JP2853452B2 - Method for producing molded article immobilized with physiologically active substance - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a polypeptide antibiotic.
Quality, click Bu Rikohe ° off DEG tide antibiotic and amino click Bu glycosidic antibiotic
The present invention relates to a method for producing a molded article on which a physiologically active substance selected from substances is immobilized.

[0002]

2. Description of the Related Art A molded article in which a physiologically active substance is immobilized on a molded article carrier is widely used for applications such as a therapeutic blood purifying agent, an antibacterial agent, a therapeutic imino modulator, and an adsorbent for affinity chromatography. There is a demand for a homogeneous and inexpensive molded article on which a physiologically active substance is immobilized. For example, an endotoxin neutralizing agent in which polymyxin is immobilized on a fibrous carrier by a covalent bond has an excellent feature that it eliminates the side effects of polymyxin while maintaining the original endotoxin neutralizing and detoxifying effect of polymyxin. It is known as a very useful neutralizing agent for use in treating patients with infectious diseases, removing endotoxins in transfusions and dialysates (Japanese Patent Application Laid-Open No. 60-5166).

[0003] Biologically active substances are usually very expensive,
It is important for industrialization to efficiently and stably immobilize the necessary and minimum physiologically active substance.

In Example 1 of JP-A-60-5166, during the immobilization reaction, 4.8 g (440 mg per 1 g of carrier) of Polymyxin B Pfizer was used for 11 g of carrier fiber, and 110 mg / g fiber was immobilized. Is becoming

In Example 1 of Japanese Patent Publication No. 1-16389, 1 g of polymyxin B sulfate was added to 10 g of a BrCN-activated crosslinked agarose carrier (wet state) during the immobilization reaction.
In Example 2, 400 mg of colistin sulfate (200 mg per 1 g of carrier) was added to 2 g of epoxidized cellulose beads during the immobilization reaction in Example 2.
The techniques used are indicated.

[0006] In these examples, the amount of the physiologically active substance to be used is 100 mg per 1 g of the molded article carrier at the minimum, and the molded article on which the physiologically active substance is immobilized is expensive.

[0007]

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for producing a highly active, homogeneous and inexpensive bioactive substance-immobilized molded article by minimizing the amount of use of a physiologically active substance having an amino group. To provide.

[0008]

The above object is achieved by the present invention described below. As the physiologically active substance having an "amino group, Poripepuchido antibiotic, click Bu Rikohe ° off DEG tide antibiotic
Few selected from substances and amino click Bu glycosidic antibiotics
In a method for producing a molded article immobilized with a physiologically active substance by causing at least one of them to undergo an immobilization reaction on a molded article alone, the amount of the physiologically active substance used for the immobilization reaction is adjusted to 2 mg / m 2 per unit surface area of the molded article carrier. ² or more, 200 mg / m ²
A method for producing a molded article immobilized with a physiologically active substance, characterized by the following. Hereinafter, the present invention will be described in detail.

The present inventors have found that in a molded article on which a physiologically active substance is immobilized, the immobilized physiologically active substance in the vicinity of the surface predominantly contributes to the development of the physiological activity. for that reason,
In the present invention, from the viewpoint of cost-effectiveness, the physiologically active substance amount having an amino group used in the immobilization reaction, moldings carrier unit surface area per 2 mg / m ² or more, 200 mg / m ²
The following is assumed.

[0010] As the physiologically active substance having the present invention the amino group, physiological activity selected Poripepuchito Bu antibiotics, from click Bu Rikohe ° off DEG Tito Bu antibiotics and Aminoku Bu Rikoshito Bu antibiotics
Substances are used .

[0011] Among them, the polypeptide antibiotics have an extremely excellent antibacterial action, but have a problem that they cannot be administered to the body due to side effects such as nephrotoxicity and neurotoxicity. It is particularly effective because it solves the problem of side effects and can effectively exert its original excellent antibacterial action as a therapeutic blood purifier.

Examples of the polypeptide antibiotics include polymyxin A, polymyxin B1, polymyxin B2, polymyxin D1, polymyxin E1 and polymyxin E2, which exhibit a strong antibacterial action against Gram-negative bacteria. And bacitracin, which exhibits a strong antibacterial action on the skin.

The glycopeptide antibiotics include vancomycin, which is effective against aerobic and anaerobic gram-positive bacteria, and the aminoglycoside antibiotics include Pseudomonas aeruginosa.
Tobramycin, dibekacin, amikacin, and the like, which are effective against deformed bacteria, etc., can be mentioned.

In the present invention, the pH of the reaction solution for immobilizing the physiologically active substance having an amino group is preferably adjusted to 7 or more. Further, in view of the balance between the immobilization reaction rate, the immobilization reaction yield, and the physiological activity of the molded article immobilized with a bioactive substance, the pH of the immobilization reaction solution containing the bioactive substance having an amino group is in the range of 8 to 10. It is particularly preferable to control the value to a constant value.

The concentration of the physiologically active substance in the reaction solution is preferably as low as possible within the range of a practical reaction rate, and more preferably from 0.05 kg / m ³ to 10 kg / m ³ .

In Example 1 of JP-A-60-5166, magnesium oxide is used as a fixing agent. In the case of such a hardly soluble base, it is difficult to control a desired pH, and the precipitated or undissolved magnesium oxide coats the surface of the molded article carrier, and a uniform immobilization reaction on the molded article carrier surface is performed. There is a problem that hinders progress.
Therefore, in the present invention, in immobilizing a physiologically active substance having an amino group on a molded article carrier, the pH of an immobilization reaction solution is increased, and a physiologically active substance having an amino group is used as a fixing agent for accelerating the reaction. A base that is readily soluble in the reaction solution containing the active substance is particularly preferably used. When the solvent of the immobilization reaction solution is water, examples of the base include sodium hydroxide, potassium hydroxide, lithium hydroxide, ammonium hydroxide, and the like, but are not limited thereto. What is necessary is just to select suitably according to the kind of solvent and a physiologically active substance. In addition to water, as a solvent, an alcohol such as methanol or ethanol, an aqueous solution thereof, or the like can be used.

As the molded article carrier, those having a functional group which is chemically and physically stable under the conditions of use and which can chemically fix a physiologically active substance having an amino group can be used. For example, a polystyrene molded article or a cross-linked polystyrene molded article having an α-halogenated acyl group, a benzyl halide group or an isocyanic acid group, an acrylic acid / acrylonitrile copolymer molded article, a polyvinyl alcohol molded article having a carboxyl group, and the like can be given. However, the present invention is not limited to these. In particular, a crosslinked polystyrene molded article having an α-chloroacetamidomethyl group is excellent in terms of expressing a physiologically active substance and is preferably used.

The shape of the molded article carrier is not particularly limited, but the fibrous molded article carrier can easily increase the surface area per unit volume and has low flow resistance of a fluid when packed in a column. And so on, which are particularly preferable.

The molded article on which the physiologically active substance is fixed according to the present invention comprises a therapeutic blood purifying agent, an antibacterial agent, a therapeutic imino modulator,
It is suitably used for applications such as adsorbents for affinity chromatography, but is not limited thereto.
Among them, specific examples of therapeutic blood purifying agents include endotoxemia in which a purifying agent obtained by immobilizing a polypeptide antibiotic such as polymyxin or bacitracin on cross-linked polystyrene fiber having an α-chloroacetamidomethyl group is packed in a column. A therapeutic blood purifier.

[0020]

The present invention will be described in more detail with reference to the following examples. The present invention is not limited to these examples. Example 1 Polypropylene (“Noblen” J3H- manufactured by Mitsui Toatsu Co., Ltd.)
G) A mixture of 46 parts by weight of polystyrene (“Stylon” 679, manufactured by Asahi Kasei Kogyo Co., Ltd.) and 4 parts by weight of polypropylene (“Noblen” J3H-G, manufactured by Mitsui Toatsu Co., Ltd.) was used as the sea component. Using a raw material to be melt-spun at 280 ° C. (16 islands), it was stretched to 3.1 times to obtain a multifilament sea-island composite fiber (single yarn denier 3).

The composite fiber processed into a knitted fabric 700
g on an open bobbin, diameter 200 mm, height 30
Packed in a 0 mm column.

Nitrobenzene 1 in a stainless steel container
5 kg, 98% concentrated sulfuric acid 15 kg, N-methylol-α-
A chloroacetamidomethylation reaction solution consisting of 2.25 kg of chloroacetamide and 32.3 g of paraformaldehyde was prepared.

This reaction solution was supplied to the hollow portion of the bobbin core in the column by a pump, and circulated in the fiber layer in the radial direction. The reaction solution flowing out of the column was circulated through a stainless steel container.

After reacting at 10 to 20 ° C. for 2 hours, the chloroacetamidomethylation reaction solution was withdrawn from the column and the stainless steel vessel, and at room temperature, 20 kg of nitrobenzene and 20 kg of pure water were passed through the column in the same manner as in the reaction in the order of the reaction. After circulating the fibers, the remaining sulfuric acid was neutralized with a 24% aqueous sodium hydroxide solution.

By the same operation, the remaining nitrobenzene was extracted with methanol and the remaining methanol was extracted and removed with warm water at 70 ° C. to obtain 980 g of purified chloroacetamidomethylated fiber (fiber A).

An immobilized reaction solution (polymyxin B sulfate concentration: 0.71 kg / m ³ ) consisting of 20 l of pure water and 14.2 g of polymyxin B sulfate was prepared in a stainless steel container.

The amount of the polymyxin B sulfate is 43 mg / m ² per unit surface area of the carrier fiber.

This reaction solution is pumped to the fiber A column by the same route as in the chloroacetamidomethylation reaction,
The pH of the immobilized reaction solution was controlled at 9.5 by continuously adding dropwise a / 10 N aqueous sodium hydroxide solution to the reaction solution. After the reaction for 1 hour, the immobilized reaction solution was withdrawn from the column and the stainless steel container, circulated and washed three times with 1/10 N hydrochloric acid (15 kg) and pure water (15 kg) four times, and polymyxin B was removed.
An immobilized fiber was obtained.

The amount of immobilized polymyxin B per unit surface area of the immobilized fiber determined by amino acid analysis was 24 mg / m 2.
² (8 mg / g of carrier fiber). 56% of the polymyxin B used in the immobilization reaction is immobilized,
It can be said that the yield of polymyxin B is extremely high.

The endotoxin removal rate in the batch adsorption test was 95%. This high removal rate indicates that the polymyxin B-immobilized fiber can exert excellent specific adsorption performance on endotoxin in competitive adsorption of a plasma system rich in protein components.

Next, an ultrathin section of the polymyxin-immobilized fiber obtained by the freeze-drying method was prepared, stained with ruthenium oxide, and then transmitted through a transmission electron microscope (JEM-12 manufactured by JEOL Ltd.).
00EX), the cross section of the immobilized fiber was observed. The cross-sectional photograph is shown in FIG. In the vicinity of the surface, polymyxin B immobilized in a band with a width of about 5,000 angstroms was observed.

The amount of polymyxin immobilized (amino acid analysis) was measured by the following method.

The immobilized fiber was hydrolyzed by a conventional method, and the produced amino acid was coupled with phenylisothiocyanate. Then, the amount of the amino acid was quantified by high performance liquid chromatography to determine the amount of immobilized polymyxin.

The perimeter of the immobilized fiber was measured by a transmission electron micrograph of the cross section of the immobilized fiber, and the surface area of the immobilized fiber was calculated.

Using this and the amount of immobilized polymyxin determined by the amino acid analysis, the amount of immobilized polymyxin per unit surface area of the carrier fiber was calculated.

The endotoxin removal rate (batch adsorption test) was measured by the following method.

0.5 g (dry weight) of the immobilized fiber was weighed into an endotoxin-free dry heat sterilized (250 ° C. for 2 hours or more) 50 ml stoppered flask, and 15 ml of physiological saline was added thereto. Steam sterilization (115 ° C, 30
Min).

After the fiber was washed with distilled water for injection, bovine serum supplemented with endotoxin (E. coli 0111: B4W, lipopolysaccharide) (endotoxin concentration 10 n
(g / ml) and shaken at 37 ° C for 2 hours.

The bovine serum after shaking was used as a sample, diluted 10-fold with distilled water for injection, heated at 70 ° C. for 10 minutes, and the gelation time was measured using a toxinometer (manufactured by Wako Pure Chemical Industries, Ltd.).

The endotoxin concentration was determined from a calibration curve obtained in the same manner.

The endotoxin removal rate was calculated by {(initial concentration-analyte concentration) / initial concentration} × 100 (%).

Example 2 Polymyxin B sulfate concentration at the time of immobilization reaction was 0.07 k
A polymyxin B-immobilized fiber was synthesized in the same manner as in Example 1 except that g / m ³ was used.

The amount of immobilized polymyxin B per unit surface area of the immobilized fiber determined by amino acid analysis was 2 mg / m ^2.
(0.7 mg per 1 g of carrier fiber).

The endotoxin removal rate in the batch adsorption test was 90%. The amount of immobilized polymyxin B is slightly less than 1/10 that of Example 1, but the adsorption performance is not much different. Therefore, immobilized polymyxin B near the surface of the immobilized fiber of polymyxin B predominantly contributes to the expression of physiological activity. Suggests that Example 3 A colistin-immobilized fiber was synthesized in the same manner as in Example 1 except that colistin (polymyxin E) was used instead of polymyxin B.

The amount of colistin immobilized per unit surface area of the immobilized fiber determined by amino acid analysis was 24 mg / m 2.
² (8 mg / g of carrier fiber).

The endotoxin removal rate in the batch adsorption test was 98%.

[0047]

Industrial Applicability According to the present invention, a high activity, which can be widely used for therapeutic blood purifying agents, antibacterial agents, therapeutic imino modulators, adsorbents for affinity chromatography, etc.
A homogeneous and inexpensive molded article immobilized with a physiologically active substance could be provided.

[Brief description of the drawings]

FIG. 1 is a cross-sectional photograph of a polymyxin B-immobilized fiber of Example 1, taken with a transmission electron microscope.

──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Hisataka Koji 1-1-1 Sonoyama, Otsu-shi, Shiga Toray Co., Ltd. Examiner at Shiga Works Co., Ltd. Shunichi Yokoo (56) References JP-A-61-114734 (JP, A JP-A-61-135674 (JP, A) JP-A-62-191219 (JP, A) (58) Fields investigated (Int. Cl. ⁶ , DB name) A61M 1/00 C07K 17/08 CA (STN )

Claims (5)

(57) [Claims] As the physiologically active substance having a 1. A amino group, Po
Rehabilitative antibiotics, glycolytic antibiotics and
By immobilization reaction in the molded article itself at least one selected from amino click Bu glycosidic antibiotics, a process for producing a physiologically active substance fixed moldings, the physiologically active substance amount to be used for the immobilization reaction, moldings A method for producing a molded article on which a physiologically active substance is immobilized, comprising ² mg / m ² or more and 200 mg / m ² or less per unit surface area of a carrier. 2. The concentration of a physiologically active substance in an immobilization reaction solution containing a physiologically active substance having an amino group is 0.05 kg / m2.
³ or process according to claim 1, wherein the physiologically active substance fixed molded article, characterized in that it is 10 kg / m ³ or less. 3. The process for producing a molded article with a physiologically active substance immobilized thereon according to claim 1, wherein a base readily soluble in an immobilization reaction solution containing a physiologically active substance having an amino group is used as a fixing agent. . 4. The bioactive substance immobilization according to claim 1, wherein the pH of the immobilization reaction solution containing the bioactive substance having an amino group is controlled to a constant value in the range of 8 to 10. Manufacturing method of chemical molded products. 5. The method for producing a physiologically active substance-immobilized molded article according to claim 1, wherein the molded article is fibrous.