WO2007061237A1 - Composition for anti-inflammation - Google Patents

Composition for anti-inflammation Download PDF

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Publication number
WO2007061237A1
WO2007061237A1 PCT/KR2006/004951 KR2006004951W WO2007061237A1 WO 2007061237 A1 WO2007061237 A1 WO 2007061237A1 KR 2006004951 W KR2006004951 W KR 2006004951W WO 2007061237 A1 WO2007061237 A1 WO 2007061237A1
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Prior art keywords
oh8dg
composition
oh8g
present
ingredients
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French (fr)
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Myung Hee Chung
Seong Won Choi
Sang Kyu Ye
Sun Hye Lee
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Seoul National University Industry Foundation
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Seoul National University Industry Foundation
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7076—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid
    • A61K31/708—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid having oxo groups directly attached to the purine ring system, e.g. guanosine, guanylic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]

Definitions

  • the present invention relates to a composition for anti-inflammation.
  • ROS Reactive oxygen species
  • oh8Gua is produced from guanine bases of dGTP (deoxyguanosine triphosphate) and GTP (guanosine triphosphate) which exist in cytoplasm as well as guanine of DNA of nucleus because cells have an oh ⁇ dGTPase (also referred to as Mut T), an enzyme hydrolasing 8-hydroxydeoxyguanosine triphosphate (oh ⁇ dGTP) and 8-hydroxyguanosine triphosphate (oh8GTP) which are produced from modification of guanine base of dGTP and GTP by POS to oh ⁇ dGMP and oh ⁇ GMP (Shinmura K, et al.
  • dGTP deoxyguanosine triphosphate
  • GTP guanosine triphosphate
  • Ras has been known to play an important role in cell reactions for oxidative stress.
  • ERK extracellular signal-regulated kinase
  • MAPK mitogen-activated protein kinase
  • oh8GTP under the presence of oxidative stress, oh8GTP is created from GTP in cytoplasm, Ras is more activated by oh8GTP than GTP, and activated Ras activates all of kinase in downstream (Yoon SH, et al. Biochem Biophys Res Commun. 2005 Feb 4;327(l):342-8). Accordingly, it has been conceived that oh8dGTP and oh8GTP in cytoplasm may be substances with physiological activity that can affect cell function, not simple by-products of disturbing genetic information by insertion to DNA or RNA.
  • Rh8GTP is a small GTP -binding protein, besides Ras.
  • Rac is a small GTP-binding protein, which produces ROS by inducing activation of NADPH oxidase in leucocytes [Blood. 2002 Oct 15; 100(8): 2692-6; J Immunol. 2004 Nov 15;173(10):5971-9; J Biol Chem. 1983 Aug 25;258(16):9631-5; J Immunol. 2005 Apr 15;174(8):4613-20.; J Biol Chem.
  • Oh8G' 8-hydroxyguanosine
  • Oh8dG' 8-hydroxydeoxyguanosine
  • Oh8dG' 8-oxo-deoxyguanosine
  • one object of the present invention is to provide a composition for anti-inflammation comprising one or more ingredients of 8-hydroxyguanosine (oh8G) and 8 -hydroxy deoxyguanosine (oh8dG) as active ingredients.
  • Another object of the present invention is to provide a use of a composition comprising one or more ingredients of oh8G and oh8dG as active ingredients for the manufacture of anti-inflammatory agent.
  • Another object of the present invention is to provide a method for treating inflammation that comprises administrating to a mammal a therapeutically effective amount of one or more ingredients of oh8G and oh8dG.
  • the present invention provides a composition for anti-inflammation comprising one or more ingredients of 8-hydroxyguanosine (oh8G) and 8-hydroxydeoxyguanosine (oh8dG) as active ingredients. Also, the present invention provides a use of a composition comprising one or more ingredients of oh8G and oh ⁇ dG as active ingredients for the manufacture of anti-inflammatory agent.
  • the present invention also provides a method for treating inflammation, comprising administrating to a mammal a therapeutically effective amount of one or more ingredients of oh8G and oh8dG.
  • the composition comprises oh8dG as an active ingredient.
  • the composition is used for treating one or more inflammatory diseases selected from rheumatic arthritis, Lupus ethrythematosus, multiple scleroderma, or adult respiratory distress syndrome (ARDS), as an anti-inflammatory agent.
  • ARDS adult respiratory distress syndrome
  • the present invention provides a composition for anti-inflammation comprising one or more ingredients of oh8G and oh ⁇ dG as active ingredients.
  • the active ingredients of the present composition, oh8G and oh8dG are nucleosides of oh8Gua which is produced from oxidation of guanine by ROS, and are excreted as body wastes in a very small amount. They can be obtained by isolating from body wastes or synthesizing according to a common method in the art, or can be used as commercial product.
  • the present composition comprising one or more ingredients of oh8G and oh8dG suppresses the activity of Rac 2 of mouse macrophage, thereby suppressing the activity of NADPH oxidase and also suppressing ROS production from mouse macrophage.
  • the composition of the present invention remarkably suppresses the rising of blood concentration of pro-inflammatory cytokine induced by LPS; the increasing of myeloperoxidase(MPO) activity and Rac 2 activity in lung tissues; and the mortality of mouse due to LPS.
  • the anti-inflammatory effect of the present composition is considerably superior to the effect of aspirin (ASA), one of representative NSAIDs.
  • ASA aspirin
  • the present composition has little side effects such as digestive system disorder, and so can be used as an excellent anti-inflammatory agent.
  • the side effects of anti-inflammatory agents of NSAIDs seem from inhibiting COX-I
  • composition of the present invention comprising one or more ingredients of oh8G and oh8dG as active ingredients can be used as a novel anti-inflammatory agent without side effects such as anti-inflammatory agents of NSAIDs.
  • the composition of the present invention can be used for treating inflammatory diseases comprising various kinds of autoimmune diseases, such as rheumatic arthritis, Lupus ethrythematosus, multiple scleroderma, adult respiratory distress syndrome (ARDS), etc.
  • autoimmune diseases such as rheumatic arthritis, Lupus ethrythematosus, multiple scleroderma, adult respiratory distress syndrome (ARDS), etc.
  • the symptom in mouse death by LPS is similar to adult respiratory distress syndrome (ARDS) that is shown in septicemia of human.
  • ARDS adult respiratory distress syndrome
  • the present composition is useful for treating adult respiratory distress syndrome (ARDS) which has no distinct therapeutic agent at present.
  • composition comprising one or more ingredients of oh8G and oh ⁇ dG as active ingredients may be formulated by using pharmaceutically acceptable additives such as excipient, disintegrating agent, sweetener, binder, coating agent, blowing agent, lubricant, glidant, flavoring agent, solublizer, etc.
  • pharmaceutically acceptable additives such as excipient, disintegrating agent, sweetener, binder, coating agent, blowing agent, lubricant, glidant, flavoring agent, solublizer, etc.
  • composition of the present invention may further comprise one or more pharmaceutically or physiologically acceptable carriers to be formulated appropriately for administration.
  • the pharmaceutical composition of the present invention may be prepared in any form such as granule, powder, tablet, coated tablet, capsule, suppository, pill, syrup, juice, solution, suspension, emulsion or injectable liquid formulation.
  • the active ingredients may be admixed with oral, nontoxic and pharmaceutically acceptable inactive carriers such as ethanol, glycerol, water, etc.
  • appropriate binder, lubricant, disintegrating agent and color coupler may be also admixed.
  • Proper binder may be, but is not limited to, natural sugar such as starch, gelatin, glucose or ⁇ -lactose; natural or synthetic gum such as corn sweetener, acacia, tragacanth or sodium oleate; sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, etc.
  • the disintegrating agent may comprise, but is not limited to, starch, methylcellulose, agar, bentonite, Xanthan gum, etc.
  • the present composition may comprise pharmaceutically acceptable carriers which have biocompatibility and are sterilized, for example, saline, sterilized water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, malto dextrin solution, glycerol, ethanol, and mixture thereof. If necessary, other common additives such as antioxidant, buffer, bacteriostatic agent, etc. may be added to the composition. Also, by further adding diluent, dispersing agent, surfactant, binder or lubricant, the composition may be formulated to injectable dosage form such as solution, suspension, emulsion, etc., pill, capsule, granule, or tablet.
  • pharmaceutically acceptable carriers which have biocompatibility and are sterilized, for example, saline, sterilized water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, malto dextrin solution, glycerol, ethanol, and mixture thereof.
  • the present composition may be formulated in any desirable forms depending on disease or ingredient, by using conventional methods or a method described in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA).
  • the present invention also provides a use of a composition comprising one or more ingredients of 8-hydroxyguanosine (oh8G) and 8 -hydroxydeoxy guano sine (oh8dG) as active ingredients for the manufacture of anti-inflammatory agent.
  • the present composition comprising one or more ingredients of oh8G and oh8dG as active ingredients can be used for the manufacture of anti-inflammatory agent.
  • the present invention provides a method for treating inflammation which comprises administrating to a mammal a therapeutically effective amount of one or more ingredients of oh8G and oh8dG.
  • mammal as used hereinbefore or hereinafter means mammal as a subject for treatment, observation, or examination, but preferably, human.
  • therapeutically effective amount used hereinbefore or hereinafter means an amount of active ingredient or pharmaceutical composition that can induce biological or medicinal response in a tissue, system, animal or human by a researcher, veterinarian, medical doctor or other clinician, and includes an amount of inducing alleviation of the symptoms of treated diseases or disorders. It is clear for a skilled artisan that the therapeutically effective amount of the active ingredients and the number of their administration are varied depending on desirable effect.
  • most suitable dosage to be administrated can be varied depending on various factors such as the kind of diseases, the kind and severity of patient's symptom, the content of active ingredient, the content and kind of other ingredients, the type of formulation, the age, body weight, health status, gender, and food of a patient, dosing time, administration route, the secretion ratio of composition, treatment period, and other co-administrated drug.
  • the dosage of the active ingredients is preferably 0.01 mg/kg - 100mg/kg.
  • the pharmaceutical composition comprising one or more ingredients of oh8G and oh8dG as active ingredients may be administered via various routes including oral, rectal, intravenous, intra-arterial, intraperitoneal, intramuscular, intrathoracic, transdermal, topical, intraocular, and intradermal administration, according to conventional administration methods.
  • the present composition comprising one or more ingredients of oh8G and oh8dG suppresses the activity of Rac 2 of macrophage, thereby suppressing the activity of NADPH oxidase and also ROS production from macrophage. Also, the composition of the present invention remarkably suppresses the rising of blood concentration of pro-inflammatory cytokine induced by LPS, the increasing of MPO activity and Rac2 activity in lung tissues, and the mortality of mouse due to LPS.
  • the composition of the present invention has considerably superior effect to aspirin (ASA), one of representative NSAIDs. Unlike anti-inflammatory agents of NSAIDs, the present composition has little side effects such as digestive system disorder, and so can be used as an excellent anti-inflammatory agent.
  • ASA aspirin
  • composition for anti-inflammation of the present invention can be used for treating various inflammatory diseases such as rheumatic arthritis, Lupus ethrythematosus, multiple scleroderma, adult respiratory distress syndrome (ARDS), etc., as an anti-inflammatory agent.
  • inflammatory diseases such as rheumatic arthritis, Lupus ethrythematosus, multiple scleroderma, adult respiratory distress syndrome (ARDS), etc.
  • ARDS adult respiratory distress syndrome
  • Fig. 1 shows that Rac activation induced by phorbol myristate acetate (PMA) in macrophage of C57B/L mouse is suppressed by administration of oh8dG in a concentration-dependent manner.
  • PMA phorbol myristate acetate
  • Fig. 2 shows that the consumption of NADPH in macrophage activated by PMA is suppressed by administration of oh8dG in a concentration-dependent manner.
  • Fig. 3 shows that the production of ROS in macrophage activated by PMA is suppressed by administration of oh ⁇ dG in a concentration-dependent manner.
  • Fig. 4 shows that the production of ROS in macrophage activated by PMA is remarkably suppressed by administration of oh ⁇ dG and oh8G in a concentration-dependent manner.
  • Fig. 5 shows that the pretreatment of oh ⁇ dG and oh8G reduces TNF- ⁇ concentration in blood in a dosage-dependent manner.
  • Fig. 6 shows that the pretreatment of oh ⁇ dG and oh8G reduces IL-6 concentration in blood in a dosage-dependent manner.
  • Fig. 7 shows that the pretreatment of oh8dG and oh8G reduces IFN - ⁇ concentration in blood in a dosage-dependent manner.
  • Fig. 8 shows that the pretreatment of oh8dG and oh8G reduces IL-12p70 concentration in blood in a dosage-dependent manner.
  • Fig. 9 shows that the pretreatment of oh8dG and oh8G reduces IL- 18 concentration in blood in a dosage-dependent manner.
  • Fig. 10 shows that the pretreatment of oh ⁇ dG and oh8G in mouse treated with lipopolysaccharide (LPS) reduces the invasion of neutrophil into lung tissues in a dosage-dependent manner.
  • Fig. 11 shows that the pretreatment of oh8dG and oh8G in mouse treated with LPS reduces Rac2 activity in a dosage-dependent manner.
  • LPS lipopolysaccharide
  • Fig. 12 shows that the pretreatment of oh8dG and oh8G suppresses mouse death due to septic shock induced by administration of LPS (50mg/kg b.w.) in Balb/c mouse.
  • mouse macrophage was used because it is easy to obtain a large number of mice, and macrophage is a representative leukocyte acting an important role in many kinds of inflammatory reactions.
  • thiol glycollate (Tg) solution macrophage activator
  • Tg thiol glycollate
  • FBS fetal bovine serum
  • Fig. 1 When mouse macrophage was treated with PMA (phorbol myristrate acetate), the activity of Rac2 was increased (Fig. 1), which is similar to the activation mechanism of Rac at the time of phagocytosis of bacteria. However, it was shown that such activation of Rac2 by PMA was inhibited by the pretreatment of oh8dG in a concentration-dependent manner (Fig. 1). It was observed that Rac2 activity was also inhibited by DPI (diphenylene iodide), Rac2 inhibitor, which was used as positive control.
  • DPI diphenylene iodide
  • Mouse macrophages obtained from Example 1-1 were seeded in 60mm of culture dish and washed with PBS. The cells were dropped in dish (about 80%), and ⁇ ml of PBS was added thereto. Then, oh ⁇ dG of several concentrations was pretreated for 15 minutes, and then ⁇ 0 ⁇ g/ml of PMA was treated for 5 minutes. After that, 85 ⁇ g/mf, of NADPH was added thereto, and the absorbance at 340nm (A340) was measured by using a spectrophotometer at each scheduled time for 30 min. As shown in Fig. 2, oh8dG reduced activation of NADPH oxidase was increased by
  • PMA in a concentration-dependent manner in peritonial exudate macrophage (PEM).
  • PEM peritonial exudate macrophage
  • Mouse macrophage cells obtained from Example 1-1 were seeded in 96 well plates in which each well was seeded with about 7x10 4 cells. Other derivatives of several concentrations, besides oh8dG, were pretreated for 15 minutes, and then lO ⁇ g/mC of PMA was treated for 5 minutes.
  • OxyBURST Green probe that is a fluorescent material capable of detecting extracellularly released ROS was treated, and then the existence of ROS was measured by using microplate fluorescence reader at 490nm/525nm.
  • oh8dG reduced ROS production induced by PMA stimulus in a concentration-dependent manner.
  • Fig. 3 shows that as the concentration of oh8dG increases, ROS production is decreased more than in the condition without adding PMA. It means that during isolation of mouse macrophage used in the examination, Rac2 has been already activated, and so ROS production has been increased.
  • Fig. 3 shows that oh8GTP- ⁇ S (non-hydrolysis form of oh8GTP) has no effect, which strongly suggests that a nucleotide of oh8Gua cannot be used as a drug.
  • Fig. 4 compares of the suppression effect of mouse macrophage to the ROS production by oh8dG and oh8G with the effects of other DNA base compounds.
  • DMSO which is used as solvent of the samples, reduced ROS production slightly. It is guessed due to ROS scanvenging action of DMSO.
  • Normal guanine (Gua), normal deoxyguanine (dG) and normal adenosine (A) also showed some suppression effects, but their effects were much weaker than the effect of oh ⁇ dG.
  • oh8G showed the same suppression action as oh8dG.
  • EXAMPLE 4 Confirmation of the suppression effect of oh8dG and oh8G to proinflammatory cytokine concentration in blood of lypopolysaccharide (LPS)-treated mouse
  • LPS lypopolysaccharide
  • EXAMPLE 4-1 Laboratory animal oh8dG (6, 30, 60 mg/kg b.w.), oh8G (6, 30, 60 mg/kg b.w.), and aspirin(ASA), dG, G,
  • A 60 mg/kg b.w. each are dissolved in physiological saline, and independently injected into abdominal capacity of 6 week-old male Balb/c mice. After 4 hours, in order to induce inflammation, lmg/kg b.w. of LPS was injected into abdominal capacity. After 2 hours, under ether anesthesia, about 600 ⁇ H, of blood per mouse was collected from vena cava under abdomen and heart. Lung, heart, liver and kidney were also collected and kept in a freezer. Each test group consisted of 10 mice.
  • Example 4-1 The blood collected in the above Example 4-1 process was kept at room temperature for 1 hour, then centrifuged at 8,00Og for 20 minutes, and the serum formed in the surfactant was collected, poured, and then kept in a freezer of -80 ° C .
  • Cytokine was measured by using two methods, i.e., Multicytokine analysis system or cytokine measurement system purchased from Cayman Co. (USA). Five mice per test group were used for the measurement.
  • Fig. 5 shows the effects to TNF- ⁇ concentration in blood.
  • oh8dG suppressed the increase of TNF- ⁇ concentration by LPS in a dosage-dependent manner. Such action was stronger than the action of aspirin (ASA).
  • ASA aspirin
  • oh8G also showed suppression action, but its effect was weaker than the effect of oh8dG.
  • normal dG did not show any action, and G and A showed insignificant effects.
  • Fig. 6 shows the effects to IL-6 concentration in blood.
  • oh8dG remarkably suppressed the increase of IL-6 concentration by LPS in a dosage-dependent manner. Such action was similar to the action of aspirin (ASA).
  • oh8G also showed strong suppression action as the action of oh8dG.
  • Fig. 7 shows the effects to INF- ⁇ concentration in blood. As shown in Fig. 7, oh8dG remarkably suppressed the increase of INF- ⁇ concentration. Other compounds also showed suppression action, but their effects were weaker than the effect of oh ⁇ dG.
  • Fig. 8 shows the effects to IL-12p70 concentration in blood. As shown in Fig. 8, the
  • IL-12p70 concentration of oh8dG group was more reduced than that of the control group in LPS group, normal mouse group, in a dosage-dependent manner.
  • oh8dG did not show strong effect.
  • ASA also reduced IL-12p70 concentration than the normal group, but its action was weaker than the action of oh ⁇ dG.
  • Fig. 9 shows the effect to IL- 18 concentration in blood.
  • oh8dG reduced IL- 18 concentration in a dosage-dependent manner.
  • oh8G also showed suppression action, but its effect was weaker than the effect of oh8dG.
  • the error in experimental values was large, but there was no significant effect.
  • Example 4-1 The mouse lung tissue obtained in Example 4-1 was dissolved in tissue dissolving solution (5OmM potassium phosphate buffer, pH 6.0, 0.5% hexadectltrimethylammonium bromide), and then pulverized by using homogenizer. Thus pulverized product was pulverized again by ultrasonicator three times (10 seconds each). The resulting product was centrifuged at 12,00Og for 10 minutes in refrigerated centrifuge of 4 ° C , and then the supernatant was taken, poured, and kept in a freezer of -80 ° C .
  • tissue dissolving solution 5OmM potassium phosphate buffer, pH 6.0, 0.5% hexadectltrimethylammonium bromide
  • the thawed protein 40 ⁇ g/40//C, 1.6mM of tetramethylbenzidine, and 0.1 mM of hydrogen peroxide were reacted in total 120 ⁇ H. volume at 37 ° C for 2 min, and then the reaction was stopped by addition of 2M sulfuric acid on ice. The color change resulted from the reaction was measured by using multiplate reader at 450nm.
  • Fig. 10 shows the MPO activity of neutrophil invaded into mouse lung tissues.
  • the MPO activity in the normal group shows about 0.17 U/mg tissue, but after the treatment of LPS, its value was increased to about 1 U/mg tissue.
  • oh8dG remarkably reduced the MPO activity in a dosage-dependent manner.
  • oh8G reduced the MPO activity in each concentration within an error range, but its effect was not significant.
  • ASA showed a reduction effect, but its effect was very weak, compared to the effect of oh8dG. The effects of other control groups were not good, compared to the effect of oh8dG.
  • EXAMPLE 6 Confirmation of the suppression effect of oh ⁇ dG and oh8G for Rac2 activity in neutrophill and macrophage in mouse lung
  • the lung tissues obtained from Example 4-1 was thawed, dissolved in dissolving solution (25mM Tris HCl pH 7.5, 15OmM NaCl, 5 mM MgC12, 1% NP-40, 1 mM DTT, 5% glycerol), pulverized with homogenizer, and pulverized by ultrasonicator three times (10 seconds each).
  • dissolving solution 25mM Tris HCl pH 7.5, 15OmM NaCl, 5 mM MgC12, 1% NP-40, 1 mM DTT, 5% glycerol
  • Fig. 11 shows the comparison of Rac2 activity of neutrophill and macrophage in mouse lung.
  • Rac2 activation was increased, but the pretreatment of oh8dG remarkably reduced Rac2 activity in a dosage-dependent manner.
  • oh8G also suppressed Rac2 activity slightly, but its effect was not significant, compared to the effect of oh8dG.
  • ASA could not suppress Rac2, and dG and G showed a little reduction effect.
  • Example 4-1 By using the method of Example 4-1, 60mg/kg b.w. of the test drugs was injected, and then 50 mg/kg b.w. of LPS was injected. Survived mice therefrom were countered at each scheduled time.
  • Fig. 12 shows the protection effect of oh8dG for septic shock by LPS, from the survival result of mice.
  • LPS was injected into abdominal capacity in 50mg/kg b.w.
  • the mice were died drastically within 24 hours.
  • nine of total ten mice were died.
  • the mouse mortality was slow at the same period of time. That is, the death of mice was delayed.
  • composition of the present invention reduces the mouse mortality due to septic shock.

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Abstract

The present invention provides a composition for anti-inflammation comprising one or more ingredients of 8 -hydroxy guano sine (oh8G) and 8-hydroxydeoxyguanosine (oh8dG) as active ingredients. The present composition comprising one or more ingredients of oh8G and oh8dG suppresses the activity of Rac 2 of macrophage, thereby suppressing the activity of NADPH oxidase and also ROS production. Furthermore, the composition of the present invention suppresses the rising of blood concentration of pro-inflammatory cytokine induced by LPS, the increasing of MPO activity and Rac 2 activity, and the mortality of mouse due to LPS. Thus, the composition for anti-inflammation of the present invention can be used for treating various inflammatory diseases such as rheumatic arthritis, Lupus ethrythematosus, multiple scleroderma, adult respiratory distress syndrome (ARDS), etc., as an anti-inflammatory agent.

Description

COMPOSITION FOR ANTI-INFLAMMATION
TECHNICAL FIELD
The present invention relates to a composition for anti-inflammation.
BACKGROUND ART
Reactive oxygen species (ROS) are constantly produced in a living body, and destroy main substances of cells. DNA is one of representative attack object materials of ROS. Among various kinds of DNA damages by ROS, it is known that one of most typical damages is generation of 8 -hydroxy guanine (8-oxoguanine, hereafter referred to as Oh8Gua') produced from hydroxylation in 8-positon of guanine base by ROS [Kasai H. Mutat Res. 1997 Dec; 387(3): 147-63; Wu LL, et al. Clin Chim Acta. 2004 Jan; 339(l-2):l-9]. However, until now, most studies for oh8Gua have been limited to its mutation or carcinogenesis action because oh8Gua has mutation of transferring guanine - cytosine linkage to thymine - adenine linkage by coupling with adenine, while guanine is always coupled with cytosine in living body (Shibutani S, et al. Nature. 1991, 349: 431-4; Kamiya H, et al. Cancer Res. 1992, 52: 3483-5). Also, it was found that in DNA of cells, the production of oh8Gua is increased by many carcinogenic substances [Cancer Res. 1996 Feb 15; 56(4): 683-8; Cancer Res. 1997 Apr l ;57(7):1281-7; Free Radic Biol Med. 1995 Mar;18(3):431-6] and radiation [Environ Health Perspect. 1993 Mar;99:261-3; FEBS Lett. 2004 Jan 2;556(l-3):104-10], and is decreased by various kinds of chemopreventive agent (Baik SC, et al. Cancer Res. 1996, 56: 1279-82.; Chung MH, et al. Free Radic Biol Med. 1992, 12: 523-5.; Fraga CG, et al. Proc Natl Acad Sci U S A. 1991, 88: 11003-6) at once. Also, it is known that there are enzymes of removing oh8Gua in DNA (Tchou J, et al. Proc Natl Acad Sci U S A. 1991, 88: 4690-4.; Lee YS, et al. Biochem Biophys Res Commun. 1993, 196: 1545-51). Such facts strongly suggest that oh8Gua in DNA has carcinogenic action, and so is a transformed base which should be removed. On the other side, it is known that oh8Gua is produced from guanine bases of dGTP (deoxyguanosine triphosphate) and GTP (guanosine triphosphate) which exist in cytoplasm as well as guanine of DNA of nucleus because cells have an ohδdGTPase (also referred to as Mut T), an enzyme hydrolasing 8-hydroxydeoxyguanosine triphosphate (ohδdGTP) and 8-hydroxyguanosine triphosphate (oh8GTP) which are produced from modification of guanine base of dGTP and GTP by POS to ohδdGMP and ohδGMP (Shinmura K, et al. Nucleic Acids Res. 2000, 28: 4912-8). This enzyme makes ohδdGTP or oh8GTP not be inserted to DNA or RNA, and so prevents disturbance of genetic information by ohδdGTP and oh8GTP in cytoplasm. As shown above, all studies for ohδGua in DNA or oh8Gua in dGTP (or GTP) of cytoplasm have been concentrated on mutation or carcinogenic action by disturbance of genetic information.
Ras has been known to play an important role in cell reactions for oxidative stress.
In other words, it was reported that ERK (extracellular signal-regulated kinase), which is known as one of MAPK (mitogen-activated protein kinase), is activated by ROS [Fialkow L, et al. J Biol Chem. 1994 Dec 9;269(49): 31234-42.; Guyton KZ, et al. J Biol Chem. 1996 Feb 23;271(8):4138-42], and Ras is involved in this procedure [Lander HM, et al. J Biol Chem. 1995 Sep 8;270(36):21195-8; Aikawa R, et al. J Clin Invest. 1997 Oct l ;100(7):1813-21]. Recently, it has been reported that under the presence of oxidative stress, oh8GTP is created from GTP in cytoplasm, Ras is more activated by oh8GTP than GTP, and activated Ras activates all of kinase in downstream (Yoon SH, et al. Biochem Biophys Res Commun. 2005 Feb 4;327(l):342-8). Accordingly, it has been conceived that oh8dGTP and oh8GTP in cytoplasm may be substances with physiological activity that can affect cell function, not simple by-products of disturbing genetic information by insertion to DNA or RNA. Hence, the inventors of the present invention have intended to test the effect of oh8GTP for Rac, which is a small GTP -binding protein, besides Ras. Rac is a small GTP-binding protein, which produces ROS by inducing activation of NADPH oxidase in leucocytes [Blood. 2002 Oct 15; 100(8): 2692-6; J Immunol. 2004 Nov 15;173(10):5971-9; J Biol Chem. 1983 Aug 25;258(16):9631-5; J Immunol. 2005 Apr 15;174(8):4613-20.; J Biol Chem. 1994 Mar 11 ;269(10):7055-8], and essentially acts in phagocytosis of leucocytes [J Immunol. 2004 Nov 15;173(10):5971-9; J Biol Chem. 2004 Oct 8;279(41):43273-84] and migration of leucocytes [J Immunol. 2005 Apr 15;174(8):4613-20.; Ridley AJ. Trends Cell Biol. 2001, 1 1 : 471-7]. Thus, if oh8GTP can suppress Rac unlike Ras, oh8GTP can suppress inflammation-inducing action of leucocytes. However, the inventors used 8-hydroxyguanosine (or 8-oxo-guanosine, hereinafter referred to as Oh8G') and 8-hydroxydeoxyguanosine (or 8-oxo-deoxyguanosine, hereinafter referred to as Oh8dG'), which are nucleosides of oh8Gua without phosphate group of oh8GTP, because oh8GTP can act in homogenate of cells or tissues, but cannot act when it reacts with a cell or is administered in a living body since it cannot pass through cell membrane. Therefore, the present inventors have examined the suppression action of Rac and anti-inflammatory action of oh8G and oh8dG which are two kinds of nucleoside of oh8Gua.
DISCLOSURE OF INVENTION
TECHNICAL PROBLEM
Accordingly, one object of the present invention is to provide a composition for anti-inflammation comprising one or more ingredients of 8-hydroxyguanosine (oh8G) and 8 -hydroxy deoxyguanosine (oh8dG) as active ingredients.
Another object of the present invention is to provide a use of a composition comprising one or more ingredients of oh8G and oh8dG as active ingredients for the manufacture of anti-inflammatory agent.
Still, another object of the present invention is to provide a method for treating inflammation that comprises administrating to a mammal a therapeutically effective amount of one or more ingredients of oh8G and oh8dG.
TECHNICAL SOLUTION
The present invention provides a composition for anti-inflammation comprising one or more ingredients of 8-hydroxyguanosine (oh8G) and 8-hydroxydeoxyguanosine (oh8dG) as active ingredients. Also, the present invention provides a use of a composition comprising one or more ingredients of oh8G and ohδdG as active ingredients for the manufacture of anti-inflammatory agent.
The present invention also provides a method for treating inflammation, comprising administrating to a mammal a therapeutically effective amount of one or more ingredients of oh8G and oh8dG.
In one embodiment of the present invention, the composition comprises oh8dG as an active ingredient. In one embodiment of the present invention, the composition is used for treating one or more inflammatory diseases selected from rheumatic arthritis, Lupus ethrythematosus, multiple scleroderma, or adult respiratory distress syndrome (ARDS), as an anti-inflammatory agent. The present invention will be described in more detail below.
Unless indicated otherwise, the terms used in the present invention may be regarded as having same meanings over the present specification.
The present invention provides a composition for anti-inflammation comprising one or more ingredients of oh8G and ohδdG as active ingredients. The active ingredients of the present composition, oh8G and oh8dG, are nucleosides of oh8Gua which is produced from oxidation of guanine by ROS, and are excreted as body wastes in a very small amount. They can be obtained by isolating from body wastes or synthesizing according to a common method in the art, or can be used as commercial product.
As confirmed in the following examples, the present composition comprising one or more ingredients of oh8G and oh8dG suppresses the activity of Rac 2 of mouse macrophage, thereby suppressing the activity of NADPH oxidase and also suppressing ROS production from mouse macrophage.
Furthermore, the composition of the present invention remarkably suppresses the rising of blood concentration of pro-inflammatory cytokine induced by LPS; the increasing of myeloperoxidase(MPO) activity and Rac 2 activity in lung tissues; and the mortality of mouse due to LPS. The anti-inflammatory effect of the present composition is considerably superior to the effect of aspirin (ASA), one of representative NSAIDs. Unlike other anti-inflammatory agents of NSAIDs, the present composition has little side effects such as digestive system disorder, and so can be used as an excellent anti-inflammatory agent. The side effects of anti-inflammatory agents of NSAIDs seem from inhibiting COX-I
(cyclooxygenase-1) as well as COX-2 (cyclooxygenase-2) of COX. One expected serious side effect of oh8G and oh8dG is disturbance of genetic information by their insertion into DNA or RNA, but it is already known that oh8G and ohδdG cannot be inserted into DNA or RNA since guanylate kinase, etc. do not transfer them to oh8GTP and oh8dGTP. Thus, the composition of the present invention comprising one or more ingredients of oh8G and oh8dG as active ingredients can be used as a novel anti-inflammatory agent without side effects such as anti-inflammatory agents of NSAIDs. The composition of the present invention can be used for treating inflammatory diseases comprising various kinds of autoimmune diseases, such as rheumatic arthritis, Lupus ethrythematosus, multiple scleroderma, adult respiratory distress syndrome (ARDS), etc. In particular, the symptom in mouse death by LPS is similar to adult respiratory distress syndrome (ARDS) that is shown in septicemia of human. Thus, the present composition is useful for treating adult respiratory distress syndrome (ARDS) which has no distinct therapeutic agent at present.
The present composition comprising one or more ingredients of oh8G and ohδdG as active ingredients may be formulated by using pharmaceutically acceptable additives such as excipient, disintegrating agent, sweetener, binder, coating agent, blowing agent, lubricant, glidant, flavoring agent, solublizer, etc.
The composition of the present invention may further comprise one or more pharmaceutically or physiologically acceptable carriers to be formulated appropriately for administration.
The pharmaceutical composition of the present invention may be prepared in any form such as granule, powder, tablet, coated tablet, capsule, suppository, pill, syrup, juice, solution, suspension, emulsion or injectable liquid formulation.
For example, in order to formulate into the form of tablet or capsule, the active ingredients may be admixed with oral, nontoxic and pharmaceutically acceptable inactive carriers such as ethanol, glycerol, water, etc. If desirable or necessary, appropriate binder, lubricant, disintegrating agent and color coupler may be also admixed. Proper binder may be, but is not limited to, natural sugar such as starch, gelatin, glucose or β-lactose; natural or synthetic gum such as corn sweetener, acacia, tragacanth or sodium oleate; sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, etc. The disintegrating agent may comprise, but is not limited to, starch, methylcellulose, agar, bentonite, Xanthan gum, etc.
In order to formulate as liquid formulation, the present composition may comprise pharmaceutically acceptable carriers which have biocompatibility and are sterilized, for example, saline, sterilized water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, malto dextrin solution, glycerol, ethanol, and mixture thereof. If necessary, other common additives such as antioxidant, buffer, bacteriostatic agent, etc. may be added to the composition. Also, by further adding diluent, dispersing agent, surfactant, binder or lubricant, the composition may be formulated to injectable dosage form such as solution, suspension, emulsion, etc., pill, capsule, granule, or tablet. Furthermore, the present composition may be formulated in any desirable forms depending on disease or ingredient, by using conventional methods or a method described in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA). The present invention also provides a use of a composition comprising one or more ingredients of 8-hydroxyguanosine (oh8G) and 8 -hydroxydeoxy guano sine (oh8dG) as active ingredients for the manufacture of anti-inflammatory agent. The present composition comprising one or more ingredients of oh8G and oh8dG as active ingredients can be used for the manufacture of anti-inflammatory agent. Also, the present invention provides a method for treating inflammation which comprises administrating to a mammal a therapeutically effective amount of one or more ingredients of oh8G and oh8dG.
The term "mammal" as used hereinbefore or hereinafter means mammal as a subject for treatment, observation, or examination, but preferably, human. The term "therapeutically effective amount" used hereinbefore or hereinafter means an amount of active ingredient or pharmaceutical composition that can induce biological or medicinal response in a tissue, system, animal or human by a researcher, veterinarian, medical doctor or other clinician, and includes an amount of inducing alleviation of the symptoms of treated diseases or disorders. It is clear for a skilled artisan that the therapeutically effective amount of the active ingredients and the number of their administration are varied depending on desirable effect. Therefore, most suitable dosage to be administrated can be varied depending on various factors such as the kind of diseases, the kind and severity of patient's symptom, the content of active ingredient, the content and kind of other ingredients, the type of formulation, the age, body weight, health status, gender, and food of a patient, dosing time, administration route, the secretion ratio of composition, treatment period, and other co-administrated drug. In the treating method of the present invention, when oh8G and/or ohδdG is administrated once or several times a day, the dosage of the active ingredients is preferably 0.01 mg/kg - 100mg/kg.
In the treating method of the present invention, the pharmaceutical composition comprising one or more ingredients of oh8G and oh8dG as active ingredients may be administered via various routes including oral, rectal, intravenous, intra-arterial, intraperitoneal, intramuscular, intrathoracic, transdermal, topical, intraocular, and intradermal administration, according to conventional administration methods.
The advantages and features of the present invention and the method of achieving them will be evident from the following examples described in detail. However, it should be clearly understood that the present invention is not limited thereto, and may be variously embodied and practiced. Also, it is clear that the following examples intended to illustrate the scope of the present invention to a skilled artisan. Thus, the present invention will be defined by the scope of the claims.
ADVANTAGEOUS EFFECTS The present composition comprising one or more ingredients of oh8G and oh8dG suppresses the activity of Rac 2 of macrophage, thereby suppressing the activity of NADPH oxidase and also ROS production from macrophage. Also, the composition of the present invention remarkably suppresses the rising of blood concentration of pro-inflammatory cytokine induced by LPS, the increasing of MPO activity and Rac2 activity in lung tissues, and the mortality of mouse due to LPS. The composition of the present invention has considerably superior effect to aspirin (ASA), one of representative NSAIDs. Unlike anti-inflammatory agents of NSAIDs, the present composition has little side effects such as digestive system disorder, and so can be used as an excellent anti-inflammatory agent. Thus, the composition for anti-inflammation of the present invention can be used for treating various inflammatory diseases such as rheumatic arthritis, Lupus ethrythematosus, multiple scleroderma, adult respiratory distress syndrome (ARDS), etc., as an anti-inflammatory agent.
DESCRIPTION OF DRAWINGS
Fig. 1 shows that Rac activation induced by phorbol myristate acetate (PMA) in macrophage of C57B/L mouse is suppressed by administration of oh8dG in a concentration-dependent manner.
Fig. 2 shows that the consumption of NADPH in macrophage activated by PMA is suppressed by administration of oh8dG in a concentration-dependent manner.
Fig. 3 shows that the production of ROS in macrophage activated by PMA is suppressed by administration of ohδdG in a concentration-dependent manner.
Fig. 4 shows that the production of ROS in macrophage activated by PMA is remarkably suppressed by administration of ohδdG and oh8G in a concentration-dependent manner.
Fig. 5 shows that the pretreatment of ohδdG and oh8G reduces TNF-α concentration in blood in a dosage-dependent manner.
Fig. 6 shows that the pretreatment of ohδdG and oh8G reduces IL-6 concentration in blood in a dosage-dependent manner.
Fig. 7 shows that the pretreatment of oh8dG and oh8G reduces IFN -γ concentration in blood in a dosage-dependent manner.
Fig. 8 shows that the pretreatment of oh8dG and oh8G reduces IL-12p70 concentration in blood in a dosage-dependent manner. Fig. 9 shows that the pretreatment of oh8dG and oh8G reduces IL- 18 concentration in blood in a dosage-dependent manner.
Fig. 10 shows that the pretreatment of ohδdG and oh8G in mouse treated with lipopolysaccharide (LPS) reduces the invasion of neutrophil into lung tissues in a dosage-dependent manner. Fig. 11 shows that the pretreatment of oh8dG and oh8G in mouse treated with LPS reduces Rac2 activity in a dosage-dependent manner.
Fig. 12 shows that the pretreatment of oh8dG and oh8G suppresses mouse death due to septic shock induced by administration of LPS (50mg/kg b.w.) in Balb/c mouse.
MODE OF INVENTION
EXAMPLE 1. Confirmation of the suppression effect of oh8dG to Rac2 activity of mouse macrophage
1-1. Isolation of macrophage from mouse In the Examples of the present invention, mouse macrophage was used because it is easy to obtain a large number of mice, and macrophage is a representative leukocyte acting an important role in many kinds of inflammatory reactions.
200//f. of thiol glycollate (Tg) solution, macrophage activator, was injected into abdominal cavity of 8 week-old C57BL/6 female mice. After 3 days, the mice were sacrificed, and then 10 m-β. of PBS was injected into abdominal cavity of the mice, and the mice were shaken mildly right and left, back and forth. After that, the injected PBS in abdominal cavity was immediately pulled out and centrifuged to sediment the cells. The numbers of cells were counted, and then the cells were seeded in cell culture dish comprising RPMI medium added with 10% fetal bovine serum (FBS).
1-2. Confirmation of the suppression of Rac2 activity
The effect of oh8dG (Barry & Associate Co., USA) was examined to Rac2 which is known to involve in ROS production, phagocytosis, and migration of leukocyte.
When mouse macrophage was treated with PMA (phorbol myristrate acetate), the activity of Rac2 was increased (Fig. 1), which is similar to the activation mechanism of Rac at the time of phagocytosis of bacteria. However, it was shown that such activation of Rac2 by PMA was inhibited by the pretreatment of oh8dG in a concentration-dependent manner (Fig. 1). It was observed that Rac2 activity was also inhibited by DPI (diphenylene iodide), Rac2 inhibitor, which was used as positive control.
EXAMPLE 2. Confirmation of the suppression effect of oh8dG to NADPH oxidase activity of mouse macrophage
The effect of ohδdG to activation of NADPH oxidase was confirmed since Rac2 activates NADPH oxidase in inflammatory reaction.
Mouse macrophages obtained from Example 1-1 were seeded in 60mm of culture dish and washed with PBS. The cells were dropped in dish (about 80%), and \ml of PBS was added thereto. Then, ohδdG of several concentrations was pretreated for 15 minutes, and then \0μg/ml of PMA was treated for 5 minutes. After that, 85μg/mf, of NADPH was added thereto, and the absorbance at 340nm (A340) was measured by using a spectrophotometer at each scheduled time for 30 min. As shown in Fig. 2, oh8dG reduced activation of NADPH oxidase was increased by
PMA in a concentration-dependent manner in peritonial exudate macrophage (PEM). In other words, it shows that NADPH oxidase oxidizes NADPH, thereby reducing A340, and the reduction of A340 is suppressed by oh8dG. It was also observed that DPI used as positive control also suppresses NADPH oxidase activity.
EXAMPLE 3. Confirmation of the suppression effect of oh8dG and oh8G to ROS production of mouse macrophage
In inflammatory reaction, NADPH oxidase activated by Rac2 induces ROS production, and so the present inventors examined the suppression effect of oh8dG to ROS production of mouse macrophage. Mouse macrophage cells obtained from Example 1-1 were seeded in 96 well plates in which each well was seeded with about 7x104 cells. Other derivatives of several concentrations, besides oh8dG, were pretreated for 15 minutes, and then lOμg/mC of PMA was treated for 5 minutes. OxyBURST Green probe that is a fluorescent material capable of detecting extracellularly released ROS was treated, and then the existence of ROS was measured by using microplate fluorescence reader at 490nm/525nm.
As shown in Fig. 3, oh8dG reduced ROS production induced by PMA stimulus in a concentration-dependent manner. Particularly, Fig. 3 shows that as the concentration of oh8dG increases, ROS production is decreased more than in the condition without adding PMA. It means that during isolation of mouse macrophage used in the examination, Rac2 has been already activated, and so ROS production has been increased.
However, Fig. 3 shows that oh8GTP-γS (non-hydrolysis form of oh8GTP) has no effect, which strongly suggests that a nucleotide of oh8Gua cannot be used as a drug.
Fig. 4 compares of the suppression effect of mouse macrophage to the ROS production by oh8dG and oh8G with the effects of other DNA base compounds. DMSO, which is used as solvent of the samples, reduced ROS production slightly. It is guessed due to ROS scanvenging action of DMSO. Normal guanine (Gua), normal deoxyguanine (dG) and normal adenosine (A) also showed some suppression effects, but their effects were much weaker than the effect of ohδdG. However, oh8G showed the same suppression action as oh8dG.
EXAMPLE 4. Confirmation of the suppression effect of oh8dG and oh8G to proinflammatory cytokine concentration in blood of lypopolysaccharide (LPS)-treated mouse In order to examine the anti-inflammatory action of oh8dG and oh8G in animal experiment, the inventors administered LPS into mouse to induce inflammation, and then confirmed the cytokine concentration in blood relating to inflammation induction.
EXAMPLE 4-1. Laboratory animal oh8dG (6, 30, 60 mg/kg b.w.), oh8G (6, 30, 60 mg/kg b.w.), and aspirin(ASA), dG, G,
A (60 mg/kg b.w.) each are dissolved in physiological saline, and independently injected into abdominal capacity of 6 week-old male Balb/c mice. After 4 hours, in order to induce inflammation, lmg/kg b.w. of LPS was injected into abdominal capacity. After 2 hours, under ether anesthesia, about 600 μH, of blood per mouse was collected from vena cava under abdomen and heart. Lung, heart, liver and kidney were also collected and kept in a freezer. Each test group consisted of 10 mice.
EXAMPLE 4-2. Confirmation of the suppression effect of oh8dG and oh8G to proinflammatory cytokine
The blood collected in the above Example 4-1 process was kept at room temperature for 1 hour, then centrifuged at 8,00Og for 20 minutes, and the serum formed in the surfactant was collected, poured, and then kept in a freezer of -80 °C . Cytokine was measured by using two methods, i.e., Multicytokine analysis system or cytokine measurement system purchased from Cayman Co. (USA). Five mice per test group were used for the measurement.
Fig. 5 shows the effects to TNF-α concentration in blood. As shown in Fig. 5, oh8dG suppressed the increase of TNF-α concentration by LPS in a dosage-dependent manner. Such action was stronger than the action of aspirin (ASA). oh8G also showed suppression action, but its effect was weaker than the effect of oh8dG. However, normal dG did not show any action, and G and A showed insignificant effects.
Fig. 6 shows the effects to IL-6 concentration in blood. As shown in Fig. 6, oh8dG remarkably suppressed the increase of IL-6 concentration by LPS in a dosage-dependent manner. Such action was similar to the action of aspirin (ASA). oh8G also showed strong suppression action as the action of oh8dG.
Fig. 7 shows the effects to INF-γ concentration in blood. As shown in Fig. 7, oh8dG remarkably suppressed the increase of INF-γ concentration. Other compounds also showed suppression action, but their effects were weaker than the effect of ohδdG. Fig. 8 shows the effects to IL-12p70 concentration in blood. As shown in Fig. 8, the
IL-12p70 concentration of oh8dG group was more reduced than that of the control group in LPS group, normal mouse group, in a dosage-dependent manner. However, oh8dG did not show strong effect. ASA also reduced IL-12p70 concentration than the normal group, but its action was weaker than the action of ohδdG.
Fig. 9 shows the effect to IL- 18 concentration in blood. As shown in Fig. 9, oh8dG reduced IL- 18 concentration in a dosage-dependent manner. oh8G also showed suppression action, but its effect was weaker than the effect of oh8dG. In the other control groups, the error in experimental values was large, but there was no significant effect.
EXAMPLE 5. Confirmation of the suppression effect of oh8dG and oh8G for MPO activity of mouse lung
The mouse lung tissue obtained in Example 4-1 was dissolved in tissue dissolving solution (5OmM potassium phosphate buffer, pH 6.0, 0.5% hexadectltrimethylammonium bromide), and then pulverized by using homogenizer. Thus pulverized product was pulverized again by ultrasonicator three times (10 seconds each). The resulting product was centrifuged at 12,00Og for 10 minutes in refrigerated centrifuge of 4 °C , and then the supernatant was taken, poured, and kept in a freezer of -80 °C .
For the measurement of MPO activity, the thawed protein 40μg/40//C, 1.6mM of tetramethylbenzidine, and 0.1 mM of hydrogen peroxide were reacted in total 120 μH. volume at 37 °C for 2 min, and then the reaction was stopped by addition of 2M sulfuric acid on ice. The color change resulted from the reaction was measured by using multiplate reader at 450nm.
Fig. 10 shows the MPO activity of neutrophil invaded into mouse lung tissues. The MPO activity in the normal group shows about 0.17 U/mg tissue, but after the treatment of LPS, its value was increased to about 1 U/mg tissue. oh8dG remarkably reduced the MPO activity in a dosage-dependent manner. oh8G reduced the MPO activity in each concentration within an error range, but its effect was not significant. ASA showed a reduction effect, but its effect was very weak, compared to the effect of oh8dG. The effects of other control groups were not good, compared to the effect of oh8dG.
EXAMPLE 6. Confirmation of the suppression effect of ohδdG and oh8G for Rac2 activity in neutrophill and macrophage in mouse lung The lung tissues obtained from Example 4-1 was thawed, dissolved in dissolving solution (25mM Tris HCl pH 7.5, 15OmM NaCl, 5 mM MgC12, 1% NP-40, 1 mM DTT, 5% glycerol), pulverized with homogenizer, and pulverized by ultrasonicator three times (10 seconds each). For the measurement of enzyme activity, 7 ill of PAK-I PBD beads (Upstate
Biotechnology Co., USA) adhered to active Rac2 was added to 500 βg of protein, and then the resulting product was kept in refrigerator under rotation condition for 1 hour. The beads were separated from the resulting products by using refrigerated centrifuge at 14,00Og for 4 seconds, and then were washed with the above dissolving solution three times. Since the activated Rac2 is adhered to the obtained beads, SDS sample solution was added to the beads, and boiled at 95 °C , and then electrophoresis was performed in 12% acrylamide gel to separate Rac2. Western blotting was performed by using an antibody of Santa Cruz Co. (USA) as a first antibody.
Fig. 11 shows the comparison of Rac2 activity of neutrophill and macrophage in mouse lung. By LPS treatment, Rac2 activation was increased, but the pretreatment of oh8dG remarkably reduced Rac2 activity in a dosage-dependent manner. oh8G also suppressed Rac2 activity slightly, but its effect was not significant, compared to the effect of oh8dG. ASA could not suppress Rac2, and dG and G showed a little reduction effect.
Example 7. Confirmation of the reduction of mouse mortality due to septic shock
By using the method of Example 4-1, 60mg/kg b.w. of the test drugs was injected, and then 50 mg/kg b.w. of LPS was injected. Survived mice therefrom were countered at each scheduled time.
Fig. 12 shows the protection effect of oh8dG for septic shock by LPS, from the survival result of mice. When LPS was injected into abdominal capacity in 50mg/kg b.w., the mice were died drastically within 24 hours. Within 24 hours, nine of total ten mice were died. However, in the case that ohδdG was pretreated, the mouse mortality was slow at the same period of time. That is, the death of mice was delayed.
After 36 hours, three mice of oh8dG treatment group (total 10) and one mouse of ASA treatment group (total 10) were survived. Thus, it could be confirmed that the composition of the present invention reduces the mouse mortality due to septic shock.

Claims

WHAT IS CLAIMED IS:
1. A composition for anti-inflammation comprising one or more ingredients of 8 -hydroxy guano sine (oh8G) and 8-hydroxydeoxyguanosine (oh8dG) as active ingredients.
2. The composition of Claim 1, wherein the composition comprises oh8dG as an active ingredient.
3. The composition of Claim 1 or 2, wherein the composition is used for treating one or more inflammatory diseases selected from rheumatic arthritis, Lupus ethrythematosus, multiple scleroderma, or adult respiratory distress syndrome (ARDS).
4. A use of a composition comprising one or more ingredients of 8-hydroxyguanosine (oh8G) and 8-hydroxydeoxyguanosine (oh8dG) as active ingredients for the manufacture of anti-inflammatory agent.
5. The use of Claim 4, wherein the composition comprises oh8dG as an active ingredient.
6. The use of Claim 4 or 5, wherein the anti-inflammatory agent is used for treating one or more inflammatory diseases selected from rheumatic arthritis, Lupus ethrythematosus, multiple scleroderma or adult respiratory distress syndrome(ARDS).
7. A method for treating inflammation which comprises administrating to a mammal a therapeutically effective amount of one or more ingredients of oh8G and oh8dG.
8. The method of Claim 7, wherein the method comprises administrating to a mammal a therapeutically effective amount of ohδdG.
9. The method of Claim 7 or 8, wherein the inflammation is one or more inflammatory diseases selected from rheumatic arthritis, Lupus ethrythematosus, multiple scleroderma or adult respiratory distress syndrome (ARDS).
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