JPS5922557A - Endotoxin blood disease treating device - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a therapeutic device that completely adsorbs and removes endotoxin from blood, plasma, and its thin body fluid components. For more details,
It purifies and regenerates body fluids by effectively adsorbing and removing entodoxy, which is a fundamental factor associated with intractable shock associated with Durham-negative bacterial infections.
The present invention relates to a simple treatment device that prevents the progression of the disease, alleviates the symptoms, and further speeds up the healing process.

Endotoxemia, which is often seen in severe infections, hepatobiliary diseases, fulminant hepatitis, etc., is often accompanied by serious complications such as impaired consciousness, circulatory failure, and respiratory failure. For endotoxemia, steroids, trasylol, etc. have been administered to prevent shock, but there are still no therapeutic devices that directly detoxify endotoxins, and treatment is difficult.

Endotoxin is a lipopolysaccharide that is a component of the cell wall of Durham-negative bacteria. Lipid A plays a central role in the toxicity of endotoxin, and its general properties are that it has a hydrophobic lipid moiety and is poorly soluble in water. . In terms of molecular weight, it forms a macromolecular structure with tens to hundreds of blades, and the thicker the molecular weight, the stronger the endotoxin toxicity.

-! In addition, endotoxemia refers to a disease in which endotoxin is detected in a patient's body fluids by the Limurne method.
Examples include biliary tract infections.

Examples of non-infectious diseases include ileus and hemorrhagic shock.

The effect of endotoxin on living organisms is so great that it is known worldwide as endotoxemia. The biological reaction causes fever, hemorrhage, necrosis, shock, or lethal effects.

Conventionally, body fluid purification treatment devices have mainly been used as artificial livers for liver diseases, using activated carbon or activated carbon coated with a hydrophilic polymer.
．． have been used.

Recently, there has been an example of applying this artificial liver to the treatment of endotoxemia, but treatment devices based entirely on activated carbon have a low adsorption capacity to adsorb and remove endotoxins with the above-mentioned properties. Because activated carbon has a small pore size, it has the disadvantage that it cannot adsorb endotoxins, which are macromolecules, and currently there are no therapeutic devices that can directly detoxify endotoxins in body fluids.

In view of the current situation, the present inventors have developed the present invention as a result of intensive research in order to solve these drawbacks, provide a treatment device that can be more generally disseminated, and that purifies and regenerates body fluids such as endotoxemia. reached.

That is, the present inventors created a therapeutic device in which an organic low-molecular-weight compound containing aromatic gold was bonded to a hydrophilic insoluble carrier, and evaluated the binding property with endotoxin. However, surprisingly, it was discovered that this therapeutic device hardly adsorbs other useful ingredients and binds endotoxin with extremely high activity, leading to the completion of the present invention.

The present invention provides an endotoxemia treatment benefit in which an adsorbent comprising a hydrophilic insoluble carrier bound to an organic low-molecular-weight compound containing an aromatic ring is housed in a container having an inlet and outlet for body fluids. .

In the present invention, the hydrophilic insoluble carrier means a carrier having a surface having a contact angle with water of 60° or less, more preferably 30° or less.

Contact angle is the angle between the solid surface and the tangent line drawn to the droplet at the intersection of the surface of a water droplet placed on a solid surface and the solid surface, and is generally the angle that contains the liquid. Hydrophilicity can be determined by the following, and a small el means hydrophilicity.

A characteristic feature of the hydrophilic insoluble carrier is that it does not adsorb and remove useful components during extracorporeal circulation; for example, it has low adsorption of blood cell components such as platelets and white blood cells, and plasma component albumin. Examples of hydrophilic insoluble carriers include natural polymer carriers such as agarose, dextran, and cellulose, synthetic polymer carriers such as polyacrylamide and polyvinyl alcohol, and inorganic carriers such as glass, silica, and activated carbon. can give.

The shape of the hydrophilic insoluble carrier used in the present invention is as follows:
Any known shape such as particulate, fibrous, hollow fiber, or membrane may be used, but particulate is preferable in view of the large surface area of the carrier and ease of handling as an adsorbent.

For particulate carriers, the average particle size is important because if the particle size is too small, pressure loss will be large when flowing viscous liquids such as body fluids, and if the particle size is too large, the surface area of the carrier will decrease. The range is preferably 25 to 5000 μm, more preferably 75 to 1000 μm.

As a particulate carrier, since it has a large surface area,
Porous particles are preferred. The porous particles used in the present invention can immobilize an organic low-molecular compound containing an aromatic ring on their surfaces, and have an average pore diameter of 100A to 900A.
0A, preferably in the range of 400 to 2000A. If the average pore size is too small, the amount of endotoxin adsorbed will be small, and if it is too large, the particle strength will decrease, which is not preferable.

Since the hydrophilic insoluble carrier of the present invention is used for therapeutic purposes,
1. Greater safety and sterilization are required.

In general, sterilization methods for medical supplies and instruments include sterilization with chemical solutions such as Polmarine, gas sterilization with ethylene oxide gas, gamma ray sterilization, and high-pressure steam sterilization, but chemical sterilization can be used for a wide range of objects. However, there are other problems, such as the need for thorough cleaning before use and the difficulty of completely removing residual chemicals. The hydrophilic insoluble carrier used in the present invention can generally be sterilized with a chemical solution. Advantages of the present invention: For treatment devices that are used in direct contact with body fluids such as blood, from the viewpoint of safety, it is preferable to apply sterilization methods that have little drug residue, and sterilization using volatile gas that is easy to remove. Gas sterilization using oxide gas, gamma ray sterilization without using chemicals, and high-pressure steam sterilization are preferred. In order to perform gas sterilization, it is necessary that the porous structure of the carrier is not destroyed by drying and that it can be restored to its original state by rewetting. The carrier needs to be stable and thermally stable in order to perform autoclaving. Examples of carriers that meet these conditions include inorganic carriers such as porous glass and porous silica, and crosslinked synthetic polymer carriers.

Porous glass, porous silica, etc. have silanol groups on their surfaces, and are compatible with various silane coupling agents, making it possible to introduce various functional groups in the same manner as organic polymer carriers.

Hydrophilic crosslinked synthetic septa molecules can be synthesized by polymerizing monomers having hydrophilic groups or introducing hydrophilic groups through chemical reaction of polymers. It is also possible to synthesize both in combination. As the polymerization method, a radical polymerization method can be used. The crosslinking agent may be introduced through copolymerization during polymerization, or may be introduced through a chemical reaction between the polymers (between polymers or between the polymer and the crosslinking agent), or both may be used in combination.

- To give an example, it can be made by copolymerizing a vinyl monomer and a vinyl or allyl crosslinking agent. In this case, vinyl monomers include (meth)acrylates such as 2-hydroxyethyl acrylate and 2-hydroxyethyl methacrylate, vinyl esters of carboxylic acids such as vinyl acetate and vinyl propionate, methyl vinyl ether, ethyl vinyl ether, etc. Examples include vinyl ethers, ethylene carbonate and its rust conductor, acrylamide, acrylic acid, N-vinylpyrrolidone, and the like.

Examples of crosslinking agents include allyl compounds such as triallyl isocyanurate and triallyl cyanurate, di(meth)acrylates such as ethylene glycol dimethacrylate, diethylene glycol dimethacrylate, and pair p-tech IJ thritol dimethacrylate, and butane. Polyvinyl ethers such as diol divinyl ether, diethylene glycol divinyl ether, and tetravinylglyoxal, polyallyl ethers such as diarylidene pentaerythritol and tetraallyloxyethane, and glycidyl acrylates such as glycidyl methacrylate can be used. Furthermore, copolymerized comonomers with other comonomers can also be used, if necessary.

Among these hydrophilic crosslinked synthetic polymers, vinyl compounds having carboxylic acid vinyl esters and incyanurate rings (allyl compounds) are commonly used because of their ease of functional group introduction and chemical and physical stability. A carrier made of a crosslinked polymer mainly composed of vinyl alcohol units is preferred, such as a crosslinked polyvinyl alcohol triallyl in cyanurate obtained by polymerization and hydrolysis of a copolymer.

In the present invention, the organic low molecular compound containing an aromatic ring to be bonded to the hydrophilic insoluble carrier is an organic low molecular compound containing at least one aromatic ring and having a functional group capable of bonding to the hydrophilic insoluble carrier. be.

In the present invention, the organic low molecular compound is a compound with a molecular weight of 10,000 or less, preferably a compound with a molecular weight of 1,000 or less. This is sibrotiin A (molecular weight 42,000)
Compared to natural polymers such as, it is easier to handle during immobilization and to store after immobilization. Further, even when the substance is eluted from the insoluble carrier, a compound with a molecular weight of 10,000 or less has negligible antigenicity to living organisms, is safe, and can be easily sterilized.

In the present invention, the aromatic ring refers to a cyclic compound having aromatic properties, and includes an aromatic swollen hydrogen ring, an aromatic heterocycle, and those into which various substituents have been introduced. The introduction of substituents includes substituting other functional groups such as alkyl groups, amine groups, and hydroxyl groups in place of hydrogen bonded to carbon or nitrogen on the ring, as well as substituting the carbon atom adjacent to the nitrogen atom on the ring. Those in which an oxygen atom is bonded through a nine double bond to form a carbonyl group are also included because the aromaticity of monkey is maintained. Aromatic rings are strongly hydrophobic, and delocalized π electrons exist on the rings, which are presumed to interact strongly and adsorb endotoxins.

Aromatic fjl consisting of hydrocarbons include, in addition to monocyclic benzene, condensed ring indene, naphthalene, fluorene, phenanthrene, and anthracenato.

Examples of aromatic heterocycles include those with one type of heteroatom, oxygen-containing heterocycles include furan, benzofuran, etc., sulfur-containing heterocycles include thiophene, benzothiophere, etc. Nitrogen-containing heterocycles include monocyclic rings such as virol and pyridine, fused rings such as quinoline, isoquinoline, indole, carbazole, phenanthridine acridine, indolizine, and those containing two nitrogen atoms. Monocycles include pyrazole, imidazole, pyridazine, pyrimidine, and pyrazine; condensed rings include cinnoline, phthalazine, quinazoline, quinoxaline, indazole, benzimidazole, phenanthroline, phenazine, naphthyridine, and carboline; and monocycles containing three or more desired atoms. ijl, 2.5-t') azole, 1,2.4-) lyazole, tetrazole, 1.3.5-triazine, 1,2,4.5-tetrazine, fused rings include purine, pteridine, etc. It will be done.

Those containing two types of heteroatoms include oxazole, isothiazole, penzoxazole, oxadiazole containing oxygen and nitrogen, thiazole containing sulfur and nitrogen, isothiazole, benzothiazole, thiadiazole, and the like. Since safety is important in therapeutic equipment such as the extracorporeal circulation system of the present invention, the organic low-molecular-weight compound containing an aromatic ring is preferably an aromatic amino acid, a nucleic acid base, etc., which are biological constituents. As, tryptophan,
Phenylalanine, tyrosine, histidine, adenine,
Examples include cytosine, thymine, uracil, guanine and their derivatives.

In the present invention, the organic low-molecular-weight compound containing an aromatic ring can be immobilized on the surface of a hydrophilic insoluble carrier by any method such as covalent bonding, ionic bonding, physical adsorption, embedding, or precipitation insolubilization on the surface of the hydrophilic insoluble carrier. Although known methods can be used, in consideration of elution properties, thermal stability, etc. of the bound ligand, it is preferable to immobilize the bound ligand using a covalent bond.

As the immobilization method, an immobilized enzyme, a known carrier activation method and immobilization method used in affinity chromatography can be used.

Examples of the activation method include a cyanogen halide method, a triazine halide method, a bromoacetyl bromide method, an epoxide activation method using epihalohydrin, bisepoxide, and the like.

Among these methods, 2-
Preferred are epoxide activation methods in which bonds containing hydroxytrimethylene groups are formed.

Epoxide derivatives react between their epoxy groups and hydroxyl, amine, and thiol groups to form chemical bonds with the 2-hydroxytrimethylene group through ether bonds, alkylamine bonds, and thioether bonds, respectively.
This reaction proceeds under mild conditions without side reactions, and the bonds produced are extremely stable chemically and thermally stable bonds. In addition, the epoxy group is stable even in water, does not generate intense heat during reaction, and can be easily handled in large quantities industrially. Furthermore, when this epoxy group is decomposed in water, it becomes a glycol derivative, which is safe and has low toxicity in terms of writing properties.

Since the 2-hydroxytrimethylene group formed by the reaction of the methyloxirane group is a hydrophilic group containing a hydroxyl group, non-specific adsorption of proteins to this group is less likely to be a problem.

Such methyloxysilane-based binders include epihalohydrin typified by epichlorohydrin, glycidyl acrylate or methacrylate,
Examples of bisepoxides include γ-glycidoxypropyltrimethoxysilane and bisepoxides, such as 1,2-bis-(2,3-epoxypropoxy)-ethane, 1,4-bisepoxide, and bisepoxides. Examples include -(2,3-epoxypropoxy)-butane, 1,6-bis-(2,5-epoxypropoxy-hexane, etc.) Examples of bonding sites between a carrier containing a 2-hydroxytrimethylene group and a ligand In addition to the above-mentioned forms in which the epoxy binder is directly bound to the carrier and the ligand, there are also methods in which the carrier is bound to an epoxy binder, such as epichlorohydrin, and then a polyfunctional compound such as phloroglucinol is bound to the carrier. The carrier and the ligand may be bound via a spacer, such as by binding a compound such as divinyl sulfone having a group capable of covalently bonding to the ligand, and then binding the ligand.

Among these epoxy-based binders, shrimp/rohydrin, which has an asymmetric structure, has a large difference in reactivity between its two functional groups, so when the carrier and binder react, It is more preferable because it is less likely to cause a crosslinking reaction due to the reaction of two functional groups.

In the present invention, the amount of the organic low molecular weight compound containing aromatic iJ bound to the hydrophilic insoluble carrier is 1 fnt of the hydrophilic insoluble carrier.
This ranges from 0.11n9 to 10Qm9.

More preferably, it is in the range of 0.5 to 50.

When the retained amount falls below o, 1mg7=@, the adsorption amount of endotoxin decreases extremely, and when it exceeds 100 da/-,
Adsorption of useful substances such as albumin occurs, which is undesirable.

The endotoxemia treatment device of the present invention is made by filling a container having an inlet and outlet for body fluids with an adsorbent to which a low-molecular-weight organic compound containing an aromatic ring as described above is bonded.

In the drawings, reference numeral 1 shows an example of the endotoxemia treatment device of the present invention, which has a body fluid inlet 5 at one end opening of a cylinder 2 via a hook 4 with a filter 3 stretched inside. A backing 4' with a cap 6 fitted with a screw and a filter 3' stretched inside the opening at the other end of the cylinder 2.
A cap 8f having a body fluid lead-out lower part via metal: This cap is screw-fitted to form the entire container, and the gap between the filters 3 and 3' is filled and held with an adsorbent to form the entire adsorption layer 9.

The volume of the adsorbent layer 9 is preferably 10 to 1000 ml, considering the adsorption capacity and priming volume.
50 to 500- is more preferred.

The shape of the treatment device may be cylindrical or box-shaped, but a cylindrical shape is preferred in order to obtain a uniform flow. The value of the length of the treatment device (L)/diameter of the treatment device (D) is:
In terms of pressure loss, it is preferably 10 or less.

The quality of the treatment device only needs to be safe, and the material does not matter.
Considering sterilization, etc., polycarbonate and polypropylene are preferred.

When using the therapeutic device of the present invention in extracorporeal circulation, there are two methods as follows: For one thing, blood taken from the body is centrifuged using a centrifugal N# machine or a model plasma separator.
After separating the plasma components and blood cell components, the plasma components are passed through the treatment device, purified, and then returned to the body together with the blood cell components.The other method is to use blood taken from the body. This is a method for purifying the treatment device by passing it directly through the treatment device.

- In addition, regarding the passage speed of blood or plasma, the adsorption efficiency of the treatment device is very high, so the particle size of the adsorbent can be adjusted to any size, and the degree of filling can be lowered, so the shape of the adsorbent layer can be changed. A high passing speed can be provided regardless of the situation. Therefore, a large amount of body fluid can be treated.

The method for passing body fluids may be either continuous or intermittent, depending on clinical needs or equipment conditions.

As described above, the therapeutic device of the present invention adsorbs and removes endotoxin in body fluids at a high rate, hardly adsorbs other useful components, is extremely compact, and at the same time,
It is simple and safe. It also has the advantage that sterilization operations can be carried out easily and reliably.

The present invention is applicable to the general use of purifying and regenerating body fluids such as blood, and is effective in treating pathological conditions such as endotoxemia associated with Durham-negative bacterial infections.

Hereinafter, embodiments of the present invention will be explained in detail using examples.

Example 1 Vinyl acetate and triallyl isocyanurate were subjected to suspension polymerization and then saponified to obtain a crosslinked polyvinyl alcohol gel. The obtained particles were decomposed and particles with a particle size of 75μ to 210μ were collected and used as a carrier. Average particle size is 150μ
Met.

The carrier was reacted with epichlorohydrin in dimethyl sulfoxide to obtain an epoxy group-bonded gel.

Using the epoxy group-bound gel, an organic low-molecular-weight compound containing an aromatic ring was bound as a ligand to prepare a complete adsorbent. Adenine, -tryptophan, and -histidine were used as organic low-molecular-weight compounds containing aromatic rings, and were dissolved in 1 pH 9 and 8 carbonate buffer, respectively. o 5
It was dissolved to a concentration of mat/l. The epoxy binding gel each 200m/! Ic each gund solution at 30 o
rRA' was added and reacted at 50°C for 16 hours to bond, and then the excess epoxy group was removed by tris(hydroxymethyl).
Blocking was performed by reacting with a solution of 0.1 mot/aminomethane at 50° C. for 5 hours.

Ligand retention at 260 nm for adenine.

For t-tryptophan, the wavelength was 280 nm, and for t-histidine, diazotized sulfanilic acid was reacted with histidine in alkaline conditions using the Boli method, and 510 nm was used for t-histidine.
As a result of determining the absorbance of m, 1-tryptophan is 53μ
mot/W11. , t-histidine is 55μmOL/m
1, adenine was retained in an amount of 4 μmot/−.

The adsorption experiment was carried out using 1ml of human plasma KB-coli UKT-B.
Endotoxin purified from the cultured bacterial cells of the strain was mixed at a ratio of 1r:1μg, and the inner diameter was 2Cr filled with a 10-mm adsorbent.
Endotoxin-containing plasma was passed through a cylindrical treatment device with a length of 3 crn at a rate of 1 to 7 minutes, and all the plasma was collected at the outlet of the treatment device, and the adsorption percentage of endotoxin was calculated.

A similar experiment was also conducted using a treatment device filled with activated carbon as a control. Note that endotoxin was detected using a Limulus test (Limulus test, manufactured by Wako (OL1 Kojunraku Kogyo Co., Ltd.)).

The method for detecting endotoxin in blood requires the removal of inhibitors from plasma, and a chloroporm treatment method was used for this removal method. That is, 1.0 ml of plasma was mixed with 0.2 me of chloroform, shaken with a mixer for 1 hour, and stirred for 2 hours.
Centrifugation was performed at 500 rpm for 10 minutes, the cloudy middle layer was collected with a pipette, and as much chloroform as possible was removed in a 37°C water bath to prepare a sample.

As a result, the plasma that had passed through the activated charcoal contained endotoxin (1), the same as the plasma before passage, but adenine and
-Tryptophan and t-histidine are identified in the plasma that passes through the treatment device, which is endotoxin (to), and there is little decrease in albumin, which is a useful component, and this treatment device effectively adsorbs and removes endotoxin at a high rate. was clear.

Example 2 Adsorption month 100m/! prepared in Example 1! i, inner diameter 4an
A cylindrical (L/D: 2) treatment device with a length of 8α was filled with the mixture, sterilized with high-pressure steam, and a dog weighing 20 kl/kg was placed under anesthesia.
IJum injection solution was continuously added to allow blood to pass through the treatment device. At this time, endotoxin Ec
Purified from cultured cells of oliUKT-B strain (z,
It is added at a rate of 1011t7kfJ, and the blood flow rate is 110
After circulating at 0 rn/min for 1 hour, blood was sampled and the amount of endotoxin in the blood was measured.

As a result, no abnormalities were observed during extracorporeal circulation in the royal palace, and there was a small decrease in white blood cells and platelets before and after circulation.

The amount of endotoxin is 7-) A therapeutic device filled with an adsorbent that completely fixes liptophan, t~histidine, and adenine shows a positive result before circulation, but a negative result after circulation. It was found that low-molecular-weight compounds adsorb and remove endotoxin more efficiently than from blood.

Example 6 Silica gel 1001 with an average pore diameter of 4ooX and a surface area of 1507777/r was immersed in a 20% solution of γ-glycidoxypropyltrimethoxysilane (acetone solvent) and heated at 50°C.
The mixture was allowed to react with shaking for 60 hours. After the reaction, the mixture was washed with 500 ml of acetone, washed with water, replaced with 0.1M sodium carbonate buffer, and dehydrated by suction to obtain an epoxy group-bound gel. Using the epoxy group-bound gel of the gel, an organic low molecular compound containing an aromatic ring is bound as a ligand,
All adsorbents were prepared. Using t-tryptophan, t-phenylalanine, and 5-aminouracil as ligands, o and o were used in carbonate buffer of 1 pH 9 and 8, respectively.
Suspended at a ratio of s mot/L. Add 30 mL of each ligand solution to the epoxy binding gel 20 mL, perform immobilization reaction at 50°C for 20 hours, and then 60 m
Q/ml of tris(hydroxyethyl)aminomethane solution (5ml) was added, and a blocking reaction was further carried out for 50'C5 hours with stirring.

Ligand retention f is 1.4-) Ributophane is 280 nm
,, L-phenylalanine was determined and calculated based on absorbance at 258 nm, and 5-aminouracil at 260 nm.

According to it, -tryptophan is 42 μmol/
ml, l-phenylalanine is 351 unot/me
An adsorbent with a retention amount of 28 μmol/me of 5-aminouracil was obtained. This adsorption interest, inner diameter 2crn
.

A total of 1 ton of endotoxin from the E-cali UKT-B strain was added to human plasma in a treatment device filled in a cylindrical container with a length of 6C1n.
Endotoxin-added plasma total 1 added to ne at a ratio of 1 μ2
．． The liquid was passed at a rate of 5171/min.

All plasma was collected at the outlet of the treatment device, and endotoxin and A'l Mff: were discharged. In addition, endotoxin measurement Halimulus test [Limulus test, Wako]
According to

As a result, in all treatment devices, albumin adsorption was slight, and other useful components were also reduced to a small extent. Engineering? While todoxin was positive before adsorption, endotoxin was negative after passing through each treatment device.

[Brief explanation of drawings]

The drawing is a sectional view showing an example of the endotoxemia treatment device of the present invention. 1... Endotoxemia treatment device 2.
......Cylinder 6.5'...Filter 4.4'...Backing 5...
・・・・・・Body fluid inlet 6・・・・・・Cap 7・・・・・・Body fluid outlet 8・・・・・・
···cap?・・・・・・・・・Adsorbent

Claims (1)

[Claims] 1. An adsorbent comprising an organic low-molecular compound containing aromatic 311 bound to a hydrophilic insoluble carrier is capable of transporting body fluids in and out D 7j
f: An endotoxemia treatment device, characterized in that it is housed in a container having: 2. The endotoxemia treatment device according to claim 1, wherein the hydrophilic insoluble carrier is a hydrophilic crosslinked synthetic polymer carrier. 3. The device for treating endotoxemia according to claim 1, wherein the hydrophilic insoluble carrier is a hydrophilic inorganic carrier. 4. The endotoxemia treatment device according to claim 2, wherein the hydrophilic crosslinked synthetic polymer carrier is a carrier consisting of a crosslinked polymer whose main constituent is vinyl alcohol units. 5. Adsorption in which an organic low-molecular compound containing an aromatic ring is bonded to a carrier consisting of a crosslinked polymer whose main constituent is a vinyl alcohol unit via a bond containing a 2-hydroxytrimethylene group as a constituent. 1. An endotoxemia treatment device, characterized in that the material is housed in a container having an inlet and outlet for body fluids.