JP2001058950A - Cytokine regulator - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a cytokine regulator capable of promoting the production of transforming growth factor-β having potent cell growth inhibitory action against epithelium-derived cells and the like and inhibiting the production of inflammatory cytokine by including a vaccinia virus-inoculated rabbit skin extract. SOLUTION: This cytokine regulator is obtained by including a vaccinia virus-inoculated rabbit skin extract as active ingredient. It is preferable that cytokine regulation means promoting the production of transforming growth factor-β and interleukin-10 and inhibiting the production of tumor necrosis factor and interferon-γ, and this regulator is used in treating diseases including wounds, burns, detached retinas, inflammatory intestinal diseases, perforative peritonitis, septicemia convalescence and articular rheumatism. The daily dose of the above extract is pref. 3.6-16 units either singly or in two to four portions, and the administration is conducted intravenously or the like.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION The present invention provides a cytokine regulator containing a vaccinia virus-inoculated rabbit skin extract as an active ingredient.

[0002]

2. Description of the Related Art Cytokines are produced from various cells including immunocompetent cells, are very small amounts of glycoproteins having biological activities indispensable for cell-cell interaction, and cytokines produced from lymphocytes are lymphokines. Cytokines produced from monocyte / macrophage cells are also called monokines. Biological activity of cytokines has both a positive regulatory mechanism and a negative regulatory mechanism. Under normal conditions, the regulatory mechanisms operate in a well-balanced manner, form a network with each other, and are responsible for the homeostasis of living organisms.

[0003] However, when this balance is lost, abnormal cytokine production appears or causes the formation or exacerbation of various diseases. For example, if the negative regulatory mechanism (regulatory mechanism) for the produced tumor necrosis factor (TNF-α) does not function sufficiently, TNF-α may become interleukin-1 (IL-1),
It promotes the production of various other inflammatory cytokines such as interleukin-6 (IL-6), interleukin-8 (IL-8), and
It is said that a vicious cycle of inflammation is caused by a chain reaction such as promoting the production of NF-α, and finally, tissues such as bones and cartilage are destroyed, leading to autoimmune diseases such as rheumatoid arthritis.

[0004]

At present, in various diseases, cytokines involved in the formation, exacerbation, and pain of various disease states have been identified. For this reason, studies that apply cytokines to disease diagnosis and treatment judgment have been made. Going further, suppressing excessive cytokine production or its activity,
Research is actively being conducted on the replacement of deficient / deficient cytokines, promotion of production, etc., as new treatments.

[0005] Among various cytokines, transforming growth factor-β (TGF-β) is a cytokine found as a growth factor of fibroblasts, but TGF-β ₁ produced by genetic engineering is used. Developing multiple inflammation or multiple organ failure in multiple organs in defective mice (Nature, 3
59 : 693, 1992) and wounds or burns based on osteoblast proliferation, promotion of extracellular matrix synthesis substrate secretion, inhibition of substrate degrading enzyme production, granulation formation of various tissues, promotion of fibrosis and cell differentiation. Repair effect on tissue damage, etc., therapeutic effect on retinal detachment based on the effect of promoting the expression of adhesion factors, suppressive effect on excessive immune response, T lymphocyte cell group
h ₂ action, etc. of shifting to cell populations are observed. Furthermore, recently, in severe arteriosclerosis, active TG in serum
A marked decrease in F-β levels was observed (Nature Medicine, 1 : 7).
4, 1995), TGF-
It is speculated that a decrease in beta levels is involved. The mechanism of action of the biological activity of TGF-β includes TGF-β
It has been emphasized that has a strong cell growth inhibitory effect on epithelial cells, vascular endothelial cells, lymphocytes, and other hematopoietic cells and suppresses the production of cytokines derived from these cells.

[0006] Interleukin-10 (IL-1)
0) has a Th ₁ T lymphocyte is an immune-competent cells Th
Of the group of cells consisting of _2, mainly in cytokines produced from Th ₂ cell group, the control action of the immune system in the intestinal, CD8 ⁺ cytotoxic T cells, antibody-producing cells, promoting action on mast cells, Th ₁ inhibitory effects on cells and macrophages, TNF-α, IL-1 is an interferon -γ cytokines (IFN-gamma) and derived macrophages is Th ₁ cell-derived cytokines,
It is known to suppress the production of IL-6 and the like. These TNF-α and IL-1 are known as cytokines whose production mechanism or biological activity often overlap,
In addition, TNF-α, IL-1, and IL-6 form diseases such as inflammatory bowel disease such as ulcerative colitis and Crohn's disease in humans, perforating peritonitis, and poor prognosis from sepsis due to gram-negative bacteria and the like. Is said to be deeply involved in exacerbation. In human ulcerative colitis and Crohn's disease, TNF-
Significantly reduced serum levels of IL-10, which suppresses the production of α, IL-1, and IL-6 ( clinical immunity , 2
7 , 16:97, 1995), that autoimmune bowel disease develops reliably in IL-10 gene-deficient mice (Cell, 75 : 263, 1993), IL-10
Reduce pain caused by these cytokines (British Journal of Pharmacology, Brit. J. Pharmacol., 115 : 6).
84,1995) for reporting such, IL-10 production promoting, it of cytokines produced from these macrophage cells and Th ₁ cells excess becomes trigger, onset, useful in disease follow the progression elapsed Has been revealed.

[0007]

Means for Solving the Problems The present inventors have added a drug substance whose active ingredient is called vaccinia virus-inoculated rabbit inflamed skin extract to epithelial cells, vascular endothelial cells, lymphocytes, and other substances. strong cytostatic effects on hematopoietic cells, promotes inflammatory or production of suppressing TGF-beta production of cytokines cytotoxic etc. derived from these cells, also macrophage cells or Th ₁ cell-derived cytokines Promotes the production of IL-10, which has the effect of inhibiting the production or activity of IFN-γ and TNF-γ.
The present inventors have found that there is an activity to suppress the production of α, and completed the present invention.

[0008] Vaccinia virus-inoculated rabbit inflamed skin extract is a known substance described, for example, in Pharmaceutical Drugs, Japan Pharmaceutical Collection, October 1997, page 1710. As an antiallergic, analgesic and sedative agent, in particular, a drug that specifically acts on nervous tissue in a stressed state, its content is represented by "unit" indicating the potency or substantially equivalent to the "unit" It is expressed in "neurotropin units", which are regarded as equivalent to each other, and is marketed as a medical drug (hereinafter, referred to as "units"). However, until now, the cytokine T
No regulatory effect on GF-β, IL-10, IFN-γ and TNF-α was known at all.

The dose of the vaccinia virus-inoculated rabbit inflamed skin extract according to the present invention varies depending on the type of disease, the sensitivity of the individual patient, and the level of the cytokine to be regulated, but varies within a range of 1 to 60 units / day. It 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.
Administer intravenously, subcutaneously, intramuscularly or orally, 6 to 16 units / day, once a day or 2 to 4 times a day.

Hereinafter, the present invention will be described in detail with reference to examples. However, these examples do not limit the scope of the present invention. Unless otherwise specified, “extract” means the vaccinia virus-inoculated rabbit inflamed skin extract of the present invention.

[0011]

Example 1 (C57BL / 6 XD of 8 to 15 weeks old)
BA / 2) _{F 1} in female mice, rabbits inoculated with vaccinia virus inflamed skin extract (extract of the present invention) 12.5 units /
Mice were bled 24 hours after administration, in which a single intraperitoneal administration of kg was administered and intraperitoneally for three consecutive days, and serum was collected. The mice in the control group received the same volume of physiological saline for injection as the administered extract. Three mice used per group. The spleen was collected from the same mouse as that from which the serum was collected, and the total RNA was immediately extracted and purified by a standard method.
C. and stored as a sample for reverse transcription reaction-polymerase chain reaction (RT-PCR). A downstream primer specific to the TGF-β1 gene was added to ₁ μg of each mouse spleen RNA, and a reverse transcriptase was allowed to act to synthesize cDNA.
The cDNA was amplified using a synthase. C for each sample
The DNA was fractionated by 2% agar gel electrophoresis, and the concentration of the 525 base pair band was measured with a densitometer. Similarly, glyceraldehyde-3-phosphate dehydrogenase (G3PD) was used as an internal standard from the RNA of each sample.
H) Regarding the gene, cDNA was amplified by reverse transcription reaction and then cDNA was amplified. The concentration of the 983 base pair G3PDH band fractionated by agar gel electrophoresis was measured with a densitometer, and then TGF was determined for each sample.
It was calculated the concentration ratio of -β ₁ band and G3PDH band.

[0012] As a result, TGF-beta ₁ band of each sample were fractionated by 2% agarose gel electrophoresis towards the band in the extract dose groups cDNA product showed dark band compared with the control group, extraction TGF-beta ₁ gene transcription is facilitated by liquid administration. TG by densitometer measurement
The average value of the concentration ratio of F-beta ₁ band and G3PDH band extract administration group is about 0.9, the control group was about 0.7
The average value of the extract administration group was higher than the average value of the control group (FIG. 1). Different time, repeating the same extract intraperitoneally experiment again, rabbits inoculated with vaccinia virus inflamed skin extract intraperitoneal administration, facilitate TGF-beta ₁ gene transcription in a statistically significant (p <0.01) (Fig. 2).

[0013]

FIG.

[0014]

FIG. 2

[0015]

Example 2 (C57BL / 6 XD of 8 to 15 weeks old)
The BA / 2) _{F 1} female mice 2 mice, inoculated with vaccinia virus inflammatory rabbit skin extract (extract) 12.5 units / kg of the present invention were administered daily intraperitoneally once daily for 3 days. Twenty-four hours later, spleens were harvested and 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 the administered extract. The culture supernatant is collected and TG
The biological activity of F-β was defined as the activity of inhibiting the growth of mink lung fibroblasts, and a sample from one animal was quantified twice, and the average value was calculated. Alternatively, after collecting the spleen, isolating the spleen cells,
After culturing with concanavalin A for 24 hours, the culture supernatant was collected. The amount of active TGF-β protein in the spleen cell culture supernatant was determined by a sandwich enzyme-linked immunosorbent assay (ELISA).

It has been confirmed that administration of the extract not only promotes TGF-β gene transcription but also promotes TGF-β protein production. The method for measuring biological activity is described in TGF
Since-beta _1, and detects the total TGF-beta activity in _{TGF-β 2, TGF-β} 3, TGF-β 1 just Not extract administered TGF-beta _2, to TGF-beta ₃ production This means that the effects can also be estimated. Biological activity and TGF-β
Calibration curve showing the correlation between protein content were prepared using genetic recombinant human TGF-beta _1. Activated TGF-β
For the measurement of, the culture supernatant is used as it is as a sample,
The total TGF-β was measured by treating the culture supernatant with hydrochloric acid to convert latent TGF-β to an active form. In the extract administration group of this experiment, active TGF-β had 907,7 pg
/ Ml and total TGF-β was 1226.4 pg / ml, whereas each of the control groups was 135.5 pg / m 2.
1, 639.6 pg / ml (FIG. 3). Thus, the administration of the extract promoted the production of both active and latent TGF-β from splenocytes.

[0017]

FIG. 3

[0018]

Example 3 (C57BL / 6 XD of 8 to 15 weeks old)
BA / 2) _{F 1} female mice, rabbits inoculated with vaccinia virus inflamed skin extract (extract of the present invention) at about the same time 25 units / kg as a single injection intraperitoneally, concanavalin A6.25Mg / kg or Physiological saline for injection was injected intradermally. Three days after administration, blood was collected from each mouse,
The serum was separated and used as a test sample. Sandwich EL
Using ISA method, active TGF-beta ₁ in the creation of a calibration curve using recombinant human TGF-beta ₁ in serum
As a result of measuring the protein amount, in the group to which concanavalin A was not administered, the active TGF-
beta ₁ is 8.76ng / ml, in the control group 7.12Ng /
ml, and the concanavalin A administration group, extract administered active TGF-beta ₁ in mouse serum 7.86ng / m
1 and 7.05 ng / ml in the control group (fourth
Figure). Thus, the administration of the extract allows concanavalin A to be administered.
Serum TGF-β levels were statistically significantly increased (p <0.05) with and without co-administration. Concanavalin A co-administration did not affect serum TGF-β levels.

[0019]

FIG. 4

[0020]

EXAMPLE 4 (C57BL / 6 XD of 8 to 15 weeks old)
BA / 2) _{F 1} in female mice, rabbits inoculated with vaccinia virus inflamed skin extract of the present invention (extract solution) 12.5 units / k
g was administered intraperitoneally once daily for 3 days, 24 hours later, the spleen was collected, and total RNA was immediately extracted and purified by a standard method.
After storage at -80 ° C, it was used as a sample for RT-PCR. The control group of mice received the same volume of physiological saline for injection as the extract. Three mice were used in each group. A downstream primer specific to the IL-10 gene was added to 1 μg of each mouse spleen RNA and cDNA was synthesized by the action of reverse transcriptase. Then, an upstream primer was further added and the cDNA was amplified using a heat-resistant DNA synthase. Similarly, for the G3PDH gene as an internal standard from the total RNA of each sample, cDNA was synthesized by a reverse transcription reaction.
Was 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. IL-10
In the 2% agar gel electrophoresis photograph of the cDNA products after the RT-PCR reaction of G3PDH and G3PDH, the cDNA products of the extract administration group (lanes 4, 5, and 6) were better than the control group (lanes 1, 2, and 3). It showed a darker band. After measuring the band concentration with a densitometer, I
As a result of comparing the concentrations of the L-10 band and the G3PDH band, when the average band concentration ratio of the control group was set to 1.0,
The average value of the extract administration group was 2.62, and the IL-10 gene transcription was significantly (p <0.01) promoted by the administration of the extract (FIG. 5).

[0021]

FIG. 5

[0022]

Example 5 An experiment confirming the promotion of IL-10 gene transcription by a single administration of 12.5 units / kg of vaccinia virus-infected rabbit inflamed skin extract (extract) of the present invention. The same procedure as in the experiment of Example 4 was repeated except that the number of administrations was changed to a single administration using two mice in each group.
As a result of performing RT-PCR on the L-10 gene and comparing the gene transcription level with the physiological saline control group, when the IL-10 and G3PDH band concentration ratio of the untreated group was set to 1.0, the physiological saline administration group was used. 1.31, extract administration group 2.2
9, the degree of promotion of IL-10 gene transcription was observed even with a single administration, although the degree was lower than that of the three administrations (No. 6).
Figure).

[0023]

FIG. 6

[0024]

Example 6 rabbits inoculated with vaccinia virus inflamed skin extract of the present invention (extract solution) of 10, 25, 50 Units / kg in (C57BL / 6 X DBA / 2 ) F 1 female mice,
After intraperitoneal administration 5 times for 3 days, spleen was collected 16 hours later, spleen cells were isolated, and concanavalin A 3.0 μg /
of IL-10 in the culture supernatant.
Was quantified by a sandwich ELISA method. Use 5 animals per group. Generating a recombinant IL mouse
-10 was used. The amount of IL-10 protein contained in the spleen cell culture supernatant of the extract administration group was 1 unit / kg in the 10 unit / kg administration group.
29.0 pg / ml, 25 units / kg 131.
13.6 pg in 1 pg / ml, 50 units / kg administration group
/ Ml, and the IL-10 protein concentration in the spleen cell culture supernatant was 107.5 p.
g / ml or physiological saline for injection group 112.3
pg / ml, significant (p
<0.05 or p <0.01 (No. 7)
Figure).

[0025]

FIG. 7

[0026]

Embodiment 7 (C57BL / 6 X DBA / 2) F ₁
A female mouse was treated with a vaccinia virus-inoculated rabbit extract of the present invention in rabbit inflamed skin extract (extract) 6.25, 12.5 units / kg.
Was administered intraperitoneally once a day for 3 consecutive days. The mice in the control group were similarly administered with physiological saline for injection, and dexamethasone 2 mg / kg / dose as a suppression positive control group. About 24 hours after the last administration, the spleen was collected, the spleen cells were cultured for 24 hours in the presence of 2.5 μg / ml of concanavalin A, and IFN-γ in the culture supernatant was subjected to a sandwich ELISA.
Quantified by the method. Genetic recombinant mouse IFN-γ was used for preparing the calibration curve. 11. Administration of the extract reduced the amount of IFN-γ production in mouse splenocytes in a dose-dependent manner.
5 units / kg, 1.78 ng of dexamethasone administration group
IFN-γ to 1.94 ng / ml, which is comparable to
The amount of production was suppressed (FIG. 8).

[0027]

FIG. 8

[0028]

Embodiment 8 (C57BL / 6 X DBA / 2) F ₁
Female mice were intraperitoneally administered 12.5 units / kg of vaccinia virus-inoculated rabbit inflamed skin extract (extract) of the present invention once a day for 3 consecutive days. Mice in the control group were similarly administered with physiological saline for injection, and 2 mg / kg of dexamethasone as a suppression positive control group. About 24 from last dose
At the time, the spleen was collected, and the spleen cells were cultured for 2 hours in the presence of 10 / μg / ml of E. coli-derived endotoxin (E. coli 026: B6), and TNF-α in the culture supernatant was quantified by a sandwich ELISA method. To prepare a calibration curve, genetically modified mouse TNF-α was used. TNF-α production of spleen cells in the extract-administered group was suppressed to the same degree as in the dexamethasone-administered group (FIG. 9).

[0029]

FIG. 9

[Brief description of the drawings]

FIG. 1 shows TGF after RT-PCR reaction in Example 1.
It is a graph which shows the density | concentration ratio of the band after the electrophoresis of cDNA of (beta) ₁ and G3PDH.

[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.

FIG. 3 is a graph showing active TGF-β activity and total TGF-β activity in a mouse spleen cell culture supernatant in Example 2.

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.

FIG. 5 shows IL- after RT-PCR reaction in Example 4.
It is a graph which shows the density ratio of the band after electrophoresis of cDNA of 10 and G3PDH.

FIG. 6 is a graph showing the concentration ratio of IL-10 and G3PDH bands after electrophoresis in an experiment in which the same experiment as in Example 4 was performed at different dates and times.

FIG. 7 is a graph showing the results obtained by measuring the amount of IL-10 protein in the culture supernatant of a mouse spleen cell stimulated with concanavalin A in Example 6 by a sandwich ELISA method.

FIG. 8 shows that the concentration of IFN-γ in the culture supernatant of the mouse spleen cells stimulated with concanavalin A in Example 7 was measured using sandwich E.
It is a graph which shows the result measured by LISA method measurement.

FIG. 9 is a graph showing the results of measuring the concentration of TNF-α in the culture supernatant stimulated with E. coli derived endotoxin of mouse spleen cells in Example 8 by sandwich ELISA.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 43/00 111 A61P 43/00 111 (72) Inventor Noriyoshi Norisada 1-3-3 Nishi-Otsuka 1-chome, Matsubara-shi, Osaka No. F-term in Fujimoto Pharmaceutical Co., Ltd. Drug Discovery Laboratory (reference) 4C087 AA01 AA02 BB48 BC83 CA06 MA01 NA14 ZA33 ZA66 ZA89 ZB02 ZB07 ZB09 ZB11 ZB13 ZB15 ZB35

Claims (6)

[Claims] 1. A cytokine regulator comprising as an active ingredient a rabbit skin extract inoculated with vaccinia virus. 2. Cytokine regulation is achieved by transforming growth factor-β and interleukin-
The cytokine modulator according to claim 1, which promotes production of No. 10 and suppresses production of tumor necrosis factor and interferon-γ. 3. Wound, burn, retinal detachment, inflammatory bowel disease,
The cytokine modulator according to claim 1, which is used for treatment of any disease in the group consisting of perforated peritonitis, prognosis of sepsis, rheumatoid arthritis and allergic symptoms. 4. Tumor necrosis factor and interferon-
The method according to claim 1, which is used for the treatment of any disease of the group consisting of wounds, burns, retinal detachment, inflammatory bowel disease, perforating peritonitis, sepsis, prognosis of rheumatoid arthritis and allergic symptoms, in which the production of γ is enhanced. 3. The cytokine modulator according to 2. 5. Tumor necrosis factor and interferon-
The cytokine modulator according to claim 1, which is used for treating pain in a disease in which γ production is enhanced. 6. Tumor necrosis factor and interferon-
Claims: Used in the treatment of pain in any of the group consisting of wounds, burns, retinal detachment, inflammatory bowel disease, perforated peritonitis, sepsis prognosis, rheumatoid arthritis and allergic symptoms, in which the production of gamma is enhanced. 3. The cytokine modulator according to claim 1.