JPH0478619B2 - - Google Patents

Description

[Detailed description of the invention]

INDUSTRIAL APPLICATION FIELD The present invention relates to enzyme inhibitors. More specifically, the present invention relates to Serratia protease 56K, Serratia protease 60K, Serratia protease 73K, Pseudomonas
Concerning enzyme inhibition of alkaline protease or Pseudomonas elastase. DISCLOSURE OF THE INVENTION Conventionally, no substance having excellent enzyme inhibitory activity against the above-mentioned enzymes has been known. The present inventors have conducted extensive research to find a substance that has an excellent enzyme inhibitory effect on the above-mentioned enzymes, and have discovered that ovomacroglobulin is a type of protease that is completely difficult to predict based on its known pharmacological effects. In other words, ovomacroglobulin has an enzyme inhibitory effect on Serratia protease.
It was found that 56K, 60K, and 73K have an excellent enzyme inhibitory effect on Pseudomonas alkaline protease or Pseudomonas elastase. The present invention was completed based on this knowledge. The enzyme inhibitor of the present invention has an excellent enzyme inhibitory effect on Serratia protease 56K, 60K, 73K, Pseudomonas alkaline protease or Pseudomonas elastase, and
Caused by infection with pathogenic bacteria that produce protease 56K, 60K, 73K, Pseudomonas alkaline protease, or Pseudomonas elastase, or the condition worsens due to infection, such as various dermatitis, keratitis, conjunctivitis, cracks, and scorching. It is useful as a preventive and therapeutic agent for trauma such as abrasions, cuts, burns, burns, frostbite, and surgical wounds, suppuration, cell destruction, cell necrosis, and inflammation such as skin ulcers, urinary tract infections, cystitis, and dermatitis. be. The enzyme inhibitor of the present invention must contain ovomacroglobulin as its active ingredient. Here, ovomacroglobulin is known as a high molecular weight glycoprotein found in egg white, and its preparation method is already known [Feeney,
RE) et al., Comparative Biochemistry and Physiology (Comp.Biochem.
Physiol.), 54A, 281 (1976), Ikai et al., Journal of Biochemistry (J.Biochem.),
92, 1679-1682 (1982), 93, 121-127 (1983),
and Nagase et al., Journal of Biological Chemistry (JBC), vol.258, No.12,
7481-7489 (1983), etc.]. Egg white as a raw material for preparing the above-mentioned ovomacroglobulin is not particularly limited, and any egg white from various animals can be used. In general, readily available egg whites from chicken, duck, quail, turkey, etc. are preferred. There are no particular restrictions on the preparation method, and various methods commonly used for separating protein components may be followed, including various operations that utilize the physicochemical properties of ovomacroglobulin, such as treatment with a protein precipitant, molecular sieve chromatography, etc. (gel filtration),
Ion exchange chromatography, centrifugation, electrophoresis, dialysis, etc. can be performed alone or in combination. For example, by mixing egg white with a water-soluble solvent such as Tris-HCl buffer or adding polyethylene glycol, etc. to remove insoluble proteins such as ovomucin, and then applying gel filtration, the molecular weight of
Ovomacroglobulin can be obtained as a glycoprotein of 600,000 to 800,000. Furthermore, in the present invention, Serratia protease
Examples of pathogenic bacteria that produce 56K, 60K, 73K, Pseudomonas alkaline protease, or Pseudomonas elastase include Pseudomonas aeruginosa and M. marcescens. The enzyme inhibitor of the present invention can be put to practical use in the form of a general pharmaceutical preparation containing ovomacroglobulin as an active ingredient. As the pharmaceutical manufacturing form, various forms can be appropriately selected depending on the intended use of the resulting preparation. Examples include liquid coating agents,
External preparations such as lotions, aerosols, liniments, ointments, and plasters, eye ointments, eye drops,
Examples include injections and oral preparations. In preparing these various forms, various commonly used diluents, excipients, etc. can be used as appropriate. For example, when preparing ointments for external use, conventional hydrophobic or hydrophilic bases such as fats, fatty oils, lanolin, petrolatum, paraffin, waxes, glycols, higher alcohols, glycerin, water, etc. are used. Can be used.
In the preparation of eye ointments, commonly used hydrophobic or hydrophilic bases such as white petrolatum, purified lanolin, liquid paraffin, higher alcohols, glycerin, and water can be used. Furthermore, when producing eye drops, for example, sterile distilled water can typically be used as a base. The enzyme inhibitor of the present invention may further contain, for example, conventional solubilizing agents, stabilizers, buffers, preservatives, fragrances, coloring agents, etc., selected as appropriate. Furthermore, in the present invention, an effective amount of a synthetic bactericidal agent, an antibacterial agent such as an antibiotic, an anti-inflammatory agent, an antihistamine, or an analgesic may be appropriately incorporated into the enzyme inhibitor of the present invention. In this case, the therapeutic effect of the present invention can be further enhanced. Furthermore, when the enzyme inhibitor of the present invention is in the form of eye drops, it is preferable that the eye drops be made isotonic with lachrymal fluid, and therefore an isotonic agent such as common salt can be added if necessary. Furthermore, the eye drops have a pH of 5.5 to 8.5, preferably
It is desirable to adjust it to 6.5-7.5. The enzyme inhibitor of the present invention can be produced by a conventional method. Specifically, it is produced by using ovomacroglobulin as an active ingredient, mixing it with a suitable base, and then shaping it as necessary. When the enzyme inhibitor of the present invention is used as an eye ointment or eye drops, it is manufactured by further sterilization. The amount of the active ingredient, ie, ovomacroglobulin, to be contained in the enzyme inhibitor of the present invention is not particularly limited and can be appropriately selected from a wide range, but is usually selected from the range of about 0.0001 to 10% by weight in the preparation. It is better. Furthermore, the amount and method of applying the enzyme inhibitor of the present invention are as follows:
It can be determined depending on the form of the preparation, the amount of active ingredient in the preparation, the age, sex and other conditions of the patient to whom it is applied, the degree of wound, etc. For example, the enzyme inhibitor of the present invention in the form of an external preparation This can be applied to the affected area by spraying, coating, etc. one to several times a day in an amount sufficient to cover the entire affected area. The enzyme inhibitor of the present invention in the form of eye drops may be instilled into the eye from a suitable drip container or sprayed into the eye using a spray device. In the case of eye ointments, it is applied to the eye. As mentioned above, the enzyme inhibitor of the present invention thus obtained includes Serratia protease 56K, Serratia protease 60K, Serratia protease 60K,
73K claims to have an excellent enzyme inhibitory effect on Pseudomonas alkaline protease or Pseudomonas elastase, and is effective against infections caused by pathogenic bacteria that produce the enzymes, such as dermatitis, keratitis, conjunctivitis, cracks, and scaling. Excellent preventive and therapeutic effects on suppuration, cell destruction, cell necrosis, and inflammation of trauma such as abrasions, cuts, burns, burns, frostbite, surgical wounds, skin ulcers, urinary tract infections, cystitis, and dermatitis. Demonstrate. The enzyme inhibitor of the present invention is also characterized by a long duration of its efficacy. In addition, Serratia protease 56K, Serratia protease 60K,
It is a fact already known by the present inventor that the production of bradykinin is promoted by 73K, Pseudomonas alkaline protease or Pseudomonas elastase, resulting in pain [J.Biochem. 96 , 739-749 (1984), INFECTION AND IMMUNITY, 48 (No.
3), 747-753 (1985)]. Therefore, since the enzyme inhibitor of the present invention has inhibitory activity against the above-mentioned enzymes, it also suppresses pain caused by infection with pathogenic bacteria that produce the above-mentioned enzymes or when the disease condition worsens due to infection. It is useful as an analgesic. EXAMPLES In order to explain the present invention in more detail, preparation examples of ovomacroglobulin as an active ingredient, various formulation examples of the present invention formulations containing the same, and pharmacological test examples are given below. Preparation example of ovomacroglobulin: 20 kg of egg white and 10 kg containing the same amount of 1% NaCl.
Suspended in mM Tris-HCl buffer (PH7.7),
This is added to polyethylene glycol (molecular weight = 8500,
(Manufactured by Tokyo Kasei Co., Ltd.) was added to a concentration of 2.5%,
The supernatant was collected by continuous centrifugation (10,000 rpm). The same polyethylene glycol as above was further added to the obtained supernatant until the concentration reached 10%, and the mixture was again subjected to continuous centrifugation (10,000 rpm) to collect the precipitate. This was dissolved in the above buffer and further centrifuged (10,000 rpm, 10 minutes) to collect the supernatant, which was applied to a column (259 x 900 mm) of Sepharose CL-6B (manufactured by Pharmacia). With buffer, flow rate
It was eluted at a rate of 3.6/hour. For each elution section, trypsin inhibitory activity using casein as a substrate was measured using the method of Kitamoto et al. [T.
Kitamoto, M. Nakashima, and A. Ikai, J.
Biochem., 92 , 1679-1682 (1982)] and trypsin inhibitory activity fractions were collected. Next, the obtained active fraction was passed through a molecular sieve membrane.
While concentrating using a Pellicon cassette (manufactured by Millipore) equipped with a 100,000 membrane, the buffer was exchanged with 5mM Tris-HCl buffer (PH7.7). The obtained sample was diluted with 10mM NaCl.
Equilibrated with mM Tris-HCl buffer (PH7.7)
It was applied to a DEAE Trisacryl M (Trisacry IM, manufactured by LBK) column (size 50 x 800 mm). After thoroughly washing the column with the same buffer, the column was washed with 675 ml of 10 mM Tris-HCl buffer (PH 7.7) containing 50 mM NaCl and 675 ml of 10 mM Tris-HCl buffer (PH 7.7) containing 150 mM NaCl for 2.5 hours each. Elution was performed over 5 hours. Under these conditions, the trypsin inhibitory activity fraction was eluted between 70 mM and 120 mM of sodium chloride concentration. The trypsin inhibitory activity fractions were collected and dialyzed against 1 mM phosphate buffer (PH7.4). After thorough dialysis, the dialyzed fluid was freeze-dried using a freeze dryer (manufactured by Labokone). As a result of the above, 5.9 to 7.1 g of a single purified ovomacroglobulin sample was obtained. For purified samples, with 4N methanesulfonic acid,
After hydrolysis at 110°C for 24 hours (in a vacuum sealed tube), it was analyzed using an amino acid analyzer (Model 835-50, Hitachi High Speed Amino Acid Analyzer, manufactured by Hitachi, Ltd.). The results are shown in Table 1 below. Table 1 Amino acid content (mol%) Asp 10.3 Thr 6.4 Ser 8.0 Glu 11.6 Pro 4.3 Gly 5.1 Ala 5.8 Cys/2 1.8 Val 8.2 Met 2.0 Ile 6.5 Tyr 3.9 Phe 4.8 Lys 4.6 His 1.8 Arg 3.6 Formulation example 1 Ovomacroglobulin 0.01g Preservative (appropriate amount) Flavor (appropriate amount) Add distilled water to bring the total volume to 100ml. Add distilled water to the above ovomacroglobulin, preservative and fragrance to make a total volume of 100ml, then sterilize.
An enzyme inhibitor of the present invention in the form of a spray solution was prepared. Prescription example 2 Ovomacroglobulin 0.05g Preservative appropriate amount Flavor appropriate amount Add distilled water to bring the total volume to 100ml. In the same manner as in Formulation Example 1, an enzyme inhibitor of the present invention in the form of a spray solution having the above composition was prepared. Prescription example 3 Preparation of hydrophilic ointment Ovomacroglobulin 0.5g White petrolatum 250g Stearyl alcohol 220g Propylene glycol 120g Sodium lauryl sulfate 15g Ethyl paraoxybenzoate or methyl paraoxybenzoate 0.25g Propyl paraoxybenzoate 0.15g Purified water Appropriate amount Total amount 1000g The above ingredients were blended to prepare an enzyme inhibitor in the form of a hydrophilic ointment of the present invention containing ovomacroglobulin. Formulation Examples 4 to 7 Preparation of Hydrophilic Ointment In Formulation Example 3, the amount of ovomacroglobulin was changed to 0.01 g, 0.05 g, 0.1 g, and 1 g, and the enzyme inhibitor in the form of hydrophilic ointment of the present invention was prepared in the same manner. Prepared. Prescription example 8 Ovomacroglobulin 10mg Benzathonium chloride 1mg Sodium chloride 30mg Sodium dihydrogen phosphate 50mg Sodium monohydrogen phosphate, 12H ₂ O 118mg Distilled water Appropriate amount Total amount 10mg Dissolve each of the above components in distilled water and apply with appropriate filter paper. The enzyme inhibitor of the present invention in the form of eye drops was prepared by sterilization using the same method. Formulation example 9 Preparation of hydrophilic ointment Ovomacroglobulin 0.5g White petrolatum 250g Stearyl alcohol 220g Propylene glycol 120g Sodium lauryl sulfate 15g Ethyl paraoxybenzoate or methyl paraoxybenzoate 0.25g Propyl paraoxybenzoate 0.15g 9-Fluoro-8-( 4-hydroxy-1-piperidyl)-5-methyl-6,7-dihydro-1
-Oxo-1H,5H-benzo[ij]quinolidine-
2-Carboxylic acid sodium salt 10g Purified water Appropriate amount Total amount 1000g The above ingredients were blended to prepare an enzyme inhibitor in the form of a hydrophilic ointment of the present invention containing ovomacroglobulin. Prescription example 10 Ovomacroglobulin 10mg Benzathonium chloride 1mg Sodium chloride 30mg Sodium dihydrogen phosphate 50mg Sodium monohydrogen phosphate 12H ₂ O 118mg 9-Fluoro-8-(4-hydroxy-1-piperidyl)-5-methyl-6 ,7-dihydro-1
-Oxo-1H,5H-benzo[ij]quinolidine-
2-carboxylic acid sodium salt 50 mg Distilled water Appropriate amount Total amount 10 ml The above components were dissolved in distilled water and sterilized using appropriate filter paper to prepare the enzyme inhibitor of the present invention in the form of eye drops. . Pharmacological test example 1 Protease inhibitory effect test of ovomacroglobulin Method of Maeda et al. [Anal.Biochem., 92 , 222-227
(1979)]. That is, 10μl of a solution of 50mM Tris-HCl buffer (PH8.0) in which the enzymes listed in Table 2 below were dissolved and 50ml of a solution in which ovomacroglobulin (hereinafter abbreviated as "OVM") was dissolved.
The mixture was mixed with 190 μl of a solution of M Tris-HCl buffer (PH7.5) and left at 37° C. for 10 minutes. This contains 50mM Tris-HCl buffer (PH8.0) containing fluorescein-isothiocyanate (FITC)-gelatin.
1800μ was added and reacted at 37°C for 20 minutes. The degree of fluorescence polarization of the reaction solution was measured using a fluorescence depolarization measuring device (manufactured by Japan Antibody Research Institute, MAC-) at excitation light of 490 m and fluorescence of 520 nm to determine enzyme inhibitory activity.

[Table] Serratia protease 56K, 60K and Serratia protease
Enzyme residual activity when using 73K
Figure 1 shows the relationship with OVM usage. FIG. 2 shows the relationship between the residual enzyme activity and the amount of OVM used when Pseudomonas alkaline protease is used. FIG. 3 shows the relationship between the residual activity of the enzyme and the amount of OVM used when Pseudomonas elastase is used. From Figures 1 to 3, OVM is Serratia protease 56K, Serratia protease 60K, Serratia protease 73K, and Pseudomonas protease.
It is clear that it has excellent enzyme inhibitory activity against alkaline protease and Pseudomonas elastase. Furthermore, Serratia protease 56K was dissolved in 50
Dissolution 10μ of mM Tris-HCl buffer (PH8.0)
(Contains 0.6 μg of Serratia protease 56K)
50mM Tris-HCl buffer (PH
8.0) 190μ (contains 18μg of OVM) (the molar ratio of Serratia protease 56K and OVM is 2:1), react at 22℃, 10 minutes, 20 minutes,
1 hour, 2 hours, 3 hours, 18 hours, 21 hours and 24 hours
The enzyme residual activity after a period of time was examined in the same manner as above. The relationship between enzyme residual activity and reaction time is shown in the fourth section.
As shown in the figure. From FIG. 4, it is clear that OVM has excellent enzyme inhibitory activity against Serratia protease 56K even after 24 hours. Pharmacological test example 2 Corneal inflammation model test Kamata et al.'s method [OPTHALMOLOGY, 92 ,
(No. 10, 1452-1459 (1985)). That is, 10μ each of the following solutions were first administered to the corneas of guinea pigs to form experimental groups 1 to 8. Experimental Group 1 : Administration of a solution in which 2 μg of Serratia protease 56K was dissolved in physiological saline Experimental group 2: A solution in which 2 μg of Serratia protease 56K and 81 μg of OVM (molar ratio of Serratia protease 56K and OVM was 1:3) dissolved in physiological saline was administered. Administration experimental group 3: A solution of 2 μg of Serratia protease 60K dissolved in physiological saline. Experimental group 4: 2 μg of Serratia protease 60K and 65 μg of OVM (molar ratio of Serratia protease 60K and OVM is 1:2.5) in physiological saline. Administration of dissolved solution Experimental group 5: Serratia protease 73K 1.6 μg
Experimental group 6: Serratia protease 73K 1.6 μg
and OVM50μg (Serratia protease 73K
and OVM (molar ratio of 1:3) in physiological saline. Experimental group 7: Administration of a solution in which 1.5 μg of Pseudomonas alkaline protease was dissolved in physiological saline. Experimental group 8: 1.5 μg of Pseudomonas alkaline protease. and 95 μg of OVM (molar ratio of Pseudomonas alkaline protease and OVM is 1:
4) dissolved in physiological saline was administered, and a control group was administered with only physiological saline. Creation of each of the above experimental groups (administering the above solution to the cornea)
The state of the cornea was histologically observed 18 hours after the surgery. Reference photo 1 is a photo showing the condition of the cornea of experimental group 1. Reference photo 2 is a photo showing the condition of the cornea of experimental group 2. Reference photo 3 is a photo showing the condition of the cornea of experimental group 3. Reference photo 4
is a photograph showing the condition of the cornea of Experimental Group 4. Reference photo 5 is a photo showing the condition of the cornea of experimental group 5. Reference photo 6 is a photo showing the condition of the cornea of experimental group 6. Reference photo 7 is a photo showing the condition of the cornea of experimental group 7. Reference photo 8 is experimental group 8.
This is a photograph showing the condition of the cornea. Reference photo 9 is
It is a photograph showing the condition of the cornea of the control group. From reference photos 1 to 9, corneal ulcers were clearly suppressed in the OVM administration groups (experimental groups 2, 4, 6, and 8) compared to the non-OVM administration groups (experimental groups 1, 3, 5, and 7). I know that there is. Pharmacological test example 3 Vascular permeability inhibition test Kamata et al.'s method [INFECTION AND
IMMUNITY, 48 (No. 3), 747-753 (1985)], the above test was conducted using albino Hartley guinea pigs weighing 400 to 600 g. First, an amount equivalent to 30 mg of Evans Blue (manufactured by Wako Pure Chemical Industries, Ltd.) was intravenously administered to a guinea pig at a concentration of 2.5% in physiological saline, and immediately 0.1 ml of the following solution was intradermally administered to the guinea pig's shaved flank. Experimental group 1~
5 and control groups 1 to 5. Experimental group 1: Serratia protease 56K 0.3 μg,
1 μg, 3 μg, or 8 μg and 360 μg of OVM were dissolved in phosphate buffered saline (PH7.4), and the resulting solution (0.1 ml) was left at 37°C for 10 minutes. Experimental group 2: Serratia protease 60K 1 μg,
3 μg or 8 μg and 203 μg of OVM were dissolved in phosphate buffered saline (PH7.4), and the resulting solution (0.1 ml) was left at 37°C for 10 minutes. Experimental group 3: Serratia protease 73K 1 μg,
Experimental group 4: Pseudomonas alkaline protease 1 μg, 3 μg or 8 μg and 380 μg of OVM was dissolved in phosphate buffered saline (PH7.4) and the solution (0.1 ml) obtained by standing at 37°C for 10 minutes was administered Experimental group 5: Pseudomonas elastase 1 μg,
3 μg or 8 μg and 313 μg of OVM were dissolved in phosphate buffered saline (PH7.4), and the resulting solution (0.1 ml) was left at 37°C for 10 minutes. Control group 1: Serratia protease 56K 0.3 μg,
Add 1μg, 3μg or 8μg to phosphate buffered saline (PH
Control group 2: Administration of a solution (0.1 ml) dissolved in 7.4) Serratia protease 60K 1 μg,
Control group 3: Serratia protease 73K 1 μg;
Control group 4: Pseudomonas alkaline protease 1 μg, 3 μg or 8 μg dissolved in phosphate buffered saline (PH7.4) Control group 5: Administration of a solution (0.1 ml) in which 1 μg, 3 μg or 8 μg of Pseudomonas alkaline protease was dissolved in phosphate buffered saline (PH7.4) Experimental group 1 was compared to control group 1. It can be seen that the amount of pigment exudation is small, and the vascular permeability caused by Serratia protease 56K is clearly suppressed (see Reference Photo 10). In addition, when 0.1ml of phosphate buffered saline (PH7.4) was administered and
When 0.1 ml of a solution in which 360 μg of OVM was dissolved in phosphoric acid dry saline (PH7.4) was administered, no exudation of the dye was observed at all. The permeation activities for experimental groups 2-5 and control groups 2-5 were determined by Ueda et al. [Proc.So.Exp.Biol.Med.,
133, 1384-1387 (1970)], the Evans blue exuded by the epidermis of the back of the guinea pig was measured (wavelength 620 nm), and the amount of exuded pigment was calculated from the standard curve of Evans blue prepared in advance. Calculated. The results obtained are shown in FIGS. 5 and 6. Fifth
From the figure and Fig. 6, experimental groups 2 to 5 had less pigment exudation than control groups 2 to 5, and Serratia
It can be seen that vascular permeability caused by protease 60K, protease 73K, Pseudomonas alkaline protease, and Pseudomonas elastase is clearly suppressed. Pharmacological test example 4 RPMI1640 medium [containing 10% fetal calf serum (FCS)] containing Serratia protease 56K at a ratio of 12.5 μg/ml and 6.25 μg/ml, Serratia protease 56K at 25 μg/ml and OVM at 400 μg/ml
Fibroblasts were cultured in PRMI1640 medium containing 56K g/ml and RPMI1640 medium without Serratia protease 56K and OVM (control), and the proliferation state of the fibroblasts was examined. The results are shown in Reference Photo 11. In RPMI1640 medium that does not contain Serratia protease 56K and OVM, fibroblasts grow as a beautiful monolayer on the bottom of the culture vessel (see Photo A). However, when Serratia protease 56K is present in the medium, fibroblasts become granular and their growth is completely killed (Photo B).
contains Serratia protease 56K at 12.5 μg/ml.
Photo C shows the growth state after 24 hours in a medium containing Serratia protease 56K at a ratio of 6.25 μg/ml. This shows that. ). On the other hand, if OVM was further present in the above medium, fibroblasts grew beautifully as a monolayer on the bottom of the culture container, and it was observed that there was no difference from the control (see Photo D).

[Brief explanation of the drawing]

FIGS. 1 to 3 are graphs showing the relationship between residual enzyme activity and the amount of OVM used. FIG. 4 is a graph showing the relationship between enzyme residual activity and reaction time. FIGS. 5 and 6 are graphs showing the relationship between the amount of dye exudation and the amount of enzyme.

Claims (1)

[Claims] 1. Serratia protease characterized by containing ovomacroglobulin as an active ingredient
56K, 60K, 73K, enzyme inhibitors against Pseudomonas alkaline protease or Pseudomonas elastase. 2. The enzyme inhibitor according to claim 1, which is used for the prevention and treatment of keratitis.