JPH11299882A - Method for stabilizing activity of immobilized fibrinolytically active enzyme - Google Patents

Abstract

PROBLEM TO BE SOLVED: To stabilize the activity of a fibrinolytically active enzyme under physiological conditions by treating a carrier immobilized with the active enzyme with an aminoglycoside antibiotic agent. SOLUTION: An antithrombotic material is formed by imobilizing, for example, the fibrinolytically active enzyme, such as plasmin, to the carrier, such as glass. The surface of the antithrombotic material is treated with a soln. contg. the animoglycoside-base antibiotic agent, by which the activity thereof is stabilized. Gentamicin sulfate, netilmicin sulfate, tobramycin, etc., are used as the animoglycoside-base antibiotic agent. These antibiotic agents are dissolved in water. An org. solvent, such as methanol, which may be mixed with the water may be added thereto at need. The concn. of the antibiotic agents of this time may be diluted and is set at about 0.001 to about 50 wt.%, more preferably about 0.01 to about 10 wt.%. The preservable stability may be improved by this constitution.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for stabilizing the activity of a fibrinolytically active enzyme immobilized on a carrier.

[0002]

2. Description of the Related Art Hitherto, fibrin (fibrin) and fibrinolytic active enzyme which is a lytic enzyme for thrombus have been widely used for treatment of various thrombosis and embolic diseases, etc., and provide excellent clinical effects. I have. In addition, antithrombotic materials in which a fibrinolytic active enzyme is immobilized on the surface of a polymer material utilizing the excellent thrombus dissolving power of the enzyme have been developed. However, the activity stability of fibrinolytic active enzyme is not always good, and when immobilizing fibrinolytic active enzyme and using it as a medical antithrombotic material in medical treatment, the storage time of enzyme-immobilized material and the time of indwelling in the body Inactivation was a major problem. When a fibrinolytic active enzyme is immobilized and used for medical treatment as an antithrombotic material, it is necessary to sterilize the material on which the enzyme is immobilized. Usually, the activity of the enzyme is lost or reduced by sterilization. Therefore, inactivation of the enzyme during sterilization was also a major problem when used as an antithrombotic material.

[0003] As a method for stabilizing the activity of the immobilized fibrinolytic active enzyme, the surface of the carrier on which the fibrinolytic active enzyme is immobilized is treated with a basic amino acid so that the fibrinolytic active enzyme during storage and sterilization is preserved. Has been reported to be able to stabilize the activity of the protein (see Japanese Patent Publication No. 4-56591).

[0004]

However, in spite of a very serious problem, inactivation of the enzyme over time when an antithrombotic material having a fibrinolytic active enzyme immobilized thereon is actually used in a living body. There was no known way to prevent this.

It is an object of the present invention to provide a method for stably maintaining the activity of a fibrinolytically active enzyme immobilized on a carrier even under physiological conditions.

[0006]

Means for Solving the Problems The inventors of the present invention have conducted intensive studies to achieve the above object, and have found that the fibrinolytically active enzyme immobilized on a carrier is preserved, sterilized, and used in a living body. The present inventors have found that treating a carrier on which a fibrinolytic active enzyme is immobilized with an aminoglycoside antibiotic can prevent inactivation and denaturation of the fibrinolytic active enzyme during storage, sterilization, and even during use. Reached.

That is, the present invention provides an immobilized fibrinolytic enzyme characterized in that the surface of an antithrombotic material on which the fibrinolytic active enzyme is immobilized is treated with a solution containing an aminoglycoside antibiotic. The activity stabilization method of the present invention is the gist.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail. The carrier used for the antithrombotic material in the present invention may be any solid as long as it is a solid, and preferred carriers are, for example, glass, kaolinite, bentonite, inorganic substances such as activated carbon, natural rubber, cellulose,
Natural polymers such as starch, collagen, agarose, dextran, proteins, polystyrene, polyamino acids,
Polyurethane, polyamide, polyester, polyvinyl chloride, polyethylene, polypropylene, polyvinyl alcohol, polymethacrylic acid ester, ethylene vinyl acetate copolymer, a synthetic polymer such as silicone resin alone, or a carrier composed of a combination thereof. Can be The shape of the carrier is not particularly limited, and various shapes such as a fiber, a hollow fiber, a tube, a film, a film, a permeable film, a bead, and a powder depending on the purpose can be used.

[0009] The fibrinolytically active enzyme in the present invention is:
Means the enzymes involved in fibrinolysis. Such enzymes include, for example, plasmin, purinolase, urokinase, streptokinase, tissue plasminogen activator, and the like.

As a method for immobilizing these fibrinolytically active enzymes on a carrier, known methods for immobilizing enzymes can be used. For example, JP-A-53-88390, JP-A-54-26394, and Immobilized enzymes ”(edited by Ichiro Chibatake, published by Kodansha) and the like can be used.

The antithrombotic material having the fibrinolytic active enzyme immobilized on the carrier is mainly placed in the body or removed from the body. For example, IVH catheters, thermodilution catheters, blood vessels It can be suitably used for medical devices to be inserted or placed in blood vessels, such as contrast catheters, vascular dilatation catheters, dilators, indwelling needles, and guide wires.

According to the activity stabilization method of the present invention, the surface of the above antithrombotic material is treated with a solution containing an aminoglycoside antibiotic.

The aminoglycoside antibiotics used in the present invention include gentamicin sulfate, netilmicin sulfate, tobramycin, amikacin sulfate, streptomycin sulfate, fradiomycin sulfate, bekanamycin sulfate, paromomycin sulfate, ribostamycin sulfate, kanamycin sulfate, dibekacin sulfate And sisomycin sulfate, micronomycin sulfate, astromycin sulfate, isepamicin sulfate, arbekacin sulfate and the like, and derivatives thereof such as amides, esters and salts may be used.

In the present invention, the above-mentioned aminoglycoside antibiotics are used after being dissolved in water. If necessary, an organic solvent which can be mixed with water may be added to this solution. Examples of the organic solvent include methanol, ethanol, acetone, dimethyl sulfoxide and the like.

The concentration of the aminoglycoside antibiotic at the time of treatment is effective even with a dilute solution, preferably 0.001 to 50% by weight, more preferably 0.01 to 1% by weight.
0% by weight.

In order to treat the surface of the antithrombotic material with an aminoglycoside antibiotic, a solution containing the above aminoglycoside antibiotic may be brought into contact with the antithrombotic material.
As a specific method of contacting, a method of spraying a solution containing an aminoglycoside antibiotic, or, in the case of a tubular medical device such as a catheter, a method of circulating the solution in a tube is also used. The simplest method is a method of immersing the antithrombotic material in a solution containing an aminoglycoside antibiotic, and it is more effective if stirring or shaking is performed as necessary.

The temperature at the time of contact is 0 to 50 ° C.
And preferably 10 to 40 ° C. The contact time is 24 hours or less, preferably 1 hour.
Minutes to 10 hours, more preferably 5 minutes to 1 hour.

The material in which the immobilized fibrinolytic enzyme is stabilized according to the present invention can be used after sterilized by various methods. Sterilization methods include, for example, ethylene oxide, propylene oxide, formaldehyde, β-
Examples include gas sterilization using a sterilizing gas such as propionlactone and methyl bromide, and radiation sterilization using X-rays, α-rays, high-speed electron beams, beta rays, and the like. The pressure and temperature of the sterilizing gas, the dose and time of radiation, and the like may be arbitrarily determined according to the number of bacteria adhered to the fibrinolytically active enzyme-immobilized carrier. In addition, the sterilization container may be any container that allows the ingress and egress of sterilization gas and does not allow microorganisms to enter or that can transmit radiation.The shape is not limited to a bag shape, and may be any shape such as a tube, a tube, or a box. Good.

[0019]

The present invention will be described below in detail with reference to examples. The activity measurement and the stability test in the examples were performed as follows.

[Activity measurement 1] The activity of urokinase is determined by subjecting a synthetic peptide containing a fluorescent substance to an enzymatic reaction using a substrate as a substrate.
The released fluorescent substance was measured by a method of quantifying the fluorescent substance using a spectrofluorometer (see Morita et al., J. Biochem., 82, p1495 (1977)).

[Activity measurement 2] The fibrinolytic activities of streptokinase and tissue plasminogen activator were determined by placing a circularly cut sample piece (5 mm in diameter) on a fibrin plate and leaving it at 37 ° C for 24 hours. The extent of fibrin dissolution around the sample piece was determined by the area of the lysis circle (m
m ² ) (see “Procedures of Clinical Laboratory Methods”, revised 27th edition, Kanbara Publishing, VI-110).

[Storage stability 1] The material was kept at 30 ° C. and a humidity of 55.
% In a constant temperature bath for a predetermined period, and then the activity was measured. [Storage Stability 2] The material was left at room temperature (25 ° C.) and left for a predetermined period, and then the activity was measured.

[Stability under physiological conditions] The material was immersed in 10 ml of Dulbecco's physiological saline, shaken at 37 ° C for a predetermined period, and the activity was measured.

Example 1, Comparative Example 1 A polyurethane tube (inner diameter 0.97 mm, outer diameter 1.52 mm, length
50 cm) in an acetone solution in which 2% (wt / v) of a maleic anhydride-methylvinyl ether copolymer having a molecular weight of about 67,000 and 0.1% (wt / v) of polyethylene glycol having a molecular weight of 4,000 were dissolved at room temperature for 10 minutes. Dipped and then heated at 70 ° C. for 20 hours. The heated tube was washed with acetone and dried. Next, the tube was immersed in a solution of urokinase in a physiological saline solution (1,000 units / ml) at room temperature for 24 hours, and then washed with water. Subsequently, the tube was immersed in a 1 mg / ml aqueous solution of dibekacin sulfate for 1 hour at room temperature, washed with water, and the tube was dried (Example 1). For comparison, a urokinase-immobilized polyurethane tube was prepared in exactly the same manner as above except that dibekacin sulfate treatment was not performed (Comparative Example 1).

For these polyurethane tubes,
The stability in the gas sterilization process was tested. The gas sterilization process
After storing the polyurethane tube in a commercially available sterilization bag (sterilized bag made of Hogi) and completely sealing it, using a sterilization gas (20% ethylene oxide gas, 80% carbon dioxide gas),
Kg / cm ² G, 40 ° C., 40% RH, 2 hours.
The urokinase activity of the polyurethane tube prepared according to the present invention was 4.9 units / cm ² before and after the sterilization treatment, while the urokinase activity of the polyurethane tube of Comparative Example 1 was 4.94 units / cm ² . It dropped greatly from 9 units / cm ² to 3.2 units / cm ² . In addition,
Both were sterile.

For each of the gas-sterilized polyurethane tubes of Example 1 and Comparative Example 1, 1 month, 3 months, 6 months and 1 month
The stability after two months was examined. The results are shown in Table 1.

[0027]

[Table 1]

The stability of the polyurethane tubes of Example 1 and Comparative Example 1 subjected to gas sterilization under physiological conditions was tested. The results are shown in FIG.

Example 2, Comparative Example 2 An acetone solution containing 2% (wt / v) of a maleic anhydride-methylvinyl ether copolymer having a molecular weight of about 67,000 and 0.05% (wt / v) of polyethylene glycol having a molecular weight of 4,000. After immersing a nylon 6 film at room temperature for 1 minute and air-drying,
The mixture was heated under reduced pressure at 0 ° C. for 2 hours. The heated film piece was washed with acetone and dried. This film piece was placed in a physiological saline solution of streptokinase (600 units / ml) at 4 ° C.
And then washed with water. After washing, the film was immersed in a 10 mg / ml gentamicin sulfate aqueous solution at room temperature for 10 minutes, and the film was air-dried (Example 2). For comparison, a streptokinase-immobilized nylon 6 film was prepared in exactly the same manner as above except that the gentamicin sulfate treatment was not performed (Comparative Example 2).

When the fibrinolytic activity of these streptokinase-immobilized film pieces was measured, both dissolved fibrin in a circular shape having a diameter of 24 mm, and the area of the dissolved circle was 452 mm ² .

These films have a storage stability of 2
According to the method described above, the stability after standing for 12 months was examined.
As a result, the activity of the film of Example 2 was 450 mm ² , and the activity during storage was hardly reduced, whereas the activity of the film of Comparative Example ² was greatly reduced to 271 mm ² .

The stability of these films under physiological conditions was examined. Circular film piece (diameter 5m
m) was placed under the above conditions for 28 days, and the activity was measured. As a result, the streptokinase activity of Example 2 was 225 mm
^In contrast to Comparative Example 2, that of Comparative Example ² was greatly reduced to 11 mm ² .

Further, the stability of these films by sterilization was examined. The sterilization treatment was performed by storing the film in a commercially available sterilization bag (sterilized bag made of Hogi), completely sealing it, and then performing radiation (Co-60, 2.5 Mrad). As a result of measuring the streptokinase activity after sterilization, the streptokinase activity of the film of Example 2 was 4
Although the activity was 48 mm ² , and the activity was hardly reduced by the sterilization treatment, the streptokinase activity of the film of Comparative Example ² was greatly reduced to 262 mm ² . The film pieces were both sterile.

Example 3, Comparative Example 3 Polyvinyl chloride was extruded at 170.degree.
A μm film was obtained. Next, the polyvinyl chloride film obtained in an acetone solution in which 2% (wt / v) of a maleic anhydride-methyl vinyl ether copolymer having a molecular weight of 67,000 and 1% (wt / v) of polyethylene glycol having a molecular weight of 400 were dissolved was added at room temperature. After immersion for 5 minutes, it was heated at 70 ° C. for 24 hours. This film was transferred to a human melanoma cell line (human melanoma c
ellline) was immersed in a physiological saline solution (600 units / ml) of tissue plasminogen activator, left at 7 ° C. for 24 hours, and then washed with water. After washing, 0.1
After immersion in a 5 mg / ml aqueous solution of arbekacin sulfate at room temperature for 5 minutes, the film was air-dried (Example 3). For comparison, a tissue plasminogen activator-immobilized film was produced in exactly the same manner as described above except that the arbekacin sulfate treatment was not performed (Comparative Example 3).

The fibrinolytic activity of these tissue plasminogen activator-immobilized films was measured in the same manner as in Example 2, and both were 400 mm ² .

The stability of these films in sterilization was examined. The sterilization process was the same gas sterilization process as in Example 1. As a result, the activity of the film of Example 3 was 400 mm ² , and the activity of the film of Comparative Example 3 was greatly reduced to 248 mm ² , while no decrease in the activity was observed by the sterilization treatment. In addition, both films were in a sterile state.

For these sterilized films,
Under the conditions of storage stability 1, stability after 12 months was examined. As a result, the activity of the film of Example 3 was 390 mm ²
, And the contrast is decreased activity during storage was hardly observed, the activity of the film of Comparative Example 3 was reduced further increased from 248mm ² to 156 mm ^2.

The sterilized films were tested for stability under physiological conditions. The activity after 28 days was measured using a circular film piece (diameter: 5 mm).
As a result, the activity of the film of Example 3 was 200 mm ² , while the activity of the film of Comparative Example 3 was 248 mm ²
From 6 to 6 mm ² .

[0039]

According to the present invention, not only the stability of the fibrinolytic activity enzyme immobilized on the carrier during storage but also during sterilization is significantly increased, and the fibrinolytic activity is maintained under physiological conditions. The stability is significantly improved. Further, the material produced according to the present invention not only has antithrombotic properties, but also has antibacterial activity based on aminoglycoside antibiotics.

[Brief description of the drawings]

FIG. 1 is a diagram showing the stability of the polyurethane tubes obtained in Example 1 and Comparative Example 1 under physiological conditions.

Claims (1)

[Claims] 1. An activity stabilization of an immobilized fibrinolytic enzyme, wherein the surface of the antithrombotic material on which the fibrinolytic enzyme is immobilized is treated with a solution containing an aminoglycoside antibiotic. Law.