JP2005194222A - Cytokine inducer - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To produce a cytokine inducer that induces cytokines within a short time. <P>SOLUTION: This cytokine inducer contains as an active ingredient at least one member selected from the group consisting of 8 to 24C unsaturated fatty acids each having 1 to 3 double bonds, such as linoleic acid, linolenic acid, oleic acid, undecylenic acid, and myristoleic acid, and monoacylglycerides thereof. The cytokine inducer is exemplified by IL-6, IL-8 (chemokine), IL-18, TNFα, IFNα, or IFNγ. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a cytokine inducer that induces the expression of cytokines.

Cytokines are proteins produced by cells, and are a generic term for those that act on cells having receptors for them to proliferate, differentiate, function, and so on. Cytokines have the following characteristics (cited from Non-Patent Document 1 below).
1) Glycoprotein.
2) Acts in trace amounts.
3) Acts mainly in the locally produced, but some cytokines are transported to remote organs via the bloodstream.
4) Has various physiological activities.
5) Different cytokines have the same physiological activity.
6) Has a cytokine network.
7) An inhibitor is present.

  As the action of cytokines, an immune response control action, an antitumor action, an antiviral action, and the like are known by expressing the proliferation, differentiation and function of the cells described above.

  By the way, Patent Literature 1 describes an immune enhancer containing diglyceride as an active ingredient. In the example of Patent Document 1, it is shown that, when orally administered to mice, delayed hypersensitivity reaction is enhanced as compared with triglyceride.

Toshihiro Nanki, Nobuyuki Miyasaka, “Diversity and Function of Cytokines and Cytokine Receptors”, Latest Medicine 57: 831-839,2002 JP-A-6-247849

  The subject of this invention is providing the cytokine inducer which can induce a cytokine in a short time.

  As a result of intensive studies to solve the above problems, the present inventors have found that a specific fatty acid or a monoacylglyceride thereof has a function of inducing the expression of cytokines, and have completed the present invention.

  That is, the cytokine-inducing agent of the present invention is at least one selected from the group consisting of unsaturated fatty acids having 1 to 8 carbon atoms, preferably 11 to 18 carbon atoms having 1 to 3 double bonds, and monoacylglycerides thereof. Is the active ingredient. There have been many reports on cytokines, but it is not known that the above fatty acids or monoacylglycerides have a function of inducing cytokines.

  The unsaturated fatty acid may be at least one selected from the group consisting of linoleic acid, linolenic acid, oleic acid, undecylenic acid and myristoleic acid. The monoacylglyceride may be a monoacylglyceride of at least one unsaturated fatty acid selected from the group consisting of linoleic acid, linolenic acid, oleic acid, undecylenic acid and myristoleic acid.

  Examples of the cytokine include IL (interleukin, hereinafter the same) -6, IL-8 (chemokine), IL-18, TNF (tumor necrosis factor) α, IFN (interferon, same hereinafter) α, and IFNγ.

  The above compound is considered to induce cytokines in a short time (usually about 1 to 24 hours) by binding to human vascular endothelial cells. For this reason, for example, macrophages are activated by cytokines induced by intravenous injection to attack a target (for example, cancer), or effects such as immune enhancement are recognized. Therefore, the cytokine inducer of the present invention is used in the form of an injection containing an infusion preparation.

  According to the present invention, there is an effect that cytokine expression can be induced in a short time by administering a specific fatty acid or a monoacylglyceride thereof.

The present invention is described in detail below.
Examples of the unsaturated fatty acid used in the present invention include trienoic acid such as linolenic acid, dienoic acid such as linoleic acid, myristoleic acid, palmitoleic acid, oleic acid, elaidic acid, undecylenic acid, cisbaccenoic acid, vaccenic acid, erucaic acid. Typical examples include unsaturated fatty acids having 8 to 24 carbon atoms, preferably 11 to 18 carbon atoms having 1 to 3 double bonds, such as monoenoic acids such as acids. Preferably, linoleic acid, linolenic acid, oleic acid, undecylenic acid or myristoleic acid is used. Unsaturated fatty acids can be used in both cis and trans forms.

  Examples of monoacyl glycerides include monoacyl glycerides of unsaturated fatty acids as described above. In particular, monoacyl glycerides of linoleic acid, linolenic acid, oleic acid, undecylenic acid or myristoleic acid are preferably used. The production method of such monoacylglyceride is not particularly limited, and for example, a method of esterifying the unsaturated fatty acid with glycerin and then isolating the monoacylglyceride can be employed. Moreover, the said unsaturated fatty acid and its monoacylglyceride can be used individually, and can mix and use 2 or more types as needed.

  The above-mentioned specific unsaturated fatty acid or monoacylglyceride thereof exerts various actions such as immunostimulation and anticancer action by inducing the expression of various cytokines to promote cell proliferation, differentiation and functional expression. To do. Below, the main cytokine induced by the cytokine inducer of this invention is illustrated.

(1) IL-6
IL-6 is produced from various cells such as fibroblasts, endothelial cells, keratinocytes, renal mesangium as well as T cells, B cells, monocytes, etc., and T cell proliferation and differentiation into CTLs, hematopoietic stem cell proliferation It is a cytokine with various physiological activities such as platelet increase due to differentiation, megakaryocyte differentiation induction, induction of acute inflammatory proteins such as CRP as a hepatocyte stimulating factor, and growth factor such as mesangial cells, keratinocytes, and osteoclasts. (Akira S., et al., Interleukin-6 in biology and medicine. Adv. Immunol. 54: 1-78, 1993).

(2) IL-8
IL-8 belongs to the CXC chemokine and induces neutrophil infiltration (Abe et al., Overview-What is a cytokine ?. Medical Journal 36: 647-652, 2000). Chemokines are basic low-molecular-weight cytokines that have leukocyte chemotaxis and activation capabilities. Many chemokine molecules with similar structures and functions have been discovered and collectively referred to as the chemokine superfamily (Yosie O., et al. Chemokines in immunity. Adv. Immunol. 78: 57-110, 2001).

(3) IL-18
IL-18 has a tertiary structure similar to IL-1 (barrel structure with β sheet), and its receptor and signal transduction pathway are similar to IL-1 and Toll-like receptor (TLR) (Masahiro Yamamura, IL-18 and inflammatory diseases. Latest medicine 57: 873-879, 2002). IL-18 acts on various cells (T cells, B cells, natural killer (NK) cells, macrophages, dendritic cells) synergistically with IL-12, as originally discovered as an IFN-γ inducer. To induce IFN-γ production in cooperation (Okamura H, Tsutsui H., Komatsu T. et al., Cloning of a new cytokine that induces IFN-γ production by T cells. Nature 378: 88-, 1995, Yoshimoto T., Okamura H., Tagawa Y. et al., Interleukin 18 together with interleukin 12 inhibits IgE productin by induction of interferon-γ production from activated B cells.Proc. Natl. Acad. Sci. USA 94: 3948-, 1997 , Yoshimoto T., Takeda K., Tanaka T. et al., IL-12 up-regulates IL-18R expression on T cells, Th1 cells and B cells: Synergism with IL-18 for IFN-γ production. J. Immunol 161: 3400-, 1998 and Nakanishi K., Yoshimoto T., H. and Okamura H., Interleukin-18 regulates both Th1 and Th2 responses. Annu. Rev. Immunol. 19: 423-, 2001).

In addition, IL-18 exhibits a completely different action in the absence of IL-12. That is, IL-18 acts on CD4 ⁺ T cells, basophils, and mast cells to induce the production of Th2 cytokines IL-4 and IL-13 (Yoshimoto T., Tsutsui H., Tominaga K. et al. al., Interleukin-18 (IL-18), although anti-allergic when administered with IL-12, stimulates IL-4 and histamine release by basophils. Proc. Natl. Acad. Sci. USA 96: 13962-, 1999 and Yoshimoto T., Mizutani H., Tsutsui H. et al., IL-18 induction of IgE: Dependence on CD4 ⁺ T cells, IL-4 and STAT6. Nat. Immunol. 1: 132-, 2000).

  Furthermore, it has been pointed out that IL-18 increases the expression of Fas-ligand on the surface of T cells and NK cells and induces apoptosis in cells expressing Fas molecules (Akira Ono, Hidetaka Mochizuki, Inter Leukine 18 (IL-18)-Role of IL-18 in terms of infection protection and tumor immunity. History of medicine 200: 1095-1096, 2002).

  Cytokines such as IL-18, which induce Th1-type cytokines, are thought to play an important role as an infection defense factor by enhancing cellular immunity, and against IL-18 It is recognized that the immune function is activated by administration, and the resistance to the subsequent infectious invasion is also increased (from Ono, Hidetaka Mochizuki, Interleukin-18 (IL-18)) The role of Mita IL-18. History of medicine 200: 1095-1096, 2002). Furthermore, it has been pointed out that IL-18 has an antitumor action against sarcoma, neuroblastoma, and melanoma (Akira Ono, Hidetaka Mochizuki, Interleukin 18 (IL -18)-Role of IL-18 from the viewpoint of infection protection and tumor immunity. History of medicine 200: 1095-1096, 2002).

(4) TNFα
TNFα activates neutrophils and vascular endothelial cells, and promotes adhesion of activated neutrophils to vascular endothelial cells.

(5) IFN
IFN has attracted attention as an antiviral cytokine and has since been shown to regulate many immune responses in the immune system. Furthermore, an antitumor effect has been clarified against many tumor cells, and it has been clinically applied as an antitumor agent to renal cancer and the like. Among hematopoietic tumors, it is particularly useful for hair cell leukemia, chronic myeloid leukemia, adult T-cell leukemia and multiple myeloma (Kitamura Sei, Clinical application of cytokines. Experimental Medicine 18: 2181 -2188, 2000). There are also reports that it is effective for refractory idiopathic thrombocytopenic purpura and rheumatic autoimmune diseases (Kitoshi Kitamura, clinical application of cytokines. Experimental Medicine 18: 2181-2188, 2000).

  Since the inducer of the present invention induces cytokines by binding to human vascular endothelial cells, it is preferably used in the form of an injection such as an infusion preparation or a one-shot injection. The injection may be any of a liquid, emulsion, suspension, etc., and these are preferably sterilized and isotonic with blood. In preparing an injection, for example, water, ethyl alcohol, macrogol, propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitan fatty acid esters and the like can be used as diluents. In this case, a sufficient amount of sodium chloride, glucose, glycerin and the like may be added to prepare an isotonic solution, and a normal solubilizing agent, buffer, soothing agent, etc. may be added. Also good. In the case of a fat emulsion, it can be used in the form of an O / W type or W / O / W type emulsion.

The inducing agent of the present invention may contain about 1 to 50% (w / v%, the same applies hereinafter), preferably 5 to 30% of the compound as an active ingredient, but is particularly limited. is not.
The dose of the inducer of the present invention is usually about 1 to 70 g / day for the above-mentioned compound as an active ingredient in adults, but it goes without saying that it may increase or decrease depending on age, weight, symptoms, etc. Absent.

  Hereinafter, the present invention will be described in more detail with reference to examples.

  This test examined cytokine mRNA expression of human aortic vascular endothelial cells maintained in culture using the compound of the present invention, and was performed as follows.

尚、対照としてハウスキーピング遺伝子であるベータアクチン(β-actin)について同一試験を実施した。 (1) Measurement item Cytokine and each mRNA of IL-6, IL-8, IL-18, TNFα, IFNα and IFNγ were measured. Each of these cytokines is registered in the gene bank (GenBank) as follows, and each base sequence thereof follows those registered. In addition, mRNA is quantified by using RT-PCR (Real-time quantitative reverse) using a primer pair and a probe of each sequence shown in Table 1 below (the position of the start codon is in accordance with each registered nucleotide sequence). Transcription-polymerase reaction). Each primer and probe were prepared using an automatic DNA synthesizer.

As a control, the same test was carried out for beta-actin, which is a housekeeping gene.

(2) Test substance and control substance
a) Control substance name: Aminofried Manufacturer: Otsuka Pharmaceutical Factory
b) Positive control name: Lipopolysaccharide (LPS)
Manufacturer: Sigma Chemical Co.

(3) Cultured human normal aortic vascular endothelial cells English name: Aortic Endothelial Cells
Origin: Aortic Source: BioWhittaker, Inc.
Agency: Asahi Techno Glass Co., Ltd.

(4) Reagents and instruments Medium for normal human aortic vascular endothelial cells: BioWhittaker, Inc., distributor: Asahi Techno Glass Co., Ltd. Saline: Otsuka Pharmaceutical Factory Co., Ltd. Water for injection: Otsuka distilled water (Otsuka Pharmaceutical Co., Ltd.) (Made by factory)
RNase-free distilled water: Wako Pure Chemical Industries, Ltd.
Rneasy Mini Kit (50): Qiagen Co., Ltd.
QIAshredder (50): Qiagen Corporation
Yeast tRNA: GIBCO BRL
TaqMan One-Step RT-PCR Master Mix Reagents Kit: Applied Biosystems
TaqManβ-actin Control Reagents: Applied Biosystems (Product No. 401846)
Micro Amp Optical 96-well Plate: Applied Biosystems
Optical Adhesive Covers: Made by Applied Biosystems
Optical Cover Compression Pads: Applied Biosystems
24-well flat bottom plate: Asahi Techno Glass Co., Ltd. (collagen type I coat)
Trypan blue: Made by Flow Laboratories LTD. (0.4% solution in 0.85% saline)
24-well flat bottom plate: Asahi Techno Glass Co., Ltd. β-mercaptoethanol: Sigma Chemical Co.

(5) Group configuration The control group was an aminofreed diluted solution as a test solution, and the positive control group was an LPS group. The test substances used are as follows.
(a) Oleic acid
(b) Linoleic acid
(c) Linolenic acid
(d) Monoacylglycerides of linoleic acid
(e) Monoacylglycerides of linolenic acid
(f) Unacylenic monoacylglyceride
(g) Myristoleic acid monoacylglyceride control group: aminofreed diluted solution containing 2% DMSO (dimethyl sulfoxide, the same shall apply hereinafter) Positive control group: amino acid containing 2% DMSO with 10 ng / mL LPS Freed Diluted Solution The test substance was dissolved in DMSO and then diluted 50-fold with an amino freed diluted solution.

(6) Preparation of test solution <Preparation of control solution (aminofreed diluted solution containing DMSO)>
Since the osmotic pressure ratio of aminofreed was about 3, 10 mL of aminofreed was dispensed into a 50 mL conical tube, and 20 mL of water for injection was added to prepare an aminofreed diluted solution. Furthermore, DMSO was added so that the concentration was 2%, and a control solution (DMSO-containing aminofreed diluted solution) was obtained.
<Preparation of solution containing LPS>
5 mg of LPS was dissolved in 50 mL of physiological saline containing 2% DMSO to obtain a 100 μg / mL solution. Further, dilute with a physiological saline solution containing 2% DMSO to prepare a 100 ng / mL solution. Thereafter, the resultant was diluted 10-fold with an amino free diluent containing 2% DMSO.
<Preparation of solution in which compound is dissolved>
Each compound was dissolved in DMSO and diluted 50-fold with an amino free diluent to prepare a 400 μM solution. However, oleic acid is a 350 μM solution.

(7) Culture of human aortic vascular endothelial cells 1 to 4 × 10 ⁴ cells / 400 μL / well are dispensed into a 24-well plate and cultured in a CO ₂ incubator at 37 ° C. Used for exposure studies when grown to ˜70%.

(8) Exposure conditions Since the cells were cultured in a medium volume of 400 μL for each well, the cells were replaced with the same volume of test liquid, and then exposed to the test liquid for 4 hours.

(9) Preparation of total RNA Total RNA was extracted as follows. All extraction operations were performed at room temperature. That is, at the time of 4 hours from the start of exposure, the medium was removed by suction from each well of the 24-well plate. Next, 400 μL each of RLT solution containing β-mercaptoethanol (RLT solution (supplied as Rneasy Mini Kit (50)): β-mercaptoethanol = 100: 1) was added to each well, pipetted, and added β -RLT solution containing mercaptoethanol was collected from each well and added to the QIAshredder column and centrifuged at 15,000 rpm for 2 minutes. 350 μL of the eluate was collected, and an equal amount of 70% ethanol solution was added thereto. The solution was stirred 3 times for 10 seconds, added to the Rneasy Mini spin column, centrifuged at 12,000 rpm for 30 seconds, and the eluate in the collection tube was removed by suction. Subsequently, 700 μL of RW1 solution (supplied as Rneasy Mini Kit (50)) was added to the collection tube, and the collection tube was replaced after centrifugation at 12,000 rpm for 30 seconds. 500 μL of RPE solution (supplied as Rneasy Mini Kit (50)) was added to the collection tube, and centrifuged at 12,000 rpm for 30 seconds, and the eluate in the collection tube was removed by suction. Add another 500 μL of RPE solution to the collection tube, centrifuge at 15,000 rpm for 2 minutes, replace with 1.5 mL collection tube, and supply 50 μL of Rnase free water (supplied as Rneasy Mini Kit (50)) in the tube. Was added and centrifuged at 10,000 rpm for 1 minute to elute the total RNA. The eluate was diluted 5-fold with 50 μg / mL Yeast tRNA solution to obtain a total RNA solution for measurement. A 50 μg / mL Yeast tRNA solution was prepared by diluting Yeast tRNA with RNase-free distilled water.

(10) mRNA measurement
Using the ABI PRISM ^™ 7700 Sequence Detection System, housekeeping gene (β-actin) and cytokine mRNAs were quantified as follows.

RT-PCR was performed in a 50 μL / tube system using TaqMan One-Step RT-PCR Master Mix Reagents Kit containing 300 nM forward primer, 900 nM reverse primer and 200 nM TaqMan probe. 5 μL of total RNA solution was used. The PCR conditions were as follows: a temperature was maintained at 48 ° C. for 30 minutes and then at 95 ° C. for 10 minutes, followed by 40 cycles of 95 ° C. for 15 seconds and 60 ° C. for 1 minute. The fluorescence intensity was measured for each cycle. The reaction vessel was a Micro Amp Optical 96-well plate, the cover was Optical Adhesive Covers, and the pad placed on the cover was Optical Cover Compression Pads.
For measurement of β-actin, TaqMan β-actin Control Reagents (300 nM Forward Primer, 300 nM Reverse Primer and 200 nM TaqMan Probe) were used.

(11) Calculation method of results and statistical processing β-actin mRNA was used as an endogenous control. The quantitative value of each mRNA was calculated by the ΔCt method (Nishimura, M., Yaguri, H., Yoshitsugu, H., Naito, S. & Satoh, T., (2003) Yakugaku Zasshi, 123, 369-375). did. The test was performed twice. The quantitative value is shown as a relative value to the mRNA value of the control group after exposure for 4 hours.

(12) Results and Discussion Tables 2 to 7 show the results obtained. The result was expressed as a relative value when the control value was 1, and a value of 1.5 times or more was regarded as a significant value. A solution containing 10 ng / mL LPS is used as a positive control.

The following can be understood from the results shown in each table. That is, the compound shown in Table 2 significantly increased the mRNA expression level of IL-6. The compound shown in Table 3 significantly increased the mRNA expression level of IL-8. The compound shown in Table 4 markedly increased IL-18 mRNA expression level. With the compounds shown in Table 5, the mRNA expression level of TNFα was significantly increased. The compound shown in Table 6 significantly increased the mRNA expression level of IFNα. The compounds shown in Table 7 significantly increased the mRNA expression level of IFNγ. Moreover, these cytokine mRNAs are expressed after 4 hours of exposure.

Claims (6)

  A cytokine-inducing agent comprising as an active ingredient at least one selected from the group consisting of unsaturated fatty acids having 1 to 2 carbon atoms and having 8 to 24 carbon atoms and monoacylglycerides thereof.   The cytokine inducer according to claim 1, wherein the unsaturated fatty acid has 11 to 18 carbon atoms.   The cytokine-inducing agent according to claim 1 or 2, wherein the unsaturated fatty acid is at least one selected from the group consisting of linoleic acid, linolenic acid, oleic acid, undecylenic acid and myristoleic acid.   The monoacylglyceride is a monoacylglyceride of at least one unsaturated fatty acid selected from the group consisting of linoleic acid, linolenic acid, oleic acid, undecylenic acid and myristoleic acid. Cytokine inducer.   The cytokine-inducing agent according to any one of claims 1 to 4, wherein the cytokine is IL-6, IL-8 (chemokine), IL-18, TNFα, IFNα or IFNγ. The cytokine inducer according to any one of claims 1 to 5, which is used in the form of an injection.