JPH1085330A - Material for removing or detoxificating pyrogen - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a material having a less affinity to useful constituents contained in blood, medicines and the like, and having an affinity to bacterial pyrogen of protein, such as staphylococcus aureus exotoxin and so forth, but not limiting to endotoxin, by containing urea or thiourea bond. SOLUTION: The substance to remove or detoxificate pyrogen that is produced by Gram-negative or Gram-positive bacteria, is manufactured from a material containing urea or thiourea bond. In this case, as a substittion group to be joined with urea or thiourea bond, such aliphatic compounds as hexyl group, octyl group, dodecyl group and the like, and such alicyclic compounds as cyclohexane, cyclopentane and the like, are used. Most preferably, however, are those having compounds both with aromatic family compounds and hydroxyl or amino group as a group substituting for urea or thiourea bond.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a material which has an affinity for pyrogen produced by Gram-negative or Gram-positive bacteria and removes or detoxifies pyrogen. In particular, as a drug that loses the toxin activity of pyrogen by binding to pyrogen present in high-concentration protein solutions such as human blood (detoxification), as a purification column for removing pyrogen or as a wound-coating material, or to detect pyrogen Alternatively, it is suitably used as a measurement material to be quantified.

[0002]

2. Description of the Related Art Pyrogen is a generic term for a substance having a pyrogenic property, and when contained in a drug or a filling solution for artificial organs, it may cause a patient to generate heat. It is also known that in a bacterial infection, pyrogen infiltrates into the body and causes fever and shock symptoms.
Bacteria are the most pyrogenic and problematic pyrogens, and lipopolysaccharide (LPS) is the most pyrogenic. LPS is a phospholipid that is a component of the cell wall of Gram-negative bacteria, and is sometimes referred to as endotoxin. Endoxin is a stable substance, and cannot be detoxified by ordinary sterilization operations when mixed with pharmaceuticals and the like. Heat detoxification requires heating to 250 ° C. or higher, and is not applicable to endotoxin mixed in medicines, blood, artificial organs and the like.

In addition to endotoxins, bacterial pyrogens such as exotoxins of Staphylococcus aureus (enterotoxin A, B, C or toxic shock syndrome toxin-1) are known. These pyrogens, unlike endotoxins, are protein exotoxins secreted by bacteria and are known to cause fever when administered to rabbits. Unlike endotoxin, it is possible to detoxify by usual sterilization operation by heating, but since the heating operation denatures medicines and blood itself, it cannot be applied to exotoxin mixed in these.

[0004] Endotoxin mixed in a dialysate or the like can be filtered by a reverse osmosis membrane or an ultrafiltration membrane to remove pyrogen, but is difficult to adapt to blood or protein preparations.

On the other hand, as a method for removing pyrogens, particularly endotoxin, from medicines or blood by adsorption, beads obtained by reacting hexamethylene diisocyanate with fibers or cross-linked agarose beads on which polymyxin B which is an antibiotic is immobilized (Japanese Patent Laid-Open No. 4-114661) or a material on which histidine or a derivative thereof is immobilized is known. However, in the case of polymyxin B-immobilized fiber, there are problems that the polymyxin B to be immobilized is expensive, and that the histidine-immobilized material has insufficient adsorption performance. Further, there is a problem that none of the adsorbents has adsorbability to pyrogens other than endotoxin.

[0006] Further, a selective adsorption material for exotoxins of Staphylococcus aureus and Streptococcus has not been known so far.

[0007]

DISCLOSURE OF THE INVENTION The present invention is intended to solve these disadvantages of the prior art, and is inexpensive and sterilizable, and has a low affinity for useful components contained in blood, pharmaceuticals and the like. Further, it is an object of the present invention to provide a material having an affinity not only for endotoxin but also for proteinaceous bacterial pyrogen such as Staphylococcus aureus exotoxin. That is, since the material of the present invention has a high affinity for bacterial pyrogens, it binds to pyrogens present in filling fluids such as pharmaceuticals and artificial organs, or body fluids such as blood and urine. The toxin activity of pyrogen can be lost (detoxification) by, for example, shielding the binding site to the in vivo receptor. Therefore, according to the present invention, it is possible to treat sepsis and the like when used as a medicine, and when the material is water-insoluble, it can be used for blood, urine, bodily fluids such as exudate from wound sites, medicines and artificial organs. It is an object of the present invention to provide a material capable of removing pyrogen from the filling liquid of the above.

[0008]

Means for Solving the Problems As a result of intensive studies, the present inventors have found that a material containing a urea bond or a thiourea bond is
The present inventors have found that they have an affinity for pyrogens such as endotoxin and Staphylococcus aureus exotoxin, and have reached the present invention. The present invention has the following configuration.

"(1) A material having an affinity for pyrogen, characterized by containing a urea bond or a thiourea bond.

"(2) A body fluid purification column using the material having an affinity for pyrogen according to the above item 1.

"(3) A wound dressing using a material having an affinity for pyrogen as described in 1 above."

[0012]

DESCRIPTION OF THE PREFERRED EMBODIMENTS In the present invention, there is no particular limitation on the substituent which binds to a urea bond or a thiourea bond. Aliphatic compounds such as hexyl, octyl and dodecyl groups, and cyclohexane and cyclopentane can be used. Alicyclic compounds, more preferably aromatic compounds such as phenyl, diphenylmethyl and naphthyl groups are used. Also,
Aminohexyl group, N-methylaminohexyl group, N,
N-dimethylaminohexyl, aminooctyl, aminododecyl, and derivatives such as tolyl, chlorophenyl, nitrophenyl, and aminodiphenylmethyl are also preferably used. Further, sugars such as monosaccharides such as glucose, glucosamine, galactosamine, maltose, cellobiose, agarose, sucrose, cellulose, chitin and chitosan, oligosaccharides and polysaccharides, and derivatives thereof are also preferably used. Most preferably, it has both an aromatic compound and a compound having a hydroxyl group or an amino group as substituents of a urea bond or a thiourea bond.

The present invention may be any of a monomer, an oligomer and a polymer, and those in which the above-mentioned substituent or a part thereof is polymerized are also included in the material of the present invention. That is, as the above substituents or a part thereof, nylon, polymethyl methacrylate, polysulfone, polystyrene, polyethylene, polyvinyl alcohol, polytetrafluoroethylene, polyallylamine, polyvinylamine, synthetic polymers such as polyethyleneimine,
A repeating unit such as a natural polymer containing cellulose, collagen, chitin, chitosan and a derivative thereof is preferably used. Further, a material obtained by coating an inorganic material such as metal, ceramics, and glass with a suitable polymer is also preferably used.

The material of the present invention can be synthesized by a generally known method. For example, when a urea bond or a thiourea bond is introduced into an aliphatic compound or an aromatic compound, a method of reacting an isocyanate derivative or an isothiocyanate derivative with an amine can be used. When a urea bond or a thiourea bond is introduced into a saccharide, a saccharide having an amino group such as chitosan or glucosamine can be reacted with an isocyanate derivative or an isothiocyanate derivative. In the case of a saccharide having no amino group such as cellulose, the hydroxyl group of the saccharide is activated using epichlorohydrin, tresyl chloride, etc., and then reacted with ammonia or diaminoethane to introduce an amino group. Utilizing this amino group, a urea bond or a thiourea bond can be introduced into the saccharide.

When the material of the present invention is an oligomer or a polymer, for example, an oligomer or a polymer having an active ester group of a carboxylic acid such as an isocyanate group, a carboxyl group or a succinimide group may be added to an amino or urea derivative or a thiourea derivative. A method of reacting a group is preferably used. As the amino group used for the reaction, the amino group at the terminal of the urea bond or the thiourea bond is low in reactivity, and therefore, an amino group present at other sites is preferable. Usually, 1 to 5 mol of amino group is preferably used per 1 mol of active ester group contained in the oligomer or polymer. Further, an oligomer having an amino group, a polymer, or an oligomer having an amino group introduced by diaminoethane or the like, phenyl isocyanate, tolyl isocyanate, chlorophenyl isocyanate, naphthyl isocyanate,
It is also a preferable method to react an organic isocyanate such as phenylisothiocyanate and chlorophenylisothiocyanate, or an organic isothiocyanate. Normal,
An organic isocyanate or an organic isothiocyanate is preferably used in an amount of 1 to 5 mol per 1 mol of the amino group contained in the oligomer or the polymer. Functional groups such as an active ester group of a carboxylic acid such as an isocyanate group, an isothiocyanate group, a carboxyl group, and a succinimide group and an amino group can be introduced into an oligomer or a polymer, if necessary.

All the above-mentioned reactions are normally carried out at a reaction temperature of 0 to 150 ° C. and a reaction time of 0.1 to 24 hours, but are not limited thereto. Although a reaction solvent is not always necessary, the reaction is generally performed in the presence of a solvent. Solvents that can be used include methanol, ethanol, isopropanol, n-butanol, hexane, acetone,
Aliphatic hydrocarbons such as N, N dimethylformamide and dimethyl sulfoxide; aromatic hydrocarbons such as benzene, toluene and xylene; dichloromethane, chloroform;
Examples include halogenated hydrocarbons such as chlorobenzene, and ethers such as diethyl ether, tetrahydrofuran, and dioxane. After completion of the reaction, the reaction solution can be purified by an operation such as column chromatography or recrystallization, if necessary, after usual treatment such as filtration and concentration. In the case of a water-insoluble material, washing with a glass filter or the like is also a preferable method.

Since the material of the present invention has an affinity for pyrogen, for example, pyrogen can be removed or detoxified from blood, medicine, or a filling solution for artificial organs by adsorbing pyrogen. It can also be used as a pharmaceutical. In particular, water-insoluble ones include superantigen removal columns, wound dressings,
Alternatively, it can be used as a material for measuring superantigen. For example, utilizing a part of the substituents of a urea bond or a thiourea bond, a synthetic polymer such as polysulfone,
It can be immobilized on a natural polymer such as cross-linked chitosan or the like and made insoluble in water. The shape is not particularly limited, but when used as a column, beads, fibers, hollow fibers, and woven fabrics are preferable, and when used for immunological measurement, the shapes of beads, plates, tubes, and the like are used. In the case of a wound covering material, a shape such as a woven fabric or a film is preferred. For example, crosslinked chitosan beads with urea or thiourea bonds introduced
It is preferably used as a pyrogen removal column or a material for measuring superantigen. Further, those obtained by introducing a urea bond or a thiourea bond into a polystyrene fiber modified with cellulose or a polyamine compound are preferably used as a wound dressing.

Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited to the examples.

[0019]

【Example】

Example 1 Introduction of urea bond to polystyrene fiber and removal of pyrogen by adsorption A sea component (a mixture of polystyrene at a ratio of 46 by weight and a polypropylene at a ratio of 4) at a weight ratio of 50 and an island component (polypropylene) at a weight ratio of 50. USP 4,661,26
50 g of N-methylol-α-chloroacetamide, 400 g of nitrobenzene, 400 g of 50 g of the sea-island composite fiber (thickness: 2.6 denier, number of islands: 16) described in No. 0
In 98% sulfuric acid and 0.85 g of paraformaldehyde, and reacted at 20 ° C. for 1 hour. The fiber was taken out of the reaction solution, poured into 5 L of ice water at 0 ° C. to stop the reaction, washed with water, and then nitrobenzene adhering to the fiber was extracted and removed with methanol. This fiber was vacuum dried at 50 ° C. to obtain 71 g of chloroacetamidomethylated crosslinked polystyrene fiber (hereinafter abbreviated as AMPSt fiber).

10 g of diaminohydroxypropane is mixed with 500 ml of dimethyl sulfoxide (hereinafter abbreviated as DMSO).
Was dissolved. To this solution, 20 g of AMPSt fiber (corresponding to a chloro content of 20 mmol) was added with stirring. The reaction was performed at 25 ° C. for 6 hours. Thereafter, the AMPSt fiber was washed on a glass filter with 500 ml of DMSO and subsequently with 50 ml of dimethylformamide (hereinafter abbreviated as DMF). After washing, 50 ml of DMF in which isocyanate or isothiocyanate derivative shown in Table 1 is dissolved
1 g of each AMPSt fiber was added to the above solution. The reaction was performed at 25 ° C. for 1 hour. Then, it was washed with 200 ml of DMF and distilled water on a glass filter.

[0021]

[Table 1] As a pyrogen to be adsorbed, E. coli as an endotoxin derived from Gram-negative bacteria. coli0111B4W
And Staphylococcus aureus enterotoxin B as an exotoxin derived from Gram-positive bacteria. In the adsorption test, a concentration of 1 ng each of pyrogen was added to 10 ml of phosphate buffered saline (hereinafter abbreviated as PBS) containing 5 mg / ml of bovine serum albumin.
of the adsorbents (a) to (c) prepared above, and shaken at 37 ° C. for 60 minutes. The pyrogen concentration in the solution after the reaction for 60 minutes was measured for endotoxin using an endotoxin-specific turbidimetric time analysis method, and for enterotoxin B using an enzyme immunological method. As a control, unreacted AMPSt fiber was used (d). As shown in Table 2 below, it was revealed that polystyrene fibers having a urea derivative in the side chain are materials having an affinity for pyrogen that can adsorb not only endotoxin but also exotoxin of S. aureus. .

[0022]

[Table 2] Example 2 Changes in exothermicity and toxicity due to adsorption The same experiment as in Example 1 was performed on pyrogen in physiological saline. AM containing a pyrogen solution 2 ml (containing 1 μg / ml endotoxin and 10 μg / ml toxic shock syndrome toxin-1) and a urea derivative
The reaction with 0.25 g of PSt fiber ((b) of the example) was 3
The reaction was performed at 7 ° C. for 60 minutes, and the solution after the reaction was administered to rabbits.
Administration was performed by subcutaneous sustained release using an osmotic pump (manufactured by alza). When the body temperature of the rabbit was measured from the rectum, the body temperature rise was small in the pyrogen solution reacted with the fiber into which the urea derivative was introduced, and it survived for 7 days. He died three days later.

Thus, it has been shown that by treating a solution containing a pyrogen with a material into which a urea derivative has been introduced, it is possible to detoxify pyrogenicity and lethal activity.

[0024]

The pyrogen-adsorbing or detoxifying material of the present invention has an affinity not only for endotoxin but also for proteinaceous pyrogen such as Staphylococcus aureus exotoxin. Further, unlike an adsorption material of a type in which an antibiotic or another physiologically active substance is immobilized as a ligand, no damage is caused by desorption of the ligand.

Therefore, by using the material of the present invention, it can be used as a material for removing pyrogen from a heat-unstable solution such as a drug or a filling solution for artificial organs, or for removing or detoxifying pyrogen in patient blood. This has made it possible to use it as an effective material for alleviating fever and shock symptoms seen in sepsis caused by Gram-negative or Gram-positive bacteria.

Claims (7)

[Claims] 1. A material for removing or detoxifying a pyrogen, which has a urea bond or a thiourea bond. 2. The material for removing or detoxifying a pyrogen according to claim 1, which has a functional group containing an aromatic ring. 3. The material for removing or detoxifying a pyrogen according to claim 1, which has a functional group containing a hydroxyl group. 4. The material for removing or detoxifying a pyrogen according to claim 1, which has a functional group containing an amino group. 5. The method according to claim 1, wherein the composition is water-insoluble.
5. The material according to any one of items 1 to 4. 6. A body fluid purifying column using the material according to claim 5. 7. A wound covering material using the material according to claim 5.