JP4612924B2 - Cytokine regulator - Google Patents

Description

[0001]
[Industrial application fields]
The present invention provides a cytokine regulatory agent comprising a rabbit skin inoculated with vaccinia virus as an active ingredient.
[0002]
[Prior art]
Cytokines are glycoproteins that are produced from various cells including immunocompetent cells and have a very small amount of biological activity that is indispensable for cell-cell interactions. Cytokines produced from lymphocytes are lymphokines, monocytes, and macrophages. Cytokines produced from cell lines are also called monokines. There are both a positive regulatory mechanism and a negative regulatory mechanism in the biological activity of cytokines. Under normal conditions, the regulatory mechanisms operate in a well-balanced manner and form a network with each other, and are responsible for homeostasis.
[0003]
However, when this balance is lost, abnormal cytokine production may occur, or various diseases may be formed or worsened. For example, when the negative regulatory mechanism (control mechanism) for the produced tumor necrosis factor (TNF-α) does not function sufficiently, TNF-α is interleukin-1 (IL-1), interleukin via a cytokine network. Chain reaction in which the production of various other inflammatory cytokines such as IL-6 (IL-6) and interleukin-8 (IL-8) is promoted, and the produced signokine promotes the production of TNF-α again. It is said that this causes a vicious cycle of inflammation and eventually destroys tissues such as bone and cartilage, leading to rheumatoid arthritis, which is an autoimmune disease.
[0004]
[Problems to be solved by the invention]
At present, cytokines involved in pathogenesis, exacerbation, and pain have been identified in various diseases. Therefore, research to apply cytokines to diagnosis and treatment of diseases, and further production of excess cytokines In addition, active research is being conducted on the suppression of the activity, supplementation of deficient / deficient cytokines, promotion of production, and the like.
[0005]
Among various cytokines, transforming growth factor-β (TGF-β) is a cytokine found as a growth factor for fibroblasts, but in genetically engineered TGF-β _1- deficient mice. Multiple inflammation in multiple organs or multiple organ failure (Nature, 359 : 693, 1992), and osteoblast proliferation, promotion of extracellular matrix synthesis substrate secretion, production of substrate degrading enzymes Inhibition, granulation of various tissues, fibrosis promotion action and tissue damage repair action such as wound or burn based on cell differentiation action, therapeutic action against retinal detachment based on adhesion factor expression promotion action, suppression action of excessive immune response, The effect of shifting the T lymphocyte cell group to the Th ₂ cell group has been observed. Further, recently, significant reduction of active TGF-beta levels in the serum was observed in the severe arteriosclerosis (Nature Medicine (Nature Medicine), 1: 74,1995 ), the formation of arteriosclerosis, even exacerbation TGF It is speculated that a decrease in -β level is involved. As the mechanism of action of the biological activity of TGF-β, TGF-β exhibits a strong cell growth inhibitory action on epithelial cells, vascular endothelial cells, lymphocytes, and other hematopoietic cells. It is important to suppress the production of cytokines derived therefrom.
[0006]
Interleukin-10 (IL-10) is a cytokine produced mainly from the Th ₂ cell group among the cell group consisting of Th ₁ and Th ₂ of T lymphocytes which are immunocompetent cells. Regulatory action of the mechanism, CD8 ⁺ cytotoxic T cell, antibody producing cell, promoting action on mast cell, inhibitory action on Th ₁ cell and macrophage, Th ₁ cell-derived cytokine interferon-γ (IFN-γ) and It is known to suppress the production of macrophage-derived cytokines such as TNF-α, IL-1, and IL-6. These TNF-α and IL-1 are known as cytokines whose production mechanism or biological activity overlaps well, and TNF-α, IL-1, and IL-6 are human ulcerative colitis. It is said to be deeply involved in the formation and exacerbation of diseases such as inflammatory bowel diseases such as Crohn's disease, perforated peritonitis, and poor prognosis from sepsis due to Gram-negative bacteria. In human ulcerative colitis and Crohn's disease, the serum level of IL-10 that suppresses the production of TNF-α, IL-1, and IL-6 is significantly reduced ( clinical immunity , 27 , 16 : 97, 1995), IL-10 gene-deficient mice are sure to develop autoimmune bowel disease (Cell, 75 : 263, 1993), and IL-10 suppresses pain caused by these cytokines. (Brit. J. Pharmacol., 115 : 684, 1995) and the like, the promotion of IL-10 production is due to the excess of cytokines produced from these macrophage cells and Th ₁ cells. It has been clarified that it is useful for diseases that cause onset and progress as a trigger.
[0007]
[Means for Solving the Problems]
The inventors of the present invention have a potent cell growth inhibitory effect on epithelial-derived cells, vascular endothelial cells, lymphocytes and other hematopoietic cells in a drug substance whose active ingredient name is called rabbit extract inoculated with vaccinia virus. , Promoting the production of TGF-β that suppresses the production of cytokines such as inflammatory or cytotoxicity derived from these cells, and also acts to suppress the production or activity of cytokines derived from macrophage cells or Th ₁ cells It was found that there is an activity to promote the production of IL-10 and suppress the production of IFN-γ and TNF-α, and the present invention was completed.
[0008]
Rabbit inflammation skin inoculated with vaccinia virus is a known substance described in, for example, page 1710 of the October 1997 edition of the Japan Medicine Collection. Analgesic and sedative, especially as a drug that acts specifically on stressed nerve tissue, the content is expressed in “units” indicating the titer, or substantially equivalent to the “units” It is expressed as “neurotropin unit” and is marketed as a medical drug (hereinafter referred to as “unit”). However, until now, there has been no known regulatory effect on cytokines TGF-β, IL-10, IFN-γ and TNF-α.
[0009]
The dose of the rabbit skin inoculated vaccinia virus inoculated according to the present invention varies depending on the type of disease, the sensitivity of the individual patient, and the level of cytokine to be adjusted, but can be increased or decreased in the range of 1 to 60 units / day. Yes, and can be administered in admixture with a pharmaceutical carrier such as a suitable organic or inorganic solid or liquid excipient. Usually, 3 to 32 units / day, preferably 3.6 to 16 units / day are administered intravenously, subcutaneously, intramuscularly or orally once a day or divided into 2 to 4 times.
[0010]
EXAMPLES The present invention will be described in detail below with reference to examples, but these examples do not limit the scope of the present invention. Unless otherwise specified, the “extract” means the inflammatory skin extract of rabbits inoculated with the vaccinia virus of the present invention.
[0011]
[Example 1]
8-15 weeks old (C57BL / 6 X DBA / 2) F ₁ female mice were given a single intraperitoneal injection of 12.5 units / kg of vaccinia virus inoculated rabbit inflammation skin extract (extract) of the present invention. In some cases and when administered intraperitoneally every day for 3 days, the mice were bled 24 hours after administration and serum was collected. A control group of mice was administered physiological saline for injection in the same volume as the administered extract. Use 3 mice in each group. Spleens were collected from the same mice from which the serum was collected, and the total RNA was immediately extracted and purified by a conventional method and stored at −80 ° C. to prepare a sample for reverse transcription reaction-polymerase chain reaction (RT-PCR). TGF-β1 gene-specific downstream primer was added to ₁ μg of each mouse spleen RNA, reverse transcriptase was allowed to act to synthesize cDNA, upstream primer was further added, and cDNA was amplified using thermostable DNA synthase. . The cDNA of each sample was fractionated by 2% agar gel electrophoresis, and the concentration of a 525 base pair band was measured with a densitometer. Similarly, the glyceraldehyde-3-phosphate dehydrogenase (G3PDH) gene was synthesized from the RNA of each sample as an internal standard by reverse transcription reaction, and then the cDNA was amplified. After measuring the concentration of the 983 base pair G3PDH band fractionated by agar gel electrophoresis with a densitometer, the concentration ratio of the TGF-β ₁ band to the G3PDH band was calculated for each sample.
[0012]
As a result, the TGF-β ₁ band of each sample fractionated by 2% agar gel electrophoresis showed a darker band in the cDNA product group of the extract-administered group than in the control group. TGF-beta ₁ gene transcription is promoted. The average value of the concentration ratio of TGF-β ₁ band and G3PDH band by densitometer measurement is about 0.9 for the extract-administered group, about 0.7 for the control group, and the average value of the extract-administered group is The value was higher than the average value of the control group (FIG. 1). Repeated peritoneal administration experiment of the same extract again at different times, and intraperitoneal administration of vaccinia virus-inoculated rabbit inflammation skin extract promoted TGF-β ₁ gene transcription statistically significantly (p <0.01) (Fig. 2).
[0013]
[Figure 1]
[0014]
[Figure 2]
[0015]
[Example 2]
Two (C57BL / 6 X DBA / 2) F ₁ female mice aged 8 to 15 weeks were given 12.5 units / kg of rabbit skin inoculated vaccinia virus-inoculated rabbit extract of the present invention (extract) per day. It was administered intraperitoneally every day for 3 days. After 24 hours, the spleen was collected and the cells were cultured for 48 hours. Two mice in the control group were similarly administered with the same volume of physiological saline for injection as that of the administered extract. The culture supernatant was recovered, and the biological activity of TGF-β was determined as mink lung fibroblast growth inhibitory activity. The sample from one animal was quantified twice, and the average value was calculated. Alternatively, after collecting the spleen, spleen cells were isolated and cultured with concanavalin A for 24 hours, and then the culture supernatant was collected. The amount of active TGF-β protein in the spleen cell culture supernatant was determined by sandwich solid-phase enzyme immunization (ELISA) assay.
[0016]
It was confirmed that administration of the extract not only promotes TGF-β gene transcription but also promotes TGF-β protein production. Since this biological activity measurement method detects the total TGF-β activity of TGF-β ₁ , TGF-β ₂ , and TGF-β ₃ , TGF-β not only for TGF-β ₁ but also for the administration of the extract is used. _2, the effect of the TGF-β ₃ production also means that it is possible to estimate. Calibration curve showing the correlation between the biological activity and TGF-beta protein content was made using a genetic recombinant human TGF-beta _1. For the measurement of active TGF-β, the culture supernatant is used as it is as a sample, and the total TGF-β is measured by treating the culture supernatant with hydrochloric acid to convert latent TGF-β to the active form. It was. In the extract administration group of this experiment, active TGF-β was 907,7 pg / ml and total TGF-β was 1226.4 pg / ml, whereas in the control group, 135.5 pg / ml, It was 639.6 pg / ml (FIG. 3). Thus, administration of the extract promoted both active and latent TGF-β production from splenocytes.
[0017]
[Fig. 3]
[0018]
[Example 3]
8 to 15-week-old (C57BL / 6 X DBA / 2) F ₁ female mice are injected once intraperitoneally with 25 units / kg of vaccinia virus-inoculated rabbit inflammatory skin extract (extract) of the present invention. At about the same time, 6.25 mg / kg of concanavalin A or physiological saline for injection was injected intradermally. On the third day after administration, blood was collected from each mouse, and serum was separated to prepare a test sample. As a result of measuring the amount of active TGF-β ₁ protein in serum using recombinant human TGF-β ₁ for preparing a calibration curve using the sandwich ELISA method, The active TGF-β _{1 in} the serum of the extract-administered group is 8.76 ng / ml, the control group is 7.12 ng / ml, and the concanavalin A-administered group is the active TGF-β in the serum of the extract-administered mouse. ₁ was 7.86 ng / ml, and 7.05 ng / ml in the control group (FIG. 4). Thus, administration of the extract resulted in a statistically significant increase in serum TGF-β level regardless of the presence or absence of concanavalin A co-administration (p <0.05). Concanavalin A co-administration did not affect serum TGF-β levels.
[0019]
[Fig. 4]
[0020]
[Example 4]
8-15 weeks old (C57BL / 6 X DBA / 2) F ₁ female mice are treated with 12.5 units / kg of vaccinia virus inoculated rabbit inflammation skin extract (extract) of the present invention once a day for 3 days. After continuous intraperitoneal administration, the spleen was collected 24 hours later, immediately extracted and purified with total RNA by a conventional method, stored at −80 ° C., and used as a RT-PCR sample. A control group of mice was administered the same volume of physiological saline for injection as the extract. Use 3 mice in each group. An IL-10 gene-specific downstream primer was added to 1 μg of each mouse spleen RNA, and a reverse transcriptase was allowed to act to synthesize cDNA. Then, an upstream primer was further added, and the cDNA was amplified using a thermostable DNA synthase. Similarly, for the G3PDH gene as an internal standard from the total RNA of each sample, cDNA was synthesized by reverse transcription reaction and then amplified. The cDNA of each sample was fractionated by 2% agar gel electrophoresis, and the concentration of the 421 base pair band of IL-10 was measured with a densitometer. In the 2% agar gel electrophoresis photograph of the cDNA product after RT-PCR reaction of IL-10 and G3PDH, the cDNA product of the extract-administered group (lanes 4, 5, 6) is more control group (lanes 1, 2). 3) A darker band was shown. After measuring the band concentration with a densitometer, the concentrations of IL-10 band and G3PDH band for each sample were compared. As a result, when the band concentration ratio average value of the control group was 1.0, the average of the extract administration group The value was 2.62, and IL-10 gene transcription was significantly (p <0.01) promoted by the extract administration (FIG. 5).
[0021]
[Figure 5]
[0022]
[Example 5]
Experiment for confirming the promotion of IL-10 gene transcription by single administration of 12.5 units / kg of rabbit skin inoculated vaccinia virus-inoculated rabbit extract of the present invention
Using two mice in each group, RT-PCR was performed on IL-10 gene from 1 μg of spleen mRNA in the same manner as in the experiment of Example 4 except that the number of administrations was single administration. As a result of comparison with the physiological saline control group, when the concentration ratio of IL-10 and G3PDH band in the untreated group was 1.0, the physiological saline-administered group 1.31 and the extract-administered group 2.29 three times Although the degree was lower than that of administration, a tendency to promote IL-10 gene transcription was observed even after single administration (FIG. 6).
[0023]
[Fig. 6]
[0024]
[Example 6]
Vaccinia virus inoculated rabbit inflammation skin extract (extract) 10, 25, 50 units / kg of the present invention was intraperitoneally administered 5 times in 3 days to (C57BL / 6 X DBA / 2) F ₁ female mice, After 16 hours, the spleen was collected, and the spleen cells were isolated and cultured in the presence of 3.0 g / ml of concanavalin A for 43 hours, and IL-10 in the culture supernatant was quantified by sandwich ELISA. One group uses 5 animals. For the preparation of the calibration curve, genetically modified mouse IL-10 was used. The amount of IL-10 protein contained in the spleen cell culture supernatant of the extract administration group was 129.0 pg / ml in the 10 unit / kg administration group, 131.1 pg / ml in the 25 unit / kg administration group, 50 units / kg. In the kg administration group, the concentration was 163.6 pg / ml, and in any group administered with the extract, the IL-10 protein concentration in the spleen cell culture supernatant was 107.5 pg / ml in the untreated group, or physiological saline for injection was administered. Compared with the IL-10 protein concentration of the group 112.3 pg / ml, it significantly increased (p <0.05 or p <0.01) (FIG. 7).
[0025]
[Fig. 7]
[0026]
[Example 7]
(C57BL / 6 X DBA / 2) F ₁ female mouse was inoculated with the vaccinia virus inoculated rabbit inflammation skin extract (extract) 6.25, 12.5 units / kg once a day for 3 days. It was administered intraperitoneally. The mice in the control group were similarly administered with physiological saline for injection and dexamethasone 2 mg / kg / dose as the suppression positive control group. About 24 hours after the last administration, the spleen was collected, and the spleen cells were cultured in the presence of concanavalin A 2.5 μg / ml for 24 hours, and IFN-γ in the culture supernatant was quantified by sandwich ELISA. For the preparation of the calibration curve, genetically modified mouse IFN-γ was used. Administration of the extract reduced the amount of IFN-γ produced by mouse spleen cells in a dose-dependent manner, with 12.5 units / kg up to 1.94 ng / ml, which is similar to 1.78 ng / ml in the dexamethasone administration group. -The amount of γ production was suppressed (Fig. 8).
[0027]
[Fig. 8]
[0028]
[Example 8]
(C57BL / 6 X DBA / 2) F ₁ female mice were intraperitoneally administered once daily for 3 days with 12.5 units / kg of vaccinia virus-inoculated rabbit inflammation skin extract (extract) of the present invention. . The mice in the control group were similarly administered with physiological saline for injection and dexamethasone 2 mg / kg as the suppression positive control group. About 24 hours after the last administration, the spleen was collected, and the spleen cells were cultured in the presence of E. coli-derived endotoxin (E. coli 026: B6) 10 / μg / ml for 2 hours, and TNF-α in the culture supernatant was removed. Quantification was performed by sandwich ELISA. For the preparation of the calibration curve, genetically modified mouse TNF-α was used. TNF-α production of splenocytes in the extract administration group was suppressed to the same extent as in the dexamethasone administration group (FIG. 9).
[0029]
FIG. 9
[Brief description of the drawings]
1 is a band graph showing the concentration ratio of after electrophoresis of the cDNA of TGF-beta ₁ and G3PDH after RT-PCR reaction in Example 1.
[2] The same experiment as in Example 1 with experiments conducted at different time, which is a graph showing a band density ratio after electrophoresis of TGF-beta ₁ and G3PDH.
3 is a graph showing active TGF-β activity and total TGF-β activity in the spleen cell culture supernatant of mice in Example 2. FIG.
4 is a graph showing the results of measuring the active TGF-beta ₁ in mouse serum was measured by sandwich ELISA in Example 3.
5 is a graph showing the concentration ratio of bands after electrophoresis of IL-10 and G3PDH cDNA after RT-PCR reaction in Example 4. FIG.
FIG. 6 is a graph showing the concentration ratio of IL-10 and G3PDH bands after electrophoresis in experiments conducted at different dates and times similar to those in Example 4.
7 is a graph showing the results of measuring the amount of IL-10 protein in the concanavalin A-stimulated culture supernatant of mouse spleen cells in Example 6 by sandwich ELISA. FIG.
FIG. 8 is a graph showing the results of measuring the IFN-γ concentration in the concanavalin A-stimulated culture supernatant of mouse splenocytes in Example 7 by sandwich ELISA measurement.
FIG. 9 is a graph showing the results of measuring the TNF-α concentration in the E. coli-derived endotoxin-stimulated culture supernatant of mouse spleen cells in Example 8 by sandwich ELISA measurement.

Claims (1)

And rabbits inoculated with vaccinia virus skin extract as an active ingredient, tumor瘍壊Shiinko and retinal detachment with enhanced production of interferon-gamma, for the treatment of any disease of the group consisting of inflammatory bowel disease and perforation peritonitis Cytokine regulators.

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