Patent Application
20050013791
The present invention relates to cytokine-inducing material and instrument for use in the cytokine-inducing therapy or the like, more particularly to cytokine-inducing material and instrument which enable effective induction of cytokines.
Cytokine is a general term for a diversity of factors of cell signal transductions. Examples of cytokines include interferon-α, interferon-β, interferon-γ (IFN-γ), interleukin 1, interleukin 18, tumor necrosis factor-α (Tumor Necrosis Factor-α, TNF-α), tumor necrosis factor-α (Tumor Necrosis Factor-β), transforming growth factor-α (Transforming Growth Factor-α), transforming growth factor-β (Transforming Growth Factor-β, TGF-β) and various cell growth factors (Special 1995 number of Clinical Immunity Vol. 27, “All about cytokines”, Kagaku Hyoron-sha, Clinical Immunity Vol. 36, 39-44, 2001).
Cytokine is known to exhibit various activities in vivo and be involved in various diseases. A cytokine-inducing therapy has been conventionally conducted which causes such activities of cytokine in vivo to thereby treat for diseases. In the cytokine-inducing therapy, a cytokine inducer is dosed to a patient to cause induction of cytokine in vivo. Various materials are known to serve as material inducing cytokine for use in such a cytokine-inducing therapy. Examples of known materials inducing cytokine include microorganism-derived materials such as OK-432, Bacillus Callete Guerin (BCG), Bestatin, Maruyama vaccine and romurtide, and basidiomycetes-derived materials such as Krestin, lentinan and sizofiran.
For example, OK-432, BCG and the like are known to induce cytokines, such as interleukin 1 and interferon-γ, from blood (Gifu University Medical Report 43:166-177, 1995, Molecular Medicine Vol. 36, extra edition, 220-229, 1999).
Although possible to induce cytokines in vivo, the above-described cytokine-inducing therapy is hard to induce cytokines in a sufficient amount and is accordingly difficult to provide their strong efficacies, which has been a problem. A dosage of a cytokine inducer may be increased to effectively induce cytokines. However, the increased dosage heightens side effects to result in the failure to achieve effective therapy.
Japanese Patent Laying-Open No. Sho 60-120821 describes a leukocyte stimulator for treatment of malignant tumor, which comprises a stimulating agent covalently bonded to an insoluble carrier. Also, Japanese Patent Laying-Open No. Sho 61-277628 describes a leukocyte stimulator for treatment of cancer, which comprises interleukin 1, OK-432, recombinant interleukin 2 or interferon-γ covalently bonded to an insoluble carrier. These leukocyte stimulators induce tumor cytotoxic cells. These prior art references provide no description as to cytokine induction.
In view of the current state of the above-described prior art, it is an object of the present invention to provide novel cytokine-inducing material and cytokine-inducing instrument which enable establishment of a more effective cytokine-inducing therapy relative to conventional cytokine-inducing therapies.
The cytokine-inducing material of the present invention contains a cytokine-inducing agent and a water-insoluble induction enhancer.
The inventors of this application have completed the present invention as a consequence of their finding that a cytokine-inducing material, either containing a cytokine-inducing agent and a water-insoluble induction enhancer or comprising an insoluble induction enhancer incorporating a cytokine-inducing agent fixed thereto, has the capability to induce a markedly large amount of cytokines.
The cytokine-inducing instrument of the present invention has a container, and a cytokine-inducing material constituted according to the present invention and accommodated in the container.
The present invention is below described in detail.
Although not limiting, the induction enhancer in the present invention comprises a water-insoluble material, either metallic, organic or inorganic. The induction enhancer preferably comprises a water-insoluble organic material, more preferably a water-insoluble polymeric material.
Examples of metallic induction enhancer include metals such as gold, gold alloys, silver, silver alloys, titanium, titanium alloys and stainless steel.
Examples of inorganic induction enhancers include active carbon, glass, glass derivatives, silica-based compositions, alumina and hydroxyapatite.
Examples of organic or polymeric induction enhancers include celluloses, agaroses, dextrans, polystyrenes, acrylic esters, polyethylene terephthalates, nylons, polyvinyl alcohols, polysulfons, polyamides, polyacrylonitriles, polyethylenes, polyurethanes, polypropylenes and polyesters.
Examples of polystyrenes include divinyl benzene-styrene copolymers. Examples of acrylic esters include polymethyl methacrylate and polyhydroxyethyl methacrylate. Particularly preferred are polymeric materials such as based on polystyrenes, acrylic esters, nylons, polyesters, celluloses and polyvinyl alcohols.
The induction enhancer is nonpolar and maybe hydrophobic. In this case, a polystyrene-based polymeric material can be used for the induction enhancer. Such an induction enhancer can be hydrophilicized at its surface as by surface modification or surface coating.
The shape or form of the induction enhancer is not particularly specified. The induction enhancer may have a generally-known form, such as a fiber, non-woven, sponge, particulate, film or hollow fiber.
The induction enhancer, if particulate, preferably has a size between 50 μm and 2 mm and, if fibrous, preferably has a diameter of up to 10 μm, more preferably up to 5 μm. If a fibrous form is selected, the induction enhancer may preferably comprise a non-woven fabric. In such a case, it is particularly preferred that a constituent fiber has a diameter of up to 3 μm.
It is particularly preferred that the induction enhancer comprises a leukocyte adsorbent material. Examples of useful leukocyte adsorbent materials include polymeric materials such as based on polystyrenes, acrylic esters, polyesters, nylons, polyvinyl alcohols and cellulose based materials, e.g., cellulose acetate; and glass-based materials.
Also preferable for use as the induction enhancer are materials having a surface roughness imparted thereto. Such materials preferably have a surface irregularity, as specified by a centerline average roughness Ra value within the range of 0.2 μm-10 μm and a mean spacing Sm value of unevenness within the range of 5 μm-200 μm.
The Ra value refers to a centerline average roughness according to JIS B 0601-1982. The mean spacing Sm value of unevenness is defined as follows.
Mean Spacing Sm Value of Unevenness
The latest JIS provides a standard as to an information of surface roughness in the height direction but provides no standard as to an information of surface roughness in the planar direction. However, the unevenness in the present invention can also be specified by the spacings of irregularity in the planar direction, as will become apparent from the below-given Examples. Accordingly, the present invention utilizes the mean spacing Sm value of unevenness in specifying the degree of irregularity in the planar direction.
The mean spacing Sm value of unevenness is given as follows.
First, an upper count level C and a lower count level D are drawn to locate above and below a center line B of a roughness curve A shown in
As shown in
That is, the mean spacing Sm value of unevenness indicates a mean value of spacing of peaks present in the standard length L. As such, the conditions of irregularity along the planar direction can be defined by the mean spacing Sm value of unevenness.
The surface roughness may be attributed to the porosity of the induction enhancer. Porous polymeric materials such as based on polystyrenes, acrylic esters, polyesters, nylons, celluloses and polyvinyl alcohols are suitable for use as the induction enhancer.
Alternatively, the surface roughness may be attributed to the fibrous form of the material used. Fibrous or non-woven materials are suitable for use as the induction enhancer. Polymeric materials in the fibrous or non-woven form are particularly preferred. Examples include fibrous and non-woven polymeric materials such as composed of polystyrenes, acrylic esters, polyesters, nylons, celluloses and polyvinyl alcohols.
As stated above, induction of cytokines are remarkably enhanced by imparting a proper surface roughness, at least 0.2 μm in terms of an Ra value, to the induction enhancer. The size of a leukocyte is 10 μm-20 μm. When these facts are taken into consideration, it is preferred that the induction enhancer has an Ra value of 0.2 μm-10 μm. Because the specified Ra value is much smaller than the size of a leukocyte, it does not seem that the cytokine induction enhancing effect of such surface roughness simply results from the increase of a contact area.
Examples of cytokine-inducing agents include bacteria and their components such as BCG, BCG-CWS, PPD (Purified protein derivatives Tuberculin), Nocardia-CWS, OK-432 and Muramyldipeptide; polysaccharides such as PSK (Klestin), lentinan and sizofiran; polymers such as poly I:C and poly A:U; and chemical substances such as Levamisole, DNCB, Azimexon, Tilorone and Bestatin. Besides the above physiologically active substances, a variety of substances can also be used as the cytokine-inducing agent, including bacteria, components of bacteria, peptides, nucleic acids, proteins, sugars and lipids.
Among the above-listed substances, bacteria and substances derived from such bacteria are preferred for use as the cytokine-inducing agent.
Acid-fast bacteria and substances derived from acid-fast bacteria are also preferred for use as the cytokine-inducing agents. Among them, tubercule bacilli and substances derived from tubercule bacilli are particularly preferred for use as the cytokine-inducing agent. BCG, an attenuated strain of mycobacterium bovis, and substances derived from BCG are also particularly preferred.
Also, hemolytic streptococci and substances derived from hemolytic streptococci are preferred for use as the cytokine-inducing agent.
Also, actinomycetes and substances derived from actinomycetes are preferred for use as the cytokine-inducing agent.
Some of the above cytokine-inducing agents, if alone, may be difficult to fully exhibit cytokine-inducing capabilities. However, they can exhibit their cytokine-inducing activities, when used in combination with the insoluble induction enhancer. This accordingly permits the use of various substances, other than the conventionally-used cytokine-inducing substances, as the cytokine-inducing agent in the present invention.
Various techniques generally known in the art, such as physical adsorption, covalent bonding and ionic bonding, can be utilized to fix the cytokine-inducing agent onto a surface of the induction enhancer. In the case of covalent bonding or the like, a spacer having an optional length may be introduced at a location where the cytokine-inducing agent is to be bonded to the induction enhancer, if necessary.
The cytokine-inducing agent, if comprised as of bacteria, may be optionally subjected to various pretreatments such as wash, fractionating and grinding operations of bacteria, before it is fixed. The cytokine-inducing agent, if comprised as of viable bacteria, can be subjected to various methods, e.g., heat, chemical, radiation and gas sterilization treatments, either before or during or after it is fixed, to kill those bacteria. The heat treatment can be illustrated by an autoclaving treatment. Examples of chemical treatments include glutaric aldehyde, formalin and ethanol treatments. The radiation and gas sterilization treatments can be illustrated by a γ-ray treatment and an ethylene oxide gas treatment, respectively.
The cytokine-inducing agent comprised of a microorganism such as BCG can be fixed to the induction enhancer by the bonding of amino acid or saccharic substances, which are contained in outer surface walls of bacteria, to a functional group in the induction enhancer such as a carboxylic, amino and/or epoxy group. During this operation, a spacer may be introduced having various chain lengths and structures, when necessary.
When an outer layer of a microorganism such as BCG is covered as with a lipid, this lipid may be optionally removed by wash, before the fixation is carried out.
The preferred fixation technique is a physical adsorption technique. The cytokine-inducing agent can be fixed onto the induction enhancer by the physical adsorption technique. Particularly when the induction enhancer has a hydrophobic surface, physical adsorption can be utilized effectively to fix BCG or the like cytokine-inducing agent onto such an induction enhancer. The cytokine-inducing agent, if comprised of a microorganism or its components, may carry a charge at its surface. In this case, such a cytokine-inducing agent can be fixed to the induction enhancer having an oppositely charged surface by physical adsorption.
The usage of the induction enhancer is not particularly specified. When the induction enhancer is used in a particulate form, a bulk volume of the induction enhancer is generally in the approximate range of 0.02%-80%, preferably 0.1%-50%, relative to a blood volume.
The amount of the cytokine-inducing agent used is not particularly specified. For example, the cytokine-inducing agent, if comprising BCG, is preferably added to blood in the concentration of 0.001mg-10 mg/mL, on a dry weight basis, and, if comprising OK-432, is preferably added to blood in the concentration of 0.0001 KE-10 KE/mL.
When in use of the cytokine-inducing instrument in accordance with the present invention, the cytokine-inducing material, which contains the induction enhancer and cytokine-inducing agent, is allowed to contact with blood, a blood component or the like whereby cytokines are induced effectively in the blood, blood component or the like. In this case, contact temperature is preferably maintained within the range of 15-42° C. This enables more effective induction of cytokines.
The induction enhancer and cytokine-inducing agent may be mixed together before they are allowed to contact with blood. Alternatively, they may be separately allowed to contact with the blood, blood component or the like.
In the present invention, a construction of the container used to accommodate the cytokine-inducing material is not particularly specified. As schematically shown in
It is particularly preferred that the container used to accommodate the cytokine-introducing material is constructed in the form of a column, a blood bag or the like.
More preferably, the cytokine-inducing instrument has a mechanism for preventing outflow of cytokine-inducing material, when blood is allowed to contact with the cytokine-inducing material and then guided to an outside of the container.
As schematically shown in
If a therapeutic need arises, a plasma or serum component may be separated from blood after it is contacted with the cytokine-inducing material.
Specifically, the cytokine-inducing material containing the induction enhancer in a particulate, fibrous or nonwoven form or the like is packed blood bag having an inlet and an outlet. Blood, a blood component or the like is passed through the inlet into the bag. If necessary, the blood, blood component or the like after induction of cytokines may be removed through the outlet for use. The blood bag preferably has a volume of 50 mL-1,000 mL, particularly preferably 100 mL-400 mL. Such a blood bag preferably constitutes the cytokine-inducing instrument by accommodating a particulate, fibrous or nonwoven cytokine-inducing material.
Examples of particularly important cytokines include interferon-γ (IFN-γ), interleukin 2 (IL-2), interleukin 10 (IL-10), interleukin 12 (IL-12), tumor necrosis factor-α (Tumor Necrosis Factor-α, TNF-α) and transforming growth factor-β (TGF-β). For example, IFN-γ is a cytokine which plays a very important role in immune diseases such as rheumatism, inflammatory diseases, allergic diseases, cancers and other various diseases and can be expected as having a therapeutic effect on these diseases.
The above-described cytokine-inducing material and cytokine-inducing instrument can induce cytokines not only from blood, a blood component or the like but also from various cytokine-producing cells, examples of which include cells collected from tissues such as of bone marrow-derived cells, epidermal cells, fibroblasts, hepatocytes, osteoblasts, blood stem cells, embryonic stem cells, cultured cells and established cells.
The following examples illustrate the present invention more specifically but are not intended to be limiting thereof.
In the following examples, IFN-γ, TNF-α, IL-2, IL-10 and IL-12 in human plasma and rat plasma were quantified by using an R&D Systems ELISA kit, an Endogen ELISA kit and a Genzyme Techne ELISA kit. TGF-β in human plasma was quantitatively determined by using a Promega ELISA kit.
An induction enhancer-1 (synthetic aromatic adsorbent, product of Mitsubishi Chemical Corp., product name: DIAION HP-50) was washed via decantation with purified water (product of Otsuka Pharmaceutical Co., Ltd.) and then with methanol (product of Wako Pure Chemicals Industries, Ltd., for HPLC use). The induction enhancer-1 was thereafter washed via decantation with physiological saline for injection (product of Otsuka Pharmaceutical Co., Ltd.). The induction enhancer-1 in the particle bulk volume of 50 μL was then packed in a sterilized tube (product of DIATRON Corp., Eppendorf tube for 1.5 ml use).
Blood was collected from a healthy human to prepare venous blood containing 15 IU/ml of heparin. BCG (product of Japan BCG Manufacturing Co., Ltd.) was added in the concentration of 1 mg/mL of blood. Here, BCG was prepared with physiological saline. A ratio by volume of the physiological saline to blood was brought to 1.25%.
About 1.45 mL of the BCG-containing blood was introduced into the tube packed with the induction enhancer-1.
The tube was tumbled to stir the blood and then attached to a rotary mixer (product of Taitech Corp.) which was subsequently rotated at 6 rpm in a constant temperature vessel to effect incubation at 37° C. for 24 hours. After incubation, the blood was centrifuged at 3,500 rpm (product of Tomy Seiko Co., Ltd., micro high-speed centrifuge MRX-150) at 4° C. for 15 minutes. Blood plasmas were then collected from the blood for cryopreservation at −20° C. The preserved plasmas were then melted and the amount of IFN-γ induced in the plasmas was determined using a Human IFN-γ ELISA kit (product of R&D Systems or ENDOGEN). The results are shown in the following Table 1.
The induction enhancer-1 was excluded and the BCG-incorporated blood was used in the amount of 1.5 mL. Otherwise, the procedure of Example 1 was followed to collect the plasmas and determine the amount of IFN-γ induced in the plasmas. The results are shown in the following Table 1.
The procedure of Example 1 was followed, except that BCG was not added to blood, to determine the amount of IFN-γ induced. The results are shown in the following Table 1.
The duration of incubation at 37° C. was altered from 24 hours to 4 hours. Otherwise, the procedure of Example 1 was followed to obtain cryopreserved plasmas. The cryopreserved plasmas were then melted and the amount of TNF-α induced in the plasmas was determined using a Human TNF-α ELISA kit (product of R&D System). The results are shown in the following Table
The procedure of Example 2 was followed, except that the induction enhancer-1 was excluded and the BCG-incorporated blood was used in the amount of 1.5 mL, to determine the amount of TNF-α induced. The results are shown in the following Table 2.
The procedure of Example 2 was followed, except that BCG was not added to blood, to determine the amount of TNF-α induced. The results are shown in the following Table 2.
The bulk volume of the induction enhancer-1 was altered from 50 μL to 5 μL and the blood was added in the amount of 1.495 mL. Otherwise, the procedure of Example 1 was followed to determine the amount of IFN-γ induced in the plasmas. The results are shown in the following Table 3.
The bulk volume of the induction enhancer-1 was altered from 50 μL to 10 μL and the blood was added in the amount of 1.49 mL. Otherwise, the procedure of Example 1 was followed to determine the amount of IFN-γ induced in the plasmas. The results are shown in the following Table 3.
The bulk volume of the induction enhancer-1 was altered from 50 μL to 20 μL and the blood was added in the amount of 1.48 mL. Otherwise, the procedure of Example 1 was followed to determine the amount of IFN-γ induced in the plasmas. The results are shown in the following Table 3.
The procedure of Example 1 was followed to determine the amount of IFN-γ induced in the plasmas. The results are shown in the following Table 3.
The bulk volume of the induction enhancer-1 was altered from 50 μL to 200 μL and the blood was added in the amount of 1.3 mL. Otherwise, the procedure of Example 1 was followed to determine the amount of IFN-γ induced in the plasmas. The results are shown in the following Table 3.
The induction enhancer-1 was excluded and the BCG-incorporated blood was used in the amount of 1.5 mL. Otherwise, the procedure of Example 3 was followed to determine the amount of IFN-γ induced. The results are shown in the following Table 3.
The procedure of Example 3 was followed, except that BCG was not added to blood, to determine the amount of IFN-γ induced. The results are shown in the following Table 3.
The procedure of Example 4 was followed, except that BCG was not added to blood, to determine the amount of IFN-γ induced. The results are shown in the following Table 3.
The procedure of Example 5 was followed, except that BCG was not added to blood, to determine the amount of IFN-γ induced. The results are shown in the following Table 3.
The procedure of Example 6 was followed, except that BCG was not added to blood, to determine the amount of IFN-γ induced. The results are shown in the following Table 3.
The procedure of Example 7 was followed, except that BCG was not added to blood, to determine the amount of IFN-γ induced. The results are shown in the following Table 3.
The procedure of Example 1 was followed, except that the bulk volume of the induction enhancer-1 was altered to 20 μL, to prepare the tube packed with the induction enhancer-1.
Picibanil (product of Chugai Pharmaceutical Co., Ltd., OK-432), in place of BCG, was added to each blood sample in a concentration of 0.1 KE/mL. This OK-432 was prepared using an RPMI medium. The ratio of the RPMI medium to blood was 20%. The OK-432 incorporated blood was poured into the tube packed with a 20 μL bulk volume of the induction enhancer-1 to a tube scale of 1.5 mL. That is, the blood was added in the amount of about 1.48 mL.
Then, the amount of IFN-γ induced was determined in the same manner as in Example 1. The results are shown in the following Table 4.
The bulk volume of the induction enhancer-1 was altered to 50 μL and the OK-432 incorporated blood was used in the amount of 1.45 mL. Otherwise, the procedure of Example 8 was followed to determine the amount of IFN-γ induced. The results are shown in the following Table 4.
The bulk volume of the induction enhancer-1 was altered to 100 μL and the OK-432 incorporated blood was used in the amount of 1.40 mL. Otherwise, the procedure of Example 8 was followed to determine the amount of IFN-γ induced. The results are shown in the following Table 4.
The induction enhancer-1 was excluded and the blood incorporated OK-432 was used in the amount of 1.5 mL. Otherwise, the procedure of Example 8 was followed to determine the amount of IFN-γ induced. The results are shown in the following Table 4.
The procedure of Example 8 was followed, except that Picibanil (OK-432) was not added, to determine the amount of IFN-γ induced. The results are shown in the following Table 4.
The procedure of Example 9 was followed, except that Picibanil (OK-432) was not added, to determine the amount of IFN-γ induced. The results are shown in the following Table 4.
The procedure of Example 10 was followed, except that Picibanil (OK-432) was not added, to determine the amount of IFN-γ induced. The results are shown in the following Table 4.
An induction enhancer-2 (synthetic acrylic ester resin, product of Organo Corp., product number: AMBERLITE XAD-7), in place of the induction enhancer-1, was used. Otherwise, the procedure of Example 1 was followed to determine the amount of IFN-γ induced in the plasmas. The results are shown in the following Table 5.
The bulk volume of the induction enhancer-2 was altered from 50 μL to 200 μL and the BCG-incorporated blood was used in the amount of 1.3 mL. Otherwise, the procedure of Example 11 was followed to determine the amount of IFN-γ induced. The results are shown in the following Table 5.
The induction enhancer-2 was excluded and the BCG-incorporated blood was used in the amount of 1.5 mL. Otherwise, the procedure of Example 11 was followed to determine the amount of IFN-γ induced. The results are shown in the following Table 5.
The procedure of Example 11 was followed, except that BCG was not added to blood, to determine the amount of IFN-γ induced. The results are shown in the following Table 5.
The procedure of Example 12 was followed, except that BCG was not added to blood, to determine the amount of IFN-γ induced. The results are shown in the following Table 5.
The following Examples and Comparative Examples illustrate induction of cytokines in rat blood.
The procedure of Example 1 was followed to obtain the sterilized tube packed with a 50 μL bulk volume of the induction enhancer-1.
Venous blood containing 15 IU/ml of heparin was collected from a Wister rat (7 weeks old, male, purchased from Japan SLC, Inc.). Picibanil (product of Chugai Pharmaceutical Co., Ltd., OK-432) was added to the blood in the concentration of 0.1 KE/mL. This OK-432 was prepared using an RPMI medium. The ratio of the RPMI medium to blood was 20%. The OK-432 incorporated blood was added to the tube, packed with the induction enhancer-1, to a tube scale of 1.5 mL.
Subsequently, the procedure of Example 1 was followed to collect blood plasmas and determine the amount of IFN-γ induced therein by using a Rat IFN-γ quantification kit (product of Genzyme Techne). The results are shown in the following Table 6.
The bulk volume of the induction enhancer-1 was altered to 500 μL and the OK-432 incorporated blood was used in the amount of 1.0 mL. Otherwise, the procedure of Example 13 was followed to determine the amount of IFN-γ induced. The results are shown in the following Table 6.
The induction enhancer-1 was excluded and the OK-432 incorporated blood was used in the amount of 1.5 mL. Otherwise, the procedure of Example 13 was followed to determine the amount of IFN-γ induced. The results are shown in the following Table 6.
The procedure of Example 13 was followed, except that OK-432 was not added to the blood, to determine the amount of IFN-γ induced. The results are shown in the following Table 6.
The procedure of Example 14 was followed, except that OK-432 was not added to the blood, to determine the amount of IFN-γ induced. The results are shown in the following Table 6.
The procedure of Example 1 was followed to obtain the sterilized tube packed with a 50 μL bulk volume of the induction enhancer-1.
Blood was collected from a Wister rat (7 weeks old, male, purchased from Japan SLC, Inc.) to obtain venous blood containing 15 IU/ml of heparin . BCG was added to this blood in the concentration of 1 mg/mL. This BCG was prepared using physiological saline. The ratio in volume of the physiological saline to the blood was 1.25%. The BCG-incorporated blood was added to the tube, packed with the induction enhancer-1, to a tube scale of 1.45 mL.
Subsequently, the procedure of Example 1 was followed to collect blood plasmas and determine the amount of IFN-γ induced therein by using a Rat IFN-γ quantification kit (product of Genzyme Techne). The results are shown in the following Table 7.
The bulk volume of the induction enhancer-1 was altered from 50 μL to 500 μL and the BCG-incorporated blood was used in the amount of 1.0 mL. Otherwise, the procedure of Example 15 was followed to determine the amount of IFN-γ induced. The results are shown in the following Table 7.
The induction enhancer-1 was excluded and the BCG-incorporated blood was used in the amount of 1.5 mL. Otherwise, the procedure of Example 15 was followed to determine the amount of IFN-γ induced. The results are shown in the following Table 7.
The procedure of Example 15 was followed, except that BCG was not added to the blood, to determine the amount of IFN-γ induced. The results are shown in the following Table 7.
The procedure of Example 16 was followed, except that BCG was not added to the blood, to determine the amount of IFN-γ induced. The results are shown in the following Table 7.
The duration of incubation at 37° C. was altered from 24 hours to 4 hours. Otherwise, the procedure of Example 15 was followed to collect blood plasmas and determine the amount of TNF-α induced therein by using a Rat Tumor Necrosis Factor-α quantification kit (product of Genzyme Techne). The results are shown in the following Table 8.
The induction enhancer-1 was excluded and the BCG-incorporated blood was used in the amount of 1.5 mL. Otherwise, the procedure of Example 17 was followed to collect blood plasmas and determine the amount of TNF-α induced therein. The results are shown in the following Table 8.
The procedure of Example 17 was followed, except that BCG was not added to the blood, to determine the amount of TNF-α induced. The results are shown in the following Table 8.
Induction Enhancer-3: A CA film (product of Artplus Corp., cellulose acetate film, product name: ACETYFILM VR-R) was used. A plasticizer was extracted from the film by soxhlet extraction for 24 hours in methyl alcohol. After removal, the film was air dried for 15 hours and further dried at 80° C. for 5 hours. Thereafter, the CA film was polished, using a Struers (Denmark) automatic polisher (product name: PLANOPOL-PEDEMAX) with a220-, 500-, 1200-, 2400- or 4000-mesh sandpaper secured thereto, to provide a polished CA film having surface roughness on its both surfaces. This film was washed with methyl alcohol and then subdivided to 2.5 mm×2.5 mm pieces. These pieces, measuring a bulk volume of about 200 μL, were washed with physiological saline for injection and then packed in a sterilized tube for 1.5 mL use.
Blood was collected from a healthy human to prepare venous blood containing 15 IU/ml of heparin. BCG (product of Japan BCG Manufacturing Co., Ltd.) was added to the blood in the concentration of 1 mg/mL. About 1.3 mL of the resulting blood was added to the tube. Subsequently, the amount of IFN-γ induced was determined in the same manner as in Example 1. The results are shown in the following Table 9.
Induction Enhancer-4: A PET film (product of Unitika Ltd., polyethylene terephthalate film, product name: EMBLET S-75) was used. The film was at its surfaces washed with methyl alcohol. Thereafter, this PET film was polished, using a Struers (Denmark) automatic polisher (product name: PLANOPOL-PEDEMAX) with a220-, 500-, 1200-, 2400- or 4000-mesh sandpaper secured thereto, to provide a polished CA film having surface roughness on its both surfaces. This film was washed with methyl alcohol and then subdivided to 2.5 mm×2.5 mm pieces. These pieces, measuring a bulk volume of about 200 μL, were washed with physiological saline for injection and then packed in a sterilized tube for 1.5 mL use. Blood was collected from a healthy human to obtain venous blood containing 15 IU/ml of heparin. BCG (product of Japan BCG Manufacturing Co., Ltd.) was added to the blood in the concentration of 1 mg/mL. About 1.3 mL of the resulting blood was added to the tube. The procedure of Example 1 was then followed to determine the amount of IFN-γ induced. The results are shown in the following Table 10.
Induction Enhancer-5: A polystyrene film (product of Mitsubishi-Monsanto Co., Ltd., product name: SANTOCLEAR) was used. The film was at its surfaces washed with purified water. Thereafter, the polystyrene film was polished, using a Struers (Denmark) automatic polisher (product name: PLANOPOL-PEDEMAX) with a 220-, 500-, 1200-, 2400- or 4000-mesh sandpaper secured thereto, to provide a polished polystyrene film having surface roughness on its both surfaces. This film was washed with purified water and then subdivided to 2.5 mm×2.5 mm pieces. These pieces, measuring a bulk volume of about 200 μL, were washed with physiological saline for injection and then packed in a sterilized tube for 1.5 mL use. Blood was collected from a healthy human to obtain venous blood containing 15 IU/ml of heparin. BCG (product of Japan BCG Manufacturing Co., Ltd.) was added to the blood in the concentration of 1 mg/mL. About 1.3 mL of the resulting blood was added to the tube. The procedure of Example 1 was then followed to determine the amount of IFN-γ induced. The results are shown in the following Table 11.
Cellulose acetate pellets (product of Artplus Corp., containing 30% acetyl triethyl citrate as a plasticizer) were injection molded to prepare 2.5 mm diameter spherical beads. The plasticizer was extracted from 50 g of these beads by soxhlet extraction at 50° C. for 24 hours in 300 mL methanol. Subsequent to extraction of the plasticizer, those beads were transferred 15 onto a stainless-steel batting, air dried for 15 hours and then further dried at 80° C. for 5 hours.
200 mL of such beads and an equal volume of an abrasive WHITE ABRAX (WA) #34 (product of Japan Abrasive Company) were introduced into a pot mill (product of Toyo Engineering Corp., product name: 51-ceramic pot mill BP-5). Also, several balls (product of Toyo Engineering Corp., product name: BB-13) for a ceramic pot mill were introduced. Polishing was applied for 5 hours by a ball polishing machine (pot mill manufactured by Nitto Kagaku Co., Ltd., product name: AN-3S). As a result, beads were obtained having an Ra value of 1.36 μm and an Sm value of 97.2 μm (induction enhancer-6).
The obtained beads were washed three times with methanol and then five times with physiological saline for injection. These beads, measuring a bulk volume of 400 μL, were packed in a sterilized tube for 1.5 mL use in the same manner as in Example 1. The procedure of Example 1 was then followed, except that the blood was added in the amount of 1.1 mL, to determine the amount of IFN-γ induced. The results are shown in the following Table 12.
The procedure of Example 36 was followed, except that polishing was not applied, to prepare the induction enhancer-6 having an Ra value of 0.186 μm and an Sm value of 298.7 μm. This induction enhancer was allowed to contact with the blood in the same manner as in Example 36. The results are shown in the following Table 12.
The induction enhancer-6 was excluded and the BCG-incorporated blood was used in the amount of 1.5 mL. Otherwise, the procedure of Example 36 was followed to determine the amount of IFN-γ induced. The results are shown in the following Table 12.
The procedure of Example 1 was followed, using the induction enhancer-1 (synthetic aromatic adsorbent, product of Mitsubishi Chemical Corp., product name: DIAION HP-50), the induction enhancer-2 (synthetic acrylic ester resin, product of Organo Corp., product number: AMBERLITE XAD-7), an induction enhancer-7 (synthetic aromatic resin, product of Organo Corp., product number: AMBERLITE XAD-2), an induction enhancer-8 (synthetic aromatic resin, product of Organo Corp., product number: AMBERLITE XAD-4) or an induction enhancer-9 (synthetic aromatic resin, product of Organo Corp., product number: AMBERLITE XAD-16HP). The results are shown in the following Table 13.
The induction enhancer was excluded and the BCG-incorporated blood was used in the amount of 1.5 mL. Otherwise, the procedure of Example 38 was followed. The results are shown in the following Table 13.
BCG was suspended in physiological saline, diluted with a phosphate buffer and centrifuged at 4,000 rpm for 15 minutes. After removal of a supernatant liquid, the resultant was again suspended in a phosphate buffer and centrifuged at 4,000 rpm (product of Tomy Seiko Co., Ltd., micro high-speed centrifuge MRX-150) for 15 minutes. This operation was repeated three times. The resultant was suspended in a phosphate buffer containing 50% ethanol and then centrifuged at 4,000 rpm for 15 minutes. Subsequently, the resultant was suspended in ethanol and centrifuged at 4,000 rpm for 15 minutes. This operation was repeated twice. Wash with a phosphate buffer was again carried out three times. Such treated BCG in the amount of 2 mg was covalently bonded to the carboxyl-introduced induction enhancer-1 (bulk volume of 1 mL) by a carbodiimide process. The remaining reactive groups were reacted with ethanolamine. The resultant was washed with a phosphate buffer and then suspended in a phosphate buffer. The thus-obtained induction enhancer, measuring 100 μL, was packed in a sterilized tube.
BCG was not added in its individual form. The blood was added in the amount of 1.4 mL. Otherwise, the procedure of Example 1 was followed to determine the amount of IFN-γ induced. The results are shown in Table 14.
The procedure of Example 43 was followed, except that the treated BCG was not covalently bonded, to determine the amount of IFN-γ induced. The results are shown in Table 14.
The induction enhancer was not added and the BCG-incorporated (1 mg/mL) blood was used in the amount of 1.5 mL. Otherwise, the procedure of Example 43 was followed to determine the amount of IFN-γ induced. The results are shown in Table 14.
The polystyrene-divinylbenzene copolymer induction enhancer-8 (product of Organo Corp., product number: AMBERLITE XAD-4), measuring a bulk volume of 1 mL, and BCG (10 mg/mL) were intimately mixed in 1 mL physiological saline at 37° C. for 20 hours, so that BCG was physically adsorbed onto particle surfaces. After the intimate mixing, these particles were fully washed with physiological saline and again suspended in physiological saline. The thus-obtained induction enhancer, measuring a bulk volume of 100 μL, were packed in a sterilized tube.
BCG was not added in its individual form. The blood was added in the amount of 1.4 mL. Otherwise, the procedure of Example 1 was followed using blood from two healthy humans to determine the amount of IFN-γ induced. The results are shown in Table 15.
The procedure of Example 44 was followed, except that BCG was not added, to prepare particles. Using thus-obtained particles, the amount of IFN-γ induced was determined in the same manner as in Example 44. The results are shown in Table
The induction enhancer was not added and the BCG-incorporated (1 mg/mL) blood was used in the amount of 1.5 mL. Otherwise, the procedure of Example 44 was followed to determine the amount of IFN-γ induced. The results are shown in Table 15.
The polystyrene-divinylbenzene copolymer induction enhancer-8, measuring a bulk volume of 1 mL, and BCG (10 mg/mL) were intimately mixed in 1 mL physiological saline containing 1% formalin (neutral buffer formalin, product of Wako Pure Chemicals Industries, Ltd.) at 4° C. for 20 hours, so that BCG was physically adsorbed onto particle surfaces. Subsequent to the intimate mixing, these particles were fully washed with physiological saline. Thereafter, those particles, measuring a bulk volume of 100 μL, were packed in a sterilized tube (product of DIATRON Corp., for 1.5 mL use).
BCG was not added in its individual form. The blood was added in the amount of 1.4 mL. Otherwise, the procedure of Example 1 was followed to determine the amount of IFN-γ induced. The results are shown in Table 16.
The procedure of Example 45 was followed, except that BCG was not added, to determine the amount of IFN-γ induced. The results are shown in Table 16.
The induction enhancer-8 was excluded and the BCG-incorporated (1 mg/mL) blood was used in the amount of 1.5 mL. Otherwise, the procedure of Example 45 was followed to determine the amount of IFN-γ. The results are shown in Table 16.
The induction enhancer-8 was treated in the same manner as in Example 45 so that BCG was physically adsorbed onto particle surfaces. The treated induction enhancer, measuring a bulk volume of 4 mL, was packed in a blood bag (product of Terumo Corp., separation bag for200 mL use). Blood was collected from a healthy human to obtain venous blood containing 15 IU/mL of heparin. 50 mL of the venous blood was introduced into the blood bag. The blood bag contents were incubated while gently stirred at 37° C. for 24 hours. The amount of IFN-γ present in the blood was determined in the same manner as in Example 1. The results are shown in Table 17.
The induction enhancer-8 was used without the physical adsorption treatment of BCG. Otherwise, the procedure of Example 46 was followed. The results are shown in Table 17.
The induction enhancer-8 was excluded and the blood containing 1 mg/mL of BCG was used in the amount of 50 mL. Otherwise, the procedure of Example 46 was followed to determine the amount of IFN-γ. The results are shown in Table 17.
The procedure of Example 1 was followed, except that interleukin 2 and interleukin 12 present in the blood were quantitatively determined. The results are shown in Table 18.
The procedure of Example 47 was followed, except that BCG was not added. The results are shown in Table 18.
The induction enhancer-1 was not added and the BCG-incorporated blood was used in the amount of 1.5 mL. Otherwise, the procedure of Example 47 was followed. The results are shown in Table 18.
The procedure of Example 1 was followed, except that the BCG concentration was altered to 0.1 mg/mL and TGF-β present in the blood was quantitatively determined. The results are shown in Table 19.
The induction enhancer-1 was not added and the BCG-incorporated blood plasma was used in the amount of 1.5 mL. Otherwise, the procedure of Example 48 was followed. The results are shown in Table 19.
The procedure of Example 48 was followed, except that OK-432, in place of BCG, was used in the amount of 0.1 KE/mL. The results are shown in Table 20.
The induction enhancer-1 was not added and the OK-432 incorporated blood was used in the amount of 1.5 mL. Otherwise, the procedure of Example 49 was followed. The results are shown in Table 20.
The procedure of Example 1 was followed, except that OK-432, in place of BCG, was used in the amount of 0.01 KE/mL and IL-10 was quantitatively determined. The results are shown in Table 21.
The induction enhancer-1 was not added and the OK-432 incorporated blood was used in the amount of 1.5 mL. Otherwise, the procedure of Example 50 was followed. The results are shown in Table 21.
The procedure of Example 1 was followed, except that BCG was added in the concentration of 0.1 mg/mL or 1 mg/mL. The results are shown in Table 22.
The procedure of Example 51 was followed, except that BCG was not added. The results are shown in Table 22.
The induction enhancer-1 was not added and the BCG-incorporated (0.1 mg/mL) blood was used in the amount of 1.5 mL. Otherwise, the procedure of Example 51 was followed. The results are shown in Table 22.
The induction enhancer-1 was not added and the BCG-incorporated (1 mg/mL) blood was used in the amount of 1.5 mL. Otherwise, the procedure of Example 52 was followed. The results are shown in Table 22.
The procedure of Example 1 was followed, except that OK-432, in place of BCG, was added in the concentration of 0.01 KE/mL or 0.1 KE/mL. The results are shown in Table 23.
The procedure of Example 53 was followed, except that OK-432 was not added. The results are shown in Table 23.
The induction enhancer-1 was not added and the OK-432 incorporated (0.01 KE/mL) blood was used in the amount of 1.5 mL. Otherwise, the procedure of Example 53 was followed. The results are shown in Table 23.
The induction enhancer-1 was not added and the OK-432 incorporated (0.1 KE/mL) blood was used in the amount of 1.5 mL. Otherwise, the procedure of Example 54 was followed. The results are shown in Table 23.
0.04 g (about 300 μL in terms of a bulk volume when packed in a 1.5 mL tube) of nylon wool (product of Wako Pure Chemicals Industries, Ltd.) was used as an induction enhancer-10. The BCG-incorporated blood was added in the amount of 1.2 mL. Otherwise, the procedure of Example 1 was followed. The results are shown in Table 24.
0.04 g (1 cm×3 cm, about 300 μL in terms of a bulk volume) of a polyester nonwoven fabric (product of Japan Vilene Co., Ltd., product designation: EL-5600) was used as an induction enhancer-11. The BCG-incorporated blood was added in the amount of 1.2 mL. Otherwise, the procedure of Example 1 was followed. The results are shown in Table 24.
0.04 g (1 cm×3 cm, about 220 μL in terms of a bulk volume) of a polyester nonwoven fabric (product of Japan Vilene Co., Ltd., product designation: EW-7180) was used as an induction enhancer-12. The BCG-incorporated blood was added in the amount of 1.28 mL. Otherwise, the procedure of Example 1 was followed. The results are shown in Table 24.
The procedure of Example 55 was followed, except that BCG was excluded. The results are shown in Table 24.
The procedure of Example 56 was followed, except that BCG was excluded. The results are shown in Table 24.
The procedure of Example 57 was followed, except that BCG was excluded. The results are shown in Table 24.
The induction enhancer was excluded and the BCG-incorporated blood was used in the amount of 1.5 mL. Otherwise, the procedure of Example 55 was followed. The results are shown in Table 24.
Actinomyces S. nobilis (JCM 4274) obtained from Institute of Physical and Chemical Research was shake cultured at 30° C. for 40 hours in 100 mL of a starch-ammonium medium containing 0.2% yeast extract to obtain bacteria. The procedure of Example 1 was followed, except that such bacteria, in place of BCG, were added to blood in the dry weight of 1 mg/mL, to determine the amount of IFN-γ. The results are shown in Table 25.
The procedure of Example 58 was followed, except that the bacteria of actinomyces were excluded. The results are shown in Table 25.
The induction enhancer-1 was excluded and the actinomyces incorporated blood was used in the amount of 1.5 mL. Otherwise, the procedure of Example 58 was followed. The results are shown in Table 25.
As an induction enhancer-13 (gel particles of polyvinyl alcohol, particle diameter of about 1.1 mm), gel particles of polyvinyl alcohol-Fe(III) complex were prepared in accordance with “Complex gels of PVA and PAA” by Hiroshi Yokoi (Polymer Processing, Vol.40, No.11, 1991). The induction enhancer-13 was suspended in physiological saline. Subsequently, gel particles of polyvinyl alcohol-Fe(III) complex, measuring a bulk volume of 300 μL, were packed in a sterilized tube for 1.5 mL use. The procedure of Example 1 was then followed, except that the blood was added in the amount of 1.2 mL. The results are shown in Table 26.
The procedure of Example 58 was followed, except that BCG was not added. The results are shown in Table 26.
The induction enhancer-13 was excluded and the BCG-incorporated blood was added in the amount of 1.5 mL. Otherwise, the procedure of Example 58 was followed. The results are shown in Table 26.
Cytokines can be induced more effectively by the use of the cytokine-inducing material in accordance with the present invention than by conventional cytokine-inducing agents. Because the cytokine-inducing material of the present invention, when allowed to contact with blood or a blood component, can induce cytokines effectively, the cytokine-inducing material and cytokine-inducing instrument in accordance with the present invention can be suitably used to treat various diseases for which induction of cytokines is therapeutically effective.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2001-338016 | Nov 2001 | JP | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP02/11335 | 10/31/2002 | WO |
