JP3363982B2 - Material for producing plasma or serum having immunosuppressive action and apparatus for producing plasma or serum having immunosuppressive action - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a material , a device, and an immunosuppressive plasma and serum for producing plasma and serum having an immunosuppressive action by bringing the material and blood into contact with each other. INDUSTRIAL APPLICABILITY The present invention is used for treatment of autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus, and allergic diseases.

[0002]

BACKGROUND OF THE INVENTION The causes of autoimmune and allergic diseases such as chronic rheumatism and systemic lupus erythematosus are
Generally, it is considered that an abnormal immune enhancement causes an immune reaction to non-self or self-antigen, which progresses to an inflammatory reaction to destroy self-organs.

Therefore, in these clinical treatments, in order to correct an abnormal immune state, conventionally, drug therapy with various steroid agents and immunosuppressive agents is performed.
However, in these drug therapies, side effects occur when an amount which can be expected to have an immunosuppressive action is used.

Therefore, as an alternative approach to these drug therapies, the following therapeutic methods aimed at removing autoantibodies, immune complexes and pathogenic substances present in bloodstream have been attempted. However, it has not been the fundamental solution to the abnormal immune status. Further, there are problems inherent in any of the following methods.

(1) Plasma exchange therapy: This method has a problem that it is difficult to obtain plasma and the risk of infection is high. (2) A therapeutic method in which a ligand such as DNA or protein A is immobilized on various materials and the immunoadsorption reaction is utilized to adsorb and remove anti-DNA antibody or immune complex. However, this method is problematic in that impurities are mixed in during purification of the ligand and desorption of the ligand.

(3) A method for adsorbing and removing a pathogenic substance using an adsorbent material in which a specific functional group is immobilized on a polymeric material without using a specific ligand has been devised, and is disclosed in Japanese Examined Patent Publication No. 2-5098. 2-5097, JP-A-64-6827
It is disclosed in Japanese Patent No. However, these methods suffer from poor selectivity.

On the other hand, it has been reported that blood cells and leukocytes are reacted with various polymer materials to produce cytokines such as interleukin 2 and TNF from blood cells and leukocytes (artificial organ 20 (1)). , p235-240, 19
91, ibid. 22 (5), p1233-1237)). However, these factors have an immunopotentiating action and an anticancer action, and do not have an immunosuppressive action suitable for treating rheumatism, allergic diseases, and autoimmune diseases.

[0008]

The object of the present invention is to solve the above is to solve the various disadvantages of the prior art described above, for the manufacture of a plasma or serum with an easily immunosuppressive effects compared to conventional methods
To provide such materials , manufacturing apparatus therefor and such immunosuppressive plasma or serum.

[0009]

[Means for Solving the Problems] In view of such circumstances, the present inventors have conducted extensive studies and as a result, induced immunosuppressive action in blood by controlling the surface roughness of a material that comes into contact with blood. As a result, they have completed the present invention. The present invention is simpler than the conventional one.
For the production of plasma and serum have immunosuppressive activity
It provides materials , devices and the plasma and serum thus obtained.

[0010] That is, an invention according to claim 1, the center line average roughness Ra value have a surface roughness which is 0.2Myuemu～10myuemu, by Rukoto is come in contact with blood, the immunosuppressive action It is a material for producing plasma or serum. The invention according to claims 2 and 3 comprises plasma and serum having an immunosuppressive action, which is composed of a container having a blood outlet and an inner wall having the above-mentioned surface roughness, or a container filled with the above material. The invention according to claim 4 is plasma or serum having an immunosuppressive action, which is obtained by contacting a material with blood .

The immunosuppressive action referred to in the present invention refers to the action of suppressing the blastogenic reaction of lymphocytes separated from human peripheral blood by mitogen stimulation. The Ra value in the present invention means
It is the center line average roughness according to JIS B0601-1982. As will be apparent from the examples described below, plasma obtained by contacting blood with a material having a surface roughness of Ra value of 0.2 μm to 10 μm is irrespective of the material, mitogen-stimulated lymphocyte immature. On the other hand, the mitogen-stimulated lymphocyte blast transformation reaction was hardly suppressed by the material that suppressed the blast reaction, while the Ra value was out of the range of 0.2 μm to 10 μm. Therefore, it is clear that plasma obtained by contacting blood with a material having a surface roughness of Ra value of 0.2 μm to 10 μm has an immunosuppressive action.

As the above-mentioned material usable in the present invention, the center line average roughness Ra value on the surface is 0.2 μm to 10 μm.
It has an unevenness of m and is harmful to the human body, for example, it does not dissolve itself when blood and material come into contact with it, and does not leak harmful metal ions or additives such as plasticizers. If it exists, it can be used regardless of its type. That is, inorganic materials such as glass and alumina, polystyrene, nylon, polytetrafluoroethylene,
Polytrifluoroethylene, polyethylene terephthalate, polyethylene, polypropylene, polyvinyl chloride,
Examples thereof include synthetic polymers such as polyurethane and acrylic resins and their copolymers, and natural organic polymer materials including cellulose, chitosan, agarose and the like and modified products thereof.

Although a polishing method is generally used as a method for imparting surface roughness, it may be configured to have the above-mentioned specific surface roughness by another method. For example, a method of physically or chemically fixing fine particles on the material surface,
A method using a material having a porous surface can also be used.

As a method of fixing fine particles on the material surface, for example, a method of coating fine particles of 0.1 μm to 10 μm can be mentioned. The fine particles used here can be prepared, for example, by emulsion-polymerizing or suspension-polymerizing a vinyl monomer such as a styrene-based or acrylic-based monomer alone or a mixture of two or more components. These fine particles are preferably those made of a copolymer with a polyfunctional monomer having two or more functional groups such as divinylbenzene as a crosslinking agent.

Next, a method of coating the fine particles will be exemplified. First, as a binder for coating, synthetic polymer or natural polymer insoluble in blood is added in an amount of 0.1-5.
These fine particles are suspended in a solution in which about% by weight is dissolved. Next, it can be produced by immersing beads, fibers or film-like material made of an organic or inorganic material, which has been formed in advance, in this suspension and then drying. The Ra value in this case can be adjusted by the size of the fine particles.

Also in the case of a porous material, the shape thereof may be any of a film shape, a fiber shape or a bead shape, and only the surface may be porous, or the entire material may be porous. . As for the surface porous material, for example, a bead, a fiber or a film carrier made of an organic or inorganic material, a synthetic polymer or a natural polymer insoluble in blood as a coating solution in a good solvent thereof in an amount of 0.1 to 5 It can be obtained by spraying a solution in which about% by weight is dissolved by spraying and heating and drying.

Further, as a method for manufacturing the material which is porous as a whole, a general manufacturing method such as a porous membrane and porous beads can be used. For example, in the case of a film, a casting polymer is prepared by dissolving a synthetic polymer insoluble in blood in a good solvent and casting it on a glass plate.
Then, the porous membrane can be prepared by washing the film with a poor solvent for the synthetic polymer and extracting the good solvent.

The Ra value in the case of a porous material can be adjusted by adjusting the pore diameter and the pore amount, and the pore diameter and the pore amount can be adjusted by changing the kind and amount of the good solvent and poor solvent used. Can be easily adjusted.

In the present invention, any method can be used as a method of bringing blood into contact with the above material as long as blood and the above material can be sufficiently brought into contact with each other. For example, a method in which the above fibrous material is packed in a column and blood is circulated in the column, or a method in which a bead-shaped material having a particle size of 50 μm to 5 mm is packed in the column and blood is circulated can be used. Further, the blood may be brought into contact with the material by suspending and suspending the material having various shapes in the blood collected in an appropriate container. Alternatively, the inner wall of the container itself may be processed to have the above-mentioned surface roughness, and the blood may be shaken in the container.

The apparatus for producing plasma or serum having an immunosuppressive action according to the present invention has a container having a blood outlet port and an inner wall of the container having the above surface roughness, or a container having the above specific surface roughness. Any device can be used as long as it has a container filled with material. For example, a commercially available extracorporeal circulation system, a blood pack or the like may be used as long as it is a device capable of performing the above-mentioned operation process.

[0021]

[Action] material according to the invention of claim 1 has a center line average roughness Ra value because it has a surface roughness of 0.2Myuemu～10myuemu, when contacted with blood, the surface shape of the material , It is considered that it interacts with some component in blood and an immunosuppressive action appears in plasma and serum.

[0022]

EXAMPLES The present invention will be described in detail below by giving Examples and Comparative Examples of the present invention.

First, the following Examples 1 to 13 and Comparative Example 1
The preparation method and test method of the samples used in Nos. 13 to 13 will be described. (1) Method for producing polishing film (Examples 1 to 6, Comparative Examples 1 to 6) A film as an immunosuppressive factor inducing material was prepared, the surface of the film was washed with methyl alcohol, and then Struers (Denmark) Manufactured by automatic polishing machine (trade name: Plano Ball Pedmax), 220, 500, 1200,
An abrasive film having surface roughness on both sides of the surface was prepared by using a sandpaper with 2400 and 4000 mesh attached.

(2) Method for producing immunosuppressive plasma using a film (Examples 1 to 6 and Comparative Examples 1 to 6) The film was cut into a size of 3 cm × 4 cm, which was further cut into a size of 5 mm × 5 mm. I cut it into pieces. These strips were washed with physiological saline for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.), and then washed with physiological saline for injection in a 2 ml tube (eppend).
orf company).

To the tube filled with the film, 2.0 ml of heparin-collected fresh blood from a healthy person was added and attached to a rotating disk, and mixed by inversion at 37 ° C. for 2 hours at a rotation speed of 26 rpm. After that, blood is collected, and 3000 rpm is used for 1
After centrifugation for 5 minutes, plasma was separated. Next, the immunosuppressive activity in plasma was measured by the method described below.

(3) Method for producing immunosuppressive plasma using beads (Examples 7 to 13 and Comparative Examples 10 to 13) After washing the beads with physiological saline for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.),
Similarly, a 2 ml tube (Eppendorf Co.) washed with a physiological saline solution for injection was filled with a prescribed number.

To a tube filled with each bead, 1.6 ml of fresh blood of a healthy person from which heparin was collected was added and attached to a rotating disc, and mixed by inversion at 37 ° C. for 2 hours at a rotation speed of 26 rpm. After that, blood is collected, and 3000 rpm is used for 1
After centrifugation for 5 minutes, plasma was separated. Next, the immunosuppressive activity in plasma was measured by the method described below.

(4) Method of measuring surface roughness Ra value of center line average roughness (cutoff value, 0.8 mm for film, described in each Example and Comparative Example)
For beads, 0.08 mm) was measured by a surface roughness measuring device (Kosaka Laboratory, trade name; Surfcoder SE-30D) or observed by a scanning laser microscope (Lasertec, trade name; 1ML21W). Image analysis system (Mitani Shoji Co., Ltd., trade name; SUPER
It processed by ASPECT) and measured.

(5) Measurement of immunosuppressive activity in plasma Peripheral blood was collected from healthy volunteers by adding heparin to obtain Ficol.
Peripheral blood mononuclear cells were separated by 1 / Paque (Pharmacia) specific gravity separation method. 2 of these peripheral blood mononuclear cells
The cells were rolled up in a microplate at a density of × 10 ⁵ cells / 100 µl / well. Pokeweed Myt
ogen (manufactured by Sigma), (final concentration 2 μg / m
l) and plasma sample (final concentration 10%), final 200
μl was added. 5% CO ₂ , 72 at 37 ° C
After culturing for 4 hours, 1 μCi / well 4 hours before the end
Of tritium thymidine ( ³ H-TdR) was added, and its uptake was measured by a liquid scintillation counter. The result of the measurement is represented by the average standard deviation. Moreover, the measured value of the control plasma-free group was 125,000 d. p.
It was m. The inhibitory activity was calculated from the following formula.

[0030]

[Equation 1]

Examples 1 to 3 and Comparative Examples 1 to 3 PET films (polyethylene terephthalate film, manufactured by Unitika Ltd., trade name: Embrette S-75) were used and polished in 5 steps according to the above-mentioned method for producing the polishing film. The produced film was produced. Then, the Ra value was determined for each of the unpolished film and the film polished in 5 steps, and then the immunosuppressive factor induction test with the above-mentioned film was performed. The results are shown in Table 1 below.

[0032]

[Table 1]

As is clear from Table 1, the Ra value is 0.6.
Above 1 μm, the immunosuppressive activity in plasma increased sharply.

Examples 4 to 6 and Comparative Examples 4 to 6 PET films were used as CA films (manufactured by Art Plus, cellulose acetate film, trade name; acetyl film VR-).
Soxhlet extraction with methyl alcohol instead of washing with methyl alcohol (24 hours)
After extracting the plasticizer and taking out the film, 1
After air-drying for 5 hours and further drying at 80 ° C. for 5 hours, unpolished and polished films were prepared in the same manner as in Examples 1 to 3 and Comparative Examples 1 to 3, and immunosuppressive factor induction test was conducted. . The results are shown in Table 2.

[0035]

[Table 2]

As is clear from Table 2, Examples 4 to 6,
That is, the immunosuppressive activity in plasma was increased in the polishing film (Ra value = 0.58 μm or more) polished with sandpaper of 1200 mesh or less.

Example 7 Cellulose acetate pellets (manufactured by Art Plus, containing 30% by weight of triethyl acetyl citrate as a plasticizer) were injection-molded to produce spherical beads having a diameter of 2.5 mm. 50 g of these beads are treated with 300 ml of methanol at 50 ° C.
Soxhlet extraction for 24 hours and the plasticizer was extracted. Then, the beads from which the plasticizer was extracted were taken out into a stainless steel vat, air-dried for 15 hours, and then at 80 ° C. for 5 hours.
Allowed to dry for hours.

Pot mill (manufactured by Toyo Engineering Co.,
Product name: 5 l-ceramic pot mill BP-5), 200 ml of the above beads and an abrasive material of the same volume; WHITE
ABRAX (WA) # 34 (manufactured by Japan Abrasives Industry Co., Ltd.) was charged, and several balls for ceramic pot mill (manufactured by Toyo Engineering Co., trade name; BB-13) were charged, and a ball polishing machine (Nitto Kagaku Co., Ltd.) was used. Pot mill, trade name;
Polished with AN-3S) for 5 hours. In this way
Beads having an Ra value of 1.36 μm were obtained.

The obtained beads were washed 3 times with methanol and 5 times with physiological saline for injection (Otsuka Pharmaceutical Co., Ltd.). Then, 2 ml washed with saline for injection as well
The tube (Eppendorf) was filled. The filling amount was 70 beads.

In addition to Comparative Example 7, an immunosuppressive factor induction experiment using the above beads was conducted. The results are shown in Table 3 below.

Comparative Example 7 Beads having an Ra value of 0.186 μm were prepared in the same manner as in Example 7, except that polishing was not performed.

As in Example 7, washing was carried out, and then a tube for 2 ml was filled with 70 of the above beads, and an immunosuppressive factor induction test using the above beads was conducted. The results are shown in Table 3 below.
Shown in.

[0043]

[Table 3]

Example 8 Nylon 66 pellets (trade name; Ube 6 manufactured by Ube Industries, Ltd.)
6 2020B) was injection-molded to produce spherical beads having a diameter of 2.5 mm.

Pot mill (manufactured by Toyo Engineering Corporation,
Product name: 5 l-ceramic pot mill BP-5), 200 ml of the above beads and an abrasive material of the same volume; WHITE
ABRAX (WA) # 34 (manufactured by Japan Abrasives Industry Co., Ltd.) was charged, and several balls for ceramic pot mill (manufactured by Toyo Engineering Co., trade name; BB-13) were charged, and a ball polishing machine (Nitto Kagaku Co., Ltd.) was used. Pot mill, trade name;
Polished with AN-3S) for 5 hours.

As described above, beads having an Ra value of 9.1 μm were obtained. Wash the beads 3 times with methanol,
It was washed 5 times with physiological saline for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.). Then, it was filled in a 2 ml tube (Eppendorf) similarly washed with physiological saline for injection. Filling amount is beads 70
I made it into an individual.

In addition to Comparative Example 8, an immunosuppressive factor induction experiment using the above beads was conducted. The results are shown in Table 4 below.

Comparative Example 8 Ra was prepared in the same manner as in Example 8 except that polishing was not performed.
Beads with a value of 0.210 μm were prepared.

As in Example 8, the beads were washed and 70 beads were filled in a 2 ml tube.

[0050]

[Table 4]

Example 9 and Comparative Example 9 The CA beads (unpolished beads) obtained in Comparative Example 7 were suspension-polymerized to prepare styrene-divinylbenzene copolymer (copolymerization ratio 1: 1) fine particles (Sekisui Immersed in a 1% by weight solution of cellulose acetate (trade name: Linter, manufactured by Daicel Chemical Industries, Ltd.) in which 2% by weight of 2% by weight made by Kagaku Kogyo Co., Ltd. is suspended in methyl chloride-ethanol (9: 1 by weight). After that, it was air-dried, and thereby fine particle-coated beads (Example 12) were prepared.

It was confirmed by an electron microscope that the fine particles were coated with cellulose acetate on the surface of the beads. An immunosuppressive factor induction test using the aforementioned beads was conducted using 70 particles each of the fine particle-coated beads (Example 12) and uncoated beads (Comparative Example 12) obtained as described above. The results are shown in Table 5.

[0053]

[Table 5]

Example 10 and Comparative Example 10 Nylon 66 pellets (manufactured by Ube Industries, Ltd.) were injection-molded to prepare beads having a particle diameter of 2.5 mm as a porous carrier used in the production induction test of immunosuppressive factors. 4.6 kg of the above beads were taken, and a 5% by weight methylene chloride / methanol (weight ratio 9: 1) solution of a cellulose acetate resin (manufactured by Daicel Chemical Industries, Ltd.) was used as a coating solution, and a flow coater (manufactured by Freund Corporation, product First name; FL0-5)
Was sprayed to prepare beads coated with a cellulose acetate resin. The coated beads were photographed by a scanning electron microscope and observed. As a result, it was confirmed that surface-porous beads having a coating layer with a thickness of 200 μm were obtained.

The coating conditions are as follows. Spray air pressure: 3.5 kg / cm ² Spray liquid temperature: Room temperature spray liquid flow rate: 100 ml / min Spray temperature: 60 ° C. Spray nozzle diameter: 1.2 mm Coated beads obtained as described above (Example 1
0) and 70 uncoated beads (Comparative Example 10) each were used for the immunosuppressive factor induction test using the beads described above. The results are shown in Table 6.

[0056]

[Table 6]

As is clear from Table 6, the coated beads (Example 10) had an Ra value of 1.18 μm,
The induction amount of the immunosuppressive factor was very large as compared with the uncoated beads (Comparative Example 10).

Examples 11, 12, and 13 Polystyrene porous particles Diaion HP-50 (manufactured by Mitsubishi Kasei), polyacrylic ester porous particles Diaion HP-1MG (manufactured by Mitsubishi Kasei), polyacrylic ester porous particles Amberlite XAD-7 (manufactured by Organo Corporation) was washed with distilled water three times, and allowed to stand overnight at room temperature. After that, it was washed with distilled water three times, washed with methanol, suspended in methanol, and allowed to stand at room temperature for 6 hours. These are washed with distilled water and physiological saline for injection, suspended in physiological saline for injection and ultrasonically cleaned,
Degas using a suction pump. Each polypropylene particle thus washed was sterilized and washed to obtain a polypropylene tube (for Eppendorf safety lock tube 2.0 ml:
(Diatron) was filled with a bulk volume of 500 μl.
Blood 1.6 taken from a healthy person using heparin in this tube
ml was added, and the mixture was gently stirred by inversion at 37 ° C. for 2 hours. Blood was taken out, plasma was separated, and immunosuppressive activity in plasma was measured. The results are shown in Table 7.

[0059]

[Table 7]

As is clear from Table 7, HP-50 and HP- having surface roughness Ra in the range of 0.2 μm to 10 μm.
In 1MG and XAD-7, remarkable induction of production of immunosuppressive factor was observed.

[0061]

As is evident from the foregoing description, according to the material of claim 1, since having the specific surface roughness, when come in contact with the blood, induction of immunosuppressive agent is effectively performed . Therefore, by using the patient's own blood, it is possible to easily obtain plasma or serum containing a patient-derived endogenous immunosuppressive factor, and to provide such plasma or serum to the patient.

Further, in the inventions described in claims 2 and 3, since the container or the material filled in the container is constituted by the material having the specific surface roughness, respectively,
It is possible to provide an optimal plasma or serum production apparatus to be used in the production method according to claim 1.

─────────────────────────────────────────────────── ─── Continuation of front page (56) Reference JP-A-1-158970 (JP, A) JP-A-62-191041 (JP, A) JP-A-57-170263 (JP, A) JP-A-57- 56039 (JP, A) JP-A-6-209992 (JP, A) (58) Fields investigated (Int.Cl. ⁷ , DB name) A61M 1/36 550 A61K 35/16 ABA

Claims (4)

(57) [Claims] 1. A center line average roughness Ra value of 0.2 μm to 1
Have a a a surface roughness 0 .mu.m, the Rukoto is come in contact with blood
More, to produce a plasma or serum having immunosuppressive activity
Material for . 2. Plasma having an immunosuppressive action, characterized in that it comprises a container having a blood inlet and outlet and an inner wall thereof having a surface roughness having a centerline average roughness Ra value of 0.2 μm to 10 μm. Or serum production equipment. 3. A container having a blood inlet / outlet port, and a material filled in the container and having a surface roughness having a center line average roughness Ra value of 0.2 μm to 10 μm. An apparatus for producing plasma or serum having a characteristic immunosuppressive action. 4. The material according to claim 1 is contacted with blood.
Plasma or serum having immunosuppressive activity, obtained by being.