylene glycol, olive oil, ethanol, polysorbate 80 (registered trademark) or the like. These injections may further contain auxiliaries such as preservatives, wetting agents, emulsifiers, dispersants, stabilizers and solubilizing agents. The above injection is usually sterilized by filtration using a bacteria-retaining filter or the like, blending of a bactericide or irradiation.
Alternatively, after these treatments, a solid composition is obtained by a method such as freeze-drying, and sterile water or a sterile diluent for injection is added immediately before use, and the composition is used.

When the preventive or therapeutic agent of the present invention is orally administered, it is administered as a solid composition or a liquid composition. Solid compositions for oral administration include tablets, pills, capsules, powders, granules and the like. In such a solid composition, propioxatin A is mixed with at least one inert diluent (lactose, mannitol, glucose, hydroxypropyl cellulose, microcrystalline cellulose, starch, polyvinylpyrrolidone, magnesium aluminometasilicate, etc.). It is used as a mixture with. These compositions include additives other than inert diluents,
For example, it may contain a lubricant, a disintegrant, a solubilizing agent, and a stabilizer. If necessary, the tablets or pills may be coated with a film of a gastric or enteric substance (sucrose, gelatin, hydroxypropylcellulose, hydroxypropylmethylcellulose phthalate, etc.). Liquid compositions for oral administration include solutions, emulsions, suspensions, syrups and elixirs. Such a liquid composition contains a commonly used inert diluent (purified water, ethanol, etc.). These compositions may contain, in addition to the inert diluent, adjuvants such as wetting agents and suspending agents, sweeteners, flavors, fragrances, and preservatives.

[0020]

INDUSTRIAL APPLICABILITY The present invention provides a specific inhibitor of seratiothioproteases comprising propioxatin A, which is Serratia marcescens .
It is useful for the prevention and treatment of corneal infections and the like caused by the pathogenic protease produced by.

[0021]

EXAMPLES The present invention will be specifically described below with reference to Reference Examples and Examples, but the present invention is not limited to these Examples. In addition, in the reference example, the total activity of the seratioprotease in each purification step was measured by the following method.

That is, the solution obtained by each purification step
Add 0.02 ml to an Eppendorf tube and then 50μ
g / ml enzyme solution [Serratia Pisukatorumu (Serratia
piscatorum ) produced seratioprotease , 0.1M
Phosphate buffer solution (pH 9.0)] 0.03 ml is mixed, and 37 ° C
After preincubating for 30 minutes at 4m as a substrate
Azoalbumin adjusted to g / ml was added to 0.2.
Mix 1 ml [0.1M phosphate buffer (pH 9.0)], 37
React at 60 ° C. for 60 minutes. After the reaction, 0.02 ml of 50% trichloroacetic acid solution (reaction stop solution) is added, and the mixture is left at 37 ° C. for 10 minutes. Then, the mixture is centrifuged (7000 g × 2 minutes), 0.12 ml of the supernatant is collected, and 0.02 ml of 10N sodium hydroxide aqueous solution is added to each microtiter plate (96 wells) added to each cell in advance. After leaving for about 20 minutes, the resulting orange coloration is changed to ELISA reader (In
450n at ternational Reagents Corporation)
It is measured as the absorbance at m. Further, the same operation as above is carried out for a solution using 0.02 ml of 0.1 M phosphate buffer (pH 9.0) instead of 0.02 ml of the solution obtained in each purification step, and the absorbance at 660 nm is measured. Next, the inhibition rate I (%) is calculated by the following formula, and 50%
The inhibitory activity when the inhibition rate of 1 was shown was defined as 1 unit.

## EQU1 ## I (%) = ((SB)-(TB)) / (S
-B) × 100 S: standard solution absorbance T: test solution absorbance B: control solution absorbance

Reference Example 1: Cultivation of strain Sakaguchi Korben of 500 ml volume, glycerol 1.0%,
Fuji Pro-R 1.5%, yeast extract 0.2%, Na ₂ HPO ₄
・ A medium similar to the above was prepared by charging 50 ml of a medium (pH 6) containing 0.3% 12H ₂ O and 0.2% adecanol, inoculating 1 platinum loop of the strain, and culturing with shaking at 30 ° C for 24 hours. (PH 6) was placed in a jar fermenter having a volume of 30 liters containing 20 liters, and cultivated with shaking at 30 ° C. for 27 hours.

Reference Example 2: Heat treatment of culture medium Steam was sent to the culture medium obtained in Reference Example 1 so that the medium temperature was 100%.
The temperature was adjusted to be 0 ° C., and heat treatment was performed for 15 minutes. At this time, the culture solution was 17 liters, and the activity recovery rate relative to the culture solution was 99%.

Reference Example 3: Filtration of Culture Solution From 17 liters of the culture solution obtained in Reference Example 2, Hyfro Super-Ce
using ll (Jones Manville Co.) as a filter aid,
The cells were removed by filtration with Toyo Filter Paper No. 2. At this time, the culture solution reached 16.2 liters, and the activity recovery rate relative to the culture solution was 96%.

Reference Example 4: Purification by HP-20 column To the 16.2 liters of the filtrate obtained in Reference Example 3, acetic acid was added to adjust the pH to 3 to 3.5, and the mixture was previously equilibrated with deionized water. HP-20 column (φ5 × 45c
m; manufactured by Mitsubishi Chemical Corporation), and the column was washed with deionized water. Then, the adsorbed substance was eluted with 60% methanol, and the active fraction was collected. This active fraction was 2.55 liters, and the activity recovery rate with respect to the culture was 76%.

Reference Example 5: Purification by Dowex 50W column 2.55 liters of the active fraction obtained in Reference Example 4 was concentrated to 0.5 liters by a rotary evaporator, and this was washed with deionized water beforehand to obtain a Dowex 50W column (φ5 × 15).
cm; manufactured by The Dow Chemical Company), and washed with deionized water. Ammonia water is added to the passing liquid.
H7 was added to remove the dark brown pigment. By this operation, the active fraction was 1 liter, and the activity recovery rate in the culture solution was 68%.

Reference Example 6: Chromatorex OD
Purification by S column 1 liter of the active fraction obtained in Reference Example 5 was concentrated to 0.1 liter by a rotary evaporator, adjusted to pH 3 to 3.5 by adding acetic acid, and preliminarily equilibrated with deionized water. After being adsorbed on an ODS column (φ7 × 9 cm; manufactured by Fuji Devison Chemical Co., Ltd.), the column was washed with deionized water. Then, the adsorbed substance was eluted by linear gradation of 0 to 70% methanol, and the active fraction was collected. This active fraction was 250 ml, and the activity recovery rate to the culture solution was 47%.

Reference Example 7: Gel Filtration 250 ml of the active fraction obtained in Reference Example 6 was concentrated to 20 ml by a rotary evaporator, and this was washed with deionized water in advance to BioGel P-2 (φ2.2 × 45 cm; Rad) and eluted with distilled deionized water to collect the active fraction. The active fraction was 80 ml, and the activity recovery rate relative to the culture solution was 35%.

Reference Example 8: Purification by TSK gel SAX HPLC 80 ml of the active fraction obtained in Reference Example 7 was concentrated to 30 ml by a rotary evaporator and preliminarily equilibrated with 5 mM disodium phosphate.
Purification was carried out by. The collected active fraction was concentrated to 30 ml by a rotary evaporator. The recovery rate of the activity with respect to the culture solution was 31%. HPLC condition column: TSK gel SAX (φ0.78 × 30 cm; manufactured by Tosoh Corporation) Developing solvent: 0-0.5 M NaCl + 5 mM disodium phosphate Flow rate: 1.0 ml / min Pressure: 51 kg / cm ² Detection: Abs 210 nm Column temperature: 27 ℃

Reference Example 9: TSK gel octadecyl 2PW
Purification by HPLC 30 ml of the active fraction obtained in Reference Example 8 was purified by HPLC under the following conditions using a column previously equilibrated with 20 mM disodium phosphate-6% methanol. The collected active fraction was concentrated to 30 ml by a rotary evaporator. The activity recovery rate for the culture broth was 20%. HPLC condition column: TSK gel octadecyl 2PW (φ0.78 × 30
cm; manufactured by Tosoh Corporation) Developing solvent: 20 mM disodium phosphate-6% methanol Flow rate: 1.0 ml / min Pressure: 100 kg / cm ² Detection: Abs 225 nm Column temperature: 25 ° C.

Reference Example 10: Nucleocyl ODS ₁₀ C
₁₈ Purification by HPLC Acetic acid was added to 30 ml of the active fraction obtained in Reference Example 9 to adjust the pH to 3.5, adsorbed on a column previously washed with double-distilled water, and then washed with double-distilled water, Under the following HPLC conditions, elution was carried out with a linear gradient of 0 to 60% methanol, and the active fraction was collected. This active fraction was concentrated to 30 ml on a rotary evaporator. The activity recovery rate for the culture broth was 16%. HPLC condition column: Nucleosil ODS ₁₀ C ₁₈ (φ0.8 × 15c
m; manufactured by Nagel Co., Ltd.) Developing solvent: 0-60% linear gradient of methanol Flow rate: 3.0 ml / min Pressure: 65 kg / cm ² Detection: Abs 225 nm Column temperature: 28 ° C.

Reference Example 11: Hitachi Gel # 3019 H
Purification by PLC In the active fraction obtained in Reference Example 10, a leak from the column was found by reverse phase chromatography using silica gel as a carrier. Therefore, a leak was removed from the column such as silica by using a porous polymer reverse phase column. I did. That is, 30 ml of the active fraction obtained in Reference Example 10 was 10 m on a rotary evaporator.
It was concentrated to 1 l, adsorbed on a column washed with redistilled water in advance, and then washed with redistilled water.
Elution with methanol collected the active fraction. This active fraction was freeze-dried to obtain 78.89 mg of white powder. The recovery rate of activity for the culture was 11.8%. HPLC condition column: Hitachi gel # 3019 (φ0.8 × 15.5 cm; manufactured by Hitachi Chemical Co., Ltd.) Developing solvent: 60% methanol Flow rate: 2.0 ml / min Pressure: 70 kg / cm ² Detection: Abs 210 nm Column temperature: 28 ° C.

Table 3 shows the activity and the like in each of the refining steps of Reference Examples 2 to 11 above.

[Table 3] Purification step Total activity (U) Yield (%) Reference example 1 Culture solution 180,000,000 100 Reference example 2 Heat treatment 179,000,000 99 Reference example 3 Celite filtration 172,000,000 96 Reference example 4 DIAION HP-20 136,000,000 76 Reference example 5 Dowex 50W 122,000,000 68 Reference Example 6 Chromatorex ODS 83,800,000 47 Reference Example 7 Bio Gel P-2 62,500,000 35 Reference Example 8 TSK Gel SAX 56,600,000 31 Reference Example 9 TSK Gel Octadecyl 2PW 36,700,000 20 Reference Example 10 Nucleosil 29,300,000 16 Reference Example 11 Hitachi Gel # 3019 21,300,000 12

The physical properties of propioxatin A produced and purified as described above are shown below. (1) Analysis Calculated: C7.82, H54.99, N11.32 (C 17 H 29 N 3 O 6) measurements: C7.77, H53.52, N11.02 (2 ) Mass spectrometry FAB- MS (m / z): 372 (M + H) ⁺ (Pog.) Glycerin matrix FAB-MS (m / z): 370 (MH) ^- (Neg.) Glycerin matrix (3) IR 3000-2900, 1760- 1700, 1700-1580, 1580-1490, 1490-1
400, 1260-1160cm ^-1 (4) ¹ H-NMR (D ₂ O): δ (ppm) 4.32 (1H, dd, J = 5.5Hz, 8.4Hz), 4.08 (1H, d, J = 5.9Hz) , 3.7
0-3.53 (2H, m), 3.05-2.95 (1H, m), 2.29-2.18 (2H, m), 2.
18-2.00 (2H, m), 1.91-1.77 (3H, m), 1.48-1.25 (2H, m),
1.24-1.14 (2H, m), 0.88-0.81 (6H, m), 0.75 (3H, t, J = 7.3H
z) (5) ¹³ C-NMR (D ₂ O): δ (ppm) CO: 175.9, 175.5, 174.2, 170.7 CH ₃ : 18.3, 17.2, 13.2 CH ₂ : 48.3, 34.6, 34.0, 29.2, 24.3, 19.4 CH: 60.0, 58.5, 39.4, 29.8 (6) Melting point 85-86 ° C (7) Optical rotation -90.2 ° (c = 0.1, 28 ° C, water)

Example: Measurement of inhibitory activity The inhibitory rate of propioxatin A thus obtained against various enzymes was measured. Examples of the enzyme include a serratioprotease produced by Serratia piscatorum , thermolysin, and subtilisin BNP ′.
α-chymotrypsin, trypsin, pepsin and papain were used. The results are shown in Table 4. The inhibition rate was measured by the following Hamalstein casein method.

[Measurement Method of Inhibition Rate] (1) Seratioprotease 50 μg / ml enzyme solution (50 mM phosphate buffer (pH
9.0)) 0.3 ml and 2 of propioxatin A obtained in Reference Example
After pre-incubating with 0.2 ml of a μg / ml solution for 30 minutes at 37 ° C., 1.5 ml of 0.7% hamalstein casein solution (at the same pH) is added and reacted at 37 ° C. for 20 minutes. After the reaction, 50% trichloroacetic acid (2 ml) was added to stop the reaction, and the mixture was allowed to stand at room temperature for 20 minutes, filtered using Toyo filter paper No. 2, and 0.5 ml of the filtrate was collected. 0.4 to this
4M sodium carbonate 1.5ml and 1N Folin reagent 0.5
After adding ml and reacting at 37 ° C for 20 minutes, 660n
Measure the absorbance at m. The inhibition rate is calculated using the absorbance measured in the same manner without adding propioxatin A as a control. (2) Thermolysin 10 μg / ml enzyme solution (50 mM phosphate buffer (pH
7.0)) 0.3 ml is used to calculate the inhibition rate in the same manner as 1 above. (3) Subtilitin BNP '50 μg / ml enzyme solution (50 mM phosphate buffer (pH
8.0)) Using 0.3 ml, calculate the inhibition rate as in 1 above. (4) α-chymotrypsin 50 μg / ml enzyme solution (50 mM phosphate buffer (pH
8.0)) Using 0.3 ml, calculate the inhibition rate as in 1 above. (5) Trypsin 50 μg / ml enzyme solution (50 mM phosphate buffer (pH
8.0)) Using 0.3 ml, calculate the inhibition rate as in 1 above. (6) Pepsin 50 μg / ml enzyme solution (50 mM phosphate buffer (pH
2.0)) 0.3 ml is used to calculate the inhibition rate in the same manner as 1 above. (7) Papain 30 μg / ml enzyme solution (50 mM containing 5 mM cysteine and 1 mM ethylenediaminetetraacetic acid (EDTA)
Using 0.3 ml of phosphate buffer (pH 7.0), the inhibition rate is calculated as in 1 above.

[0038]

[Table 4] Enzyme concentration (μg / ml) pH inhibition rate (%) Seratiothioprotease 50 9.0 89 Thermolysin 10 7.0 10 Subtilisin BNP ′ 50 8.0 0 α-chymotrypsin 50 8.0 0 Trypsin 50 8.0 0 Pepsin 50 2.0 0 Papain 30 7.0 0

As is clear from Table 4, propioxatin A has a strong inhibitory action on seratioproteases. It also shows some inhibitory effects on thermolysin, which is classified into the same metalloproteases as serathioproteases, but this is due to the chelate-forming ability of hydroxamic acid (-NH-OH) that propioxatin A has in the molecule. It is considered to be inhibited and its inhibitory effect is weak. In addition, trypsin, which is a serine protease, α-
It does not inhibit chymotrypsin and subtilisin BPN ', acidic protease pepsin, thiol protease papain and the like.

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Claims (2)

[Claims] [Claim 1] The following formula: A seratioprotease inhibitor consisting of propioxatin A represented by: 2. The following formula: A prophylactic and / or therapeutic agent for corneal infections containing propioxatin A as an active ingredient.