Classifications

• • 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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