JP2002035117A - Column for treating inflammatory disease - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a safe column for treating inflammatory disease which can efficiently treat inflammatory diseases by selectively adsorbing an inflammatory protein, such as the CRP, SAA, etc., and an inflammatory leukocyte directly from blood with high efficiency and, in addition, does not cause pressure reduction shocks. SOLUTION: This column for treating inflammatory disease is filled up with an inflammatory agent adsorbent which can remove both an inflammation responsive protein and an inflammation responsive leukocyte. The inflammatory agent adsorbing material is composed of a water-insoluble polymer prepared by bonding N-substituted poly(alkylene imine) residues to side chains.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a column for treating an inflammatory disease.

[0002]

2. Description of the Related Art Ischemic reperfusion injury of organs, vascular stenosis, ulcerative colitis, Crohn's disease, rheumatism, diabetic nephropathy, SI
In inflammatory diseases such as RS (systemic inflammatory response syndrome; hereinafter simply referred to as SIRS) and infectious diseases, CRP (C
It has long been known that inflammatory response proteins such as reactive proteins (hereinafter simply referred to as CRP) and inflammatory leukocytes such as granulocytes increase. Recently, CRP specifically binds to neutrophils (J. Immunol., Vol 136, 2202-2207 (1986)).
Therefore, it has been said that neutrophil activation is suppressed, but CRP specifically increases the production of reactive oxygen species of neutrophils and monocytes stimulated with immunoglobulin G (J. Leukocyte Biol., Vo
l 52, 449-455 (1992)), and its significance was uncertain whether it was bad or good.

However, it has recently been confirmed that CRP, in concert with complement, binds to damaged cells and tissues and activates neutrophils and monocytes to spread the damage (J. Exp. . Me
d, vol 190, 1733-1739 (1999)). In the case of infectious disease or myocardial infarction, the blood concentration rapidly rises, arteriosclerosis is high in dialysis patients, it is high when rheumatoid arthritis patients worsen, and SAA (serum amyloid protein A; below Simply referred to as SAA) correlates with worsening Crohn's disease (Hepato-Gastroenterology, vol 44, 90-107 (1997)).
In combination with the above facts, inflammatory proteins and inflammatory leukocytes have the property of strengthening each other, so it is considered better to exclude them at the same time.

An adsorbent having the property of adsorbing CRP is immobilized on dextran sulfate-immobilized cellulose gel which is an adsorbent for low-density lipoprotein (Japanese Patent Laid-Open No. 1-2804).
No. 69) A column is known, but it is intended for treating plasma and does not adsorb inflammatory leukocytes. In addition, when such an adsorbent having a functional group is circulated extracorporeally, it has the disadvantage of activating bradykinin and causing hypotensive shock.

[0005] As described above, there is no known protein which can simultaneously remove inflammatory proteins and inflammatory leukocytes and can be used for extracorporeal circulation treatment.

[0006]

SUMMARY OF THE INVENTION In view of the background of the prior art, the present invention has been made to address CRP and SAA directly from blood.
It is an object of the present invention to provide a column for the treatment of inflammatory diseases, which is capable of selectively adsorbing inflammatory proteins and inflammatory leukocytes with high efficiency, efficiently treating the inflammatory protein and preventing the occurrence of hypotensive shock.

[0007]

The present invention employs the following means in order to solve the above-mentioned problems. That is, the column for treating inflammatory diseases of the present invention is a column filled with an inflammatory substance adsorbent capable of removing both inflammatory response proteins and inflammatory leukocytes in blood,
As the inflammatory substance adsorbent, a water-insoluble polymer having an N-substituted poly (alkyleneimine) residue bonded to a side chain is used.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention has been made to solve the above-mentioned problem, that is, to selectively and efficiently adsorb inflammatory proteins such as CRP and SAA and inflammatory leukocytes directly from blood, thereby treating efficiently and Investigating a column for the treatment of inflammatory diseases that does not cause a hypotensive shock, and using a water-insoluble polymer bound to a specific nitrogen-substituted residue as an adsorbent for inflammatory substances. It was determined that both inflammatory response proteins and inflammatory leukocytes could be removed.

The inflammatory response protein referred to in the present invention means C-reactive protein or serum amyloid protein A.

The inflammatory leukocytes referred to in the present invention mean granulocytes or monocytes and activated lymphocytes.

The inflammatory substance-adsorbing material of the present invention capable of removing both the inflammatory response protein and inflammatory leukocytes is obtained by bonding an N-substituted poly (alkylimine) residue to a water-insoluble polymer by means such as grafting. Can be provided.

The water-insoluble polymer is not particularly limited as long as it is insoluble in water and can immobilize an N-substituted poly (alkyleneimine) by a covalent bond. Examples thereof include aromatic polyvinyl compounds and polysulfone-based polymers. , Polyetherimide, polyimide, polyamide, polyether, polyphenylene sulfide and the like, and a water-insoluble polymer having a reactive functional group capable of immobilizing an N-alkylated polyalkyleneimine by a covalent bond.

As such a water-insoluble polymer, particularly,
Lisulfone-based polymers can be preferably used,
For example,-｛(p-C₆H_Four) -SO_Two− (P−C₆H
_Four) -O-poly (p-phenylene ethers)
Rufon) unit or-｛(p-C₆H_Four) -SO_Two− (P
-C₆H_Four) -O- (p-C₆H_Four) -C (CH_Three) _Two
− (P−C₆H_Four) -O｝-,-｛(p-C₆H_Four)-
SO_Two− (P−C₆H _Four) -O- (p-C₆H_Four)-
O｝-,-｛(p-C₆H_Four) -SO_Two− (P−C₆H
_Four) -O- (p-C₆H_Four) -C (CF_Three)_Two− (P−
C₆H_Four) -U｝ -polyether sulfone
Use a polymer represented by a unit, etc.
Can be.

The reactive functional group includes an active halogen group such as a halomethyl group, a haloacetyl group, a haloacetamidomethyl group, a halogenated alkyl group, an epoxide group, a carboxyl group, an isocyanate group, a thioisocyanate group,
Acid anhydride groups and the like can be used,
Active halogen groups are easy to produce, highly reactive,
It is preferably used because the immobilization reaction of the N-substituted poly (alkylimine) residue can be performed under mild conditions and the covalent bond formed at this time is chemically stable.

Specific examples of such a water-insoluble polymer having a reactive functional group include chloroacetamidomethyl polystyrene, chloroacetamidomethylated Udel polysulfone, and chloroacetamidomethylated polyetherimide. . Further, among these water-insoluble polymers, those soluble in an organic solvent are particularly preferably used because they are easy to mold.

The term "N-substituted poly (alkylenimine)" as used in the present invention means a poly (alkylenimine) obtained by substituting a hydrogen atom of a nitrogen atom with at least one selected from an alkyl group and an acyl group. .

Examples of such poly (alkyleneimine) include polyethyleneimine, polypropyleneimine, polybutyleneimine, polyhexamethyleneimine,
A poly (ethylene imine / decamethylene imine) copolymer or the like can be used. Further, as the alkyl group as the N-substituent, n-butyl group, n-hexyl group,
An n-decanyl group, a lauryl group, a myristyl group, an n-hexadecyl group, an n-stearyl group, a benzyl group and the like can be used.As the acyl group as the N-substituent, a butyroyl group, an n-hexinoyl group, An n-decanoyl group, a lauroyl group, a myristoyl group, an n-hexadecyloyl group, an n-stearyloyl group, a benzoyl group, and halogen-substituted products thereof can be used.

The adsorption performance and processability of the N-substituted poly (alkyleneimine) are determined by the length of the alkylene group in the main chain, the number of alkyl carbon atoms in the N-substituent, for example, the N-alkyl group, and It varies depending on the N-alkylation rate. The parameters include the ratio of the number of carbon atoms to the number of nitrogen atoms in the molecule as a whole (hereinafter abbreviated as C / N ratio),
It can be determined using the N-substitution rate. That is,
When the C / N ratio is too small, the hydrophilicity becomes too high,
If it is difficult to process, on the other hand, if it is too large, the adsorbing ability will decrease, so it is preferably 2.3 to 26, more preferably 2.
The range is preferably 5 to 14. The N-substitution rate is
If the absorption capacity is too small or too large, the adsorption capacity is reduced, so that it is preferably 30% or more and 60% or less, and more preferably 40% or less.
The content of 60% or less is preferable from the viewpoint of the function of removing both inflammatory response proteins and inflammatory leukocytes in blood. In addition, the higher the degree of polymerization (n) of poly (alkyleneimine), the higher the adsorption performance for a high molecular weight substance is.
If the degree of polymerization is too large, it becomes difficult to immobilize the polymer in a form in which the graft chain is extended.
000, more preferably 20 to 300. Further, a part of the alkylene group in the main chain portion of the N-alkylated polyalkyleneimine may be substituted with a cyclohexane ring, a benzene ring, or the like, or may be substituted with an amide group, an ether group, or the like.

The immobilization density of the N-substituted poly (alkyleneimine) group in the adsorbent of the present invention depends on the C / N ratio of the N-substituted poly (alkylenimine), the size of n, and the water-insoluble polymer serving as the trunk. Although it depends on the chemical structure and use of the polymer, if the amount is too small, the function does not appear.On the other hand, if the amount is too large, the moldability of the polymer after immobilization becomes poor, and the function as an adsorbent also decreases. The density is preferably 0.0001 per repeating unit of the water-insoluble polymer.
0.5 to 0.5 mol, more preferably 0.001 to 0.1 mol.

The method for preparing the N-substituted poly (alkyleneimine) is as follows.
It can be easily adjusted by mixing the required amount of the acid chloride or acid anhydride of the alkyl bromide or carboxylic acid at a temperature of 00 ° C. or less. This reaction proceeds quantitatively in a polar solvent such as ethyl alcohol, tetrahydrofuran or dimethylformamide even at room temperature. When the molecular weight of the poly (alkyleneimine) is high and the N-alkylation is high, the N-alkylated polyalkyleneimine becomes difficult to dissolve in the solvent. Therefore, the one having a low alkylation rate was reacted with the water-insoluble polymer. Thereafter, it is convenient to further add a predetermined amount of an acid chloride or acid anhydride of an alkyl bromide or carboxylic acid to the reaction system to obtain a target alkylation ratio. In particular, when the substituent is an acyl group, the latter is preferably used because it is difficult to dissolve in a solvent.

The method for producing the adsorbent of the present invention includes a homogeneous reaction method in which a water-insoluble polymer and an N-substituted poly (alkyleneimine) are reacted in a solution, and a method in which a water-insoluble polymer is molded into a water-insoluble polymer. Any of the methods of heterogeneous reaction of contacting a substituted poly (alkyleneimine) solution can be employed.

An example of a method for producing an adsorbent by a homogeneous reaction is as follows. A corresponding polyamine is added to a solution of chloroacetamidomethylated polysulfone, and the solution is added at 0 to 100 ° C.
It is easily manufactured by reacting at a temperature of The amount is not particularly limited, but is preferably at least 1 mole per haloacetamide methyl group in order to obtain a soluble polymer. In particular, in the case of a branched polyamine, it is preferable to use a large excess of the polyamine in order to obtain a soluble polymer.

When the reaction is carried out in a homogeneous system, tetrahydrofuran, dimethyl sulfoxide, N, N-dimethylformamide (DMF), N, N
N-dimethylacetamide, N-methylpyrrolidone and the like are preferably used. In addition, a method of surface-treating a molded product such as a membrane or a fiber can be employed. For this purpose, a solvent that does not dissolve polysulfone such as water, methanol, and ethanol but dissolves polyamine is preferably used.

As an example of a method for producing an adsorbent by a heterogeneous reaction, a molded article such as chloroacetamidomethylated polysulfone fiber or hollow fiber is immersed in an N-alkylated polyalkyleneimine isopropanol solution.
It is easily manufactured by reacting at a temperature of 0 to 100 ° C.

It is practically preferable to coat the surface of a molded article such as nylon fiber with such an adsorbent, since a high-order molded article having a large surface area can be easily obtained. As a coating method, a nylon knitted fabric or woven fabric is immersed in a solution in which the adsorbent is dissolved in a low boiling point solvent such as methylene chloride or tetrahydrofuran, and then the solvent can be easily evaporated. Also,
A wet coating method in which a substance dissolved in a solvent such as N, N-dimethylformamide is placed in water or the like can also be used. The polymer of the molded article to be coated may be any polymer having good adhesiveness with the adsorbent of the present invention, such as polyamide, polyurethane, polyimide, polysulfone, polyvinyl chloride, and polyester, and the type thereof is not particularly limited. However, since amide polymers such as nylon and polyetherimide have particularly good adhesion,
It is preferably used.

When the adsorbent of the present invention is used for extracorporeal circulation,
It is preferable to use the anticoagulant treated with heparin, since the blood compatibility is improved and the adsorbability to LDL is improved. In particular, when the C / N ratio is high, the adsorbent becomes hydrophobic, but heparin treatment is preferred because it becomes hydrophilic and the adsorbing ability increases.

In the present invention, the specific adsorbent thus obtained is packed in a column and used. That is, such a column has a blood inflow portion for flowing blood into the adsorption portion filled with the adsorbent, and a blood outflow portion for flowing out the blood that has flowed into the adsorption portion. Any structure that does not cause drift is used without particular limitation.

[0028]

EXAMPLES The present invention will be described more specifically with reference to experimental examples.

The evaluation method in the present embodiment was as follows. 1. Analysis of components in blood Neutral fat (TG) is determined by enzyme method (free glycerol elimination method)
I asked for it. β-lipoprotein (LDL) was determined by cellulose acetate membrane electrophoresis. Total protein was determined by the BCG method. HDL-C was determined by the heparin Ca2 +, Ni2 + precipitation method. CRP and SAA were determined by latex agglutination immunoassay.

The blood cell count is measured by Nihon Kohden's fully automatic hemocytometer M.
It was determined using EK-6208.

Examples 1 and 2 Nitrobenzene 16 mL
After cooling a mixed solution of the mixture and 32 mL of sulfuric acid to 0 ° C., 4.2 g of N-methylol-α-chloroacetamide was added and dissolved, and this was dissolved at 10 ° C. in Udelpolysulfone P350.
0 in a 3 L nitrobenzene solution (300 g / 3 L)
The mixture was added with good stirring, and further stirred at room temperature for 3 hours. Thereafter, the reaction mixture was placed in a large excess of cold methanol to precipitate the polymer. The precipitate was thoroughly washed with methanol and dried, and 300 g of α-chloroacetamidomethylated polysulfone (substitution ratio: 0.05; polymer-
A) was obtained.

Polyethyleneimine (average molecular weight 1000
0: Wako Pure Chemical Industries, Ltd.) A solution of 20 g in 50 mL of DMF was added with 11.4 g of lauryl bromide, and the mixture was stirred at 50 ° C. for 5 hours. Then, 10 g of polymer A was added to 100 ml of DM.
The solution dissolved in F was added and heated at 50 ° C. for 5 hours. After adding 22.8 g of lauryl bromide in the reaction mixture, and further stirring at 50 ° C. for 8 hours, the mixture was added to isopropanol, and the precipitated polymer was collected by filtration. The polymer is dried under vacuum, dissolved in DMF, reprecipitated with isopropanol, dried under vacuum, and N-alkylated polyalkyleneimine-bonded polysulfone having an alkylation ratio of 30% (the adsorbent of Example 1; hereinafter referred to as L-30). Abbreviation) was prepared.

In addition, polyethyleneimine (average molecular weight 1
0000: Wako Pure Chemical Industries, Ltd.) To a solution of 20 g in 50 mL of DMF was added 11.4 g of lauryl bromide, and the mixture was added
After stirring for 10 hours, 10 g of polymer A was added to 100 ml.
Was added and the mixture was heated at 50 ° C. for 5 hours.

To the reaction mixture was added 57 g of lauryl bromide, and the mixture was further stirred at 50 ° C. for 8 hours, then poured into isopropanol, and the precipitated polymer was collected by filtration. The polymer is dried in vacuo, dissolved in DMF, reprecipitated with isopropanol, and dried in vacuo to obtain an N-alkylated polyalkyleneimine-bonded polysulfone having an alkylation ratio of 60% (the adsorbent of Example 2; hereinafter L-60). Abbreviation).

A 20% dimethylacetamide solution of these polymers is applied on a glass plate and put in water for 10 minutes.
The film was formed to a thickness of 0 μm. This was hollowed out into a 4.2 cm diameter disc, and 0.5 mg / mL in heparin / PBS solution was added.
After treating at 0 ° C. for 20 hours, it was washed with physiological saline and used for the following evaluation.

The above-mentioned three membranes were placed in 6 mL of human high neutral fat serum, and heated at 37 ° C. for 4 hours.
The results in Table 1 were obtained.

However, Comparative Example 1 in the table is a polysulfone film (a film prepared by applying a dimethylacetamide solution containing 20% polysulfone and 2% K30-polyvinylpyrrolidone on a glass plate and placing the film in water).

[0038]

[Table 1]

From Table 1, it can be seen that the columns (L-30, L-60) comprising the adsorbents of Examples 1 and 2 adsorb CRP well.

[Examples 3 and 4] The two membranes of Examples 1 and 2 were placed in another 6 mL of human high-neutral fat serum, heated at 37 ° C. for 4 hours, and the serum was analyzed. Was obtained.

[0041]

[Table 2]

From the table, it can be seen that the columns comprising the adsorbents of Examples 1 and 2 adsorb CRP and SAA well.

However, Comparative Example 2 was a polysulfone (C / N ratio of 13.6) membrane in which laurylated tetraethylenepentamine having a laurylation ratio of 100% was immobilized on polymer-A.
Comparative Example 3 was obtained by polymerizing polymer-A with ethylated polyethyleneimine having an ethylation ratio of 100% (polyethyleneimine having a molecular weight of 7).
This is a polysulfone (C / N ratio of 4.0) membrane having immobilized thereon.

[Examples 5 and 6] Each of the adsorbents L30 and L60 obtained in Examples 1 and 2 was dissolved in tetrahydrofuran at a concentration of 5% in 250 ml of a solution having a single fiber fineness of 0.1%.
20 g of a 7 denier nylon 6 tube was immersed, and after 20 hours, the tube was removed, drained, air-dried, and dried.
g coated knitted fabric (L-30 fiber, L-
60 fibers).

For each of these knitted fabrics, 0.
After treating 2 g with a heparin solution at 37 ° C. for 4 hours, 4 m
When the adsorption capacity was evaluated (4 hours at 37 ° C.) with 1 l of human serum, the results in Table 3 were obtained.

[0046]

[Table 3]

However, in Comparative Example 4, uncoated nylon 6 was used.
It is a knitted fabric.

Further, the adsorptivity of granulocytes to these fibers was examined, and the results shown in Table 4 were obtained.

To a New Zealand White rabbit (3.5 kg in weight), 5 μg of LPS (E. coli O111: B4 dissolved at a concentration of 10 ng / mL) was administered from the ear vein per kg of body weight, and 14 days later, the same amount was administered. 2 days after administration of LPS, blood was collected from the ear artery and used for the following evaluation.

After 100 mg of the adsorbent was placed in 2 ml of the above blood and shaken at 37 ° C. for 4 hours, the composition of the blood cells was examined by an automatic hematology analyzer, and the ratio of granulocytes to lymphocytes was determined by a flow cytometer. When the number of each blood cell was calculated, the results in Table 4 were obtained.

However, the granulocyte concentration is a value corrected with erythrocytes as 550, and the reduction rate was calculated from the corrected value.

[0052]

[Table 4]

From Tables 3 and 4, it can be seen that Samples L-30 and L-60 of the present invention adsorb both inflammatory proteins and granulocytes well.

Example 7 1.0 g of the fiber of Example 5 was
The column was packed in a 10 ml polypropylene cylindrical column, washed with 500 ml of physiological saline, and sterilized in an autoclave at 120 ° C. for 25 minutes to obtain a column.

The column and the peristaltic pump were connected by a silicon tube, and 200 ml of physiological saline was passed through the column. Next, heparin sodium 0.5mg / ml PBS
(-) 100 ml of the solution was flowed at a rate of 0.5 ml / min, and 50 ml of physiological saline was flowed at the same speed.

Heparin-collected blood (40 ml) from a volunteer having a high neutral fat is divided into two parts, and 20 ml is divided into 50 ml.
It was placed in a 1-volume polypropylene sterile tube, immersed in a 37 ° C. water bath, and refluxed for 1 hour at a rate of 1 ml / min in the previous circuit.

Blood cell count before and after perfusion and LDL in plasma,
CRP and SAA were measured.

As Comparative Example 5, a column packed with 1.0 g of Comparative Example 4 was similarly evaluated with the remaining 20 ml of blood, and the results in Tables 5 and 6 were obtained.

[0059]

[Table 5]

[0060]

[Table 6]

From Tables 5 and 6, it can be seen that the column of Example 7 adsorbs both inflammatory proteins and granulocytes, whereas the column of Comparative Example 5 adsorbs granulocytes but does not adsorb inflammatory proteins. You can see that.

[0062]

Industrial Applicability According to the present invention, inflammatory proteins such as CRP and SAA and inflammatory leukocytes can be selectively adsorbed with high efficiency and can be treated efficiently. It can treat inflammatory diseases in various ways.

Continuation of the front page F term (reference) 4C077 AA12 BB03 KK04 KK13 MM08 MM09 NN02 NN04 PP13 PP15 PP30

Claims (12)

[Claims] 1. A column for treating inflammatory diseases, which is filled with an inflammatory substance adsorbent capable of removing both inflammatory response proteins and inflammatory leukocytes in blood. 2. The column for treating inflammatory diseases according to claim 1, wherein the inflammatory substance adsorbent is a water-insoluble polymer having an N-substituted poly (alkyleneimine) residue bonded to a side chain. 3. The method according to claim 2, wherein the N-substituted poly (alkyleneimine) residue has a basic nitrogen atom, and the ratio of the number of carbon atoms to the number of basic nitrogen atoms is 2.3 to 26. Column for treating inflammatory diseases. 4. The N-substituted poly (alkylimine) residue, wherein the N-substituent is an alkyl group or an acyl group having 4 or more carbon atoms, and the number of N-substituents is the same as the number of nitrogen atoms. 3
The column for treating an inflammatory disease according to claim 2, which is 0% or more and 60% or less. 5. The poly (alkyleneimine) moiety of the N-substituted poly (alkyleneimine) residue has a degree of polymerization of 4 or more and 1
The column for treating an inflammatory disease according to any one of claims 2 to 4, which has a molecular weight of 0000 or less. 6. The column for treating an inflammatory disease according to claim 2, wherein the water-insoluble polymer is a polysulfone polymer having an amine binding group. 7. The column for treating an inflammatory disease according to claim 2, wherein the water-insoluble polymer is a poly (vinyl aromatic compound) having an amine-binding group. 8. The column for treating an inflammatory disease according to claim 2, wherein the water-insoluble polymer is a polyimide polymer. 9. The method according to claim 1, wherein the polysulfone-based polymer is poly (p-
7. The column for treating an inflammatory disease according to claim 6, which is phenylene ether sulfone). 10. The polysulfone-based polymer, wherein-｛(p
_{-C 6 H 4) -SO 2 -} (p-C 6 H 4) -O- (p-
_{C 6 H 4) -C (CH} 3) 2 - (p-C 6 H 4) -O}
The inflammatory response protein adsorbent according to claim 6 or 9, which is a polymer composed of a unit represented by-. 11. The adsorbent is at least one selected from a membrane, a fiber, a hollow fiber, and a granular material.
0. The column for treating an inflammatory disease according to any one of 0. 12. The column for treating an inflammatory disease according to claim 1, wherein the adsorbent has adsorbed heparin.