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WO2016129760A1 - Composition pour la prévention et le traitement de l'hépatotoxicité induite par acétaminophène, contenant du tnp (n2- (m-trifluorobenzyl) et n6-(p-nitrobenzyl) en tant qu'ingrédients actifs - Google Patents

Composition pour la prévention et le traitement de l'hépatotoxicité induite par acétaminophène, contenant du tnp (n2- (m-trifluorobenzyl) et n6-(p-nitrobenzyl) en tant qu'ingrédients actifs Download PDF

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Publication number
WO2016129760A1
WO2016129760A1 PCT/KR2015/006876 KR2015006876W WO2016129760A1 WO 2016129760 A1 WO2016129760 A1 WO 2016129760A1 KR 2015006876 W KR2015006876 W KR 2015006876W WO 2016129760 A1 WO2016129760 A1 WO 2016129760A1
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WIPO (PCT)
Prior art keywords
tnp
preventing
acetaminophen
acceptable salt
pharmaceutically acceptable
Prior art date
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Ceased
Application number
PCT/KR2015/006876
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English (en)
Korean (ko)
Inventor
김세윤
한용만
김영란
이슬기
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Korea Advanced Institute of Science and Technology KAIST
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Korea Advanced Institute of Science and Technology KAIST
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Priority claimed from KR1020150088220A external-priority patent/KR101666605B1/ko
Application filed by Korea Advanced Institute of Science and Technology KAIST filed Critical Korea Advanced Institute of Science and Technology KAIST
Priority to US15/032,295 priority Critical patent/US9636343B2/en
Priority to JP2016539060A priority patent/JP6145223B2/ja
Publication of WO2016129760A1 publication Critical patent/WO2016129760A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/52—Purines, e.g. adenine

Definitions

  • TNP (N2- (m-Tr if luorobenzy 1), N6- (-nitr Whyzy 1) ur i ne) 3 ⁇ 4 ⁇
  • the present invention provides a pharmaceutical composition for the prevention and treatment of liver toxicity, containing TNP (N2- (m-Tr if luorobenzy 1), N6- (pn it robenzyl) purine) or a salt acceptable by the pharmaceutical work as an active ingredient, and It relates to health functional food.
  • TNP N2- (m-Tr if luorobenzy 1), N6- (pn it robenzyl) purine
  • a salt acceptable by the pharmaceutical work as an active ingredient
  • TNP (N2- (m-Tr i ⁇ 1 iaor Plumbingzy 1), N6-()-ni : tr Anlagenzy 1-) ur ie) is a commercially available chemical and is known so far as inosyl pentakiphosphate. key, the better (pentakisphosphate inositol kinase) is a specific drug inhibition (J Biol Chem Apr 17, 2009 ; 284 (16):. 1057110582). TNP inhibits the biosynthesis of inositol pyrophosphate, which in turn lowers the level of polyphosphate in 5-inosine and increases the hepatic insulin signaling response. (Cell. 2010; 143 (6): 897910).
  • Acetaminophen developed in the United States in the 1950s and well known as Tylenol, is one of the world's most commonly used drugs for analgesic and antipyretic drugs (25 billion won in domestic annual sales of Tylenol), and its chemical name is N-acetyl. - ⁇ -aminophenol (N-acetyl_p-aminophenol), also called paracetamol (Dargan PI et al., Crit Care. 6 (2): 108-10. 2002). Acetaminophen is known to be administered orally up to 150 mg / kg body weight and up to 4 g / day in adults, and is classified as a safe over-the-counter drug without a doctor's prescription. However, acetaminophen can cause acute hepatic failure, liver necrosis, nephrotoxicity, and liver stiffness (1 iver cirrhosis) if overdose, causing severe death. It is a substance.
  • acetaminophen turns into a semi-ungotic substance that destroys cells by the action of some hepatic oxidase, which is usually not a problem because it is detoxified even when these substances are produced.
  • the body's detoxification material is altered, resulting in the destruction of liver cells.
  • a low concentration of acetaminophen is administered, it is combined with non-toxic glucononic acid and sulphate through biotransformation to remove toxicity and release into bile or plasma, 5- 10% Is metabolized by P450 (CYP), in particular CYP2E1 (Ray SD et al., J Pharmacol Exp Ther. 1996; 279: 1470-83).
  • acetaminophen is converted to N-acetyl-p-benzoquinoneimine (NAPQI) in the liver, and the converted metabolite is not toxic in combination with glutathione (Hazai E, et al. , Biochem Biophys Res Commun. 291 (4): 1089-1094. 2002).
  • NAPQI N-acetyl-p-benzoquinoneimine
  • gluconic acid and sulfate in the liver lose their ability to show nontoxicity, accumulating high semi-ungular NAPQI, causing fatal damage to the cell membrane, leading to death and incapacity of liver cells. Hepatotoxicity, eventually causing death (Webster PA et. Al., J Clin Pharmacol.
  • mice and hamsters are well known as liver damage models by acetaminophen administration, and similar phenomena have been reported in humans (Tee LB et a., Toxicol Appl Pharmacol. 83 (2): 294-314. 1986).
  • liver damage In addition, if you drink alcohol together, Toxic damage is more severe, and the US Food and Drug Administration (FDA) says that people who drink more than three drinks daily in 1998 can cause liver toxicity, so you should consult your doctor if you need to take this medicine. Mandatory notice.
  • FDA US Food and Drug Administration
  • the liver is the main organ that controls the blood death of the human body and detoxifies the detoxification function. When the liver is damaged, it is very important to develop a drug that can protect the liver damage because it causes inflammatory reactions and various diseases in the human body. Accordingly, the present inventors have tried to find a substance that is effective in the prevention and treatment of liver damage.
  • An object of the present invention is a pharmaceutical composition for the prevention and treatment of liver toxicity containing TNP (N2- (m-Tri f luorobenzyl), N6- (p-ni trobenzyl) pur ine) or a pharmaceutically acceptable salt thereof as an active ingredient. It is to provide a composition.
  • the present invention is TNP (N2- (m-Tr if 1 uorobenzy 1), N6- ( ⁇ - ⁇ itr Anlagenzy 1) pur i ne) or a pharmaceutically acceptable salt thereof as an active ingredient
  • a pharmaceutical composition for preventing and treating liver toxicity is provided.
  • the present invention is TNP or its.
  • the present invention also provides a method for treating hepatotoxicity comprising administering a pharmaceutically effective ⁇ TNP or a pharmaceutically acceptable salt thereof to an individual suffering from hepatotoxic disease.
  • the present invention also provides a method for preventing hepatotoxicity comprising administering to a subject a pharmaceutically effective amount of TNP or a pharmaceutically acceptable salt thereof.
  • the present invention also provides the use of TNP or a pharmaceutically acceptable salt thereof for use as a composition for preventing and treating liver toxicity.
  • the present invention also provides the use of TNP or a pharmaceutically acceptable salt thereof for use as a dietary supplement for preventing and improving liver toxicity.
  • the present invention provides a pharmaceutical composition for preventing and treating liver disease, which contains TNP or a pharmaceutically acceptable salt thereof as an active ingredient.
  • the present invention provides a health functional food for preventing and improving liver disease containing TNP or a pharmaceutically acceptable salt thereof as an active ingredient.
  • the present invention also provides a method for treating liver disease comprising administering a pharmaceutically effective amount of TNP or a pharmaceutically acceptable salt thereof to an individual suffering from liver disease.
  • the present invention also provides a method for preventing liver disease comprising administering to a subject a pharmaceutically effective amount of TNP or a pharmaceutically acceptable salt thereof.
  • the present invention also provides the use of TNP or a pharmaceutically acceptable salt thereof for use as a composition for preventing and treating liver disease.
  • the present invention also provides the use of TNP or a pharmaceutically acceptable salt thereof for use as a dietary supplement for preventing and improving liver disease. [Favorable effect]
  • the present invention relates to a composition for the prevention and treatment of liver toxicity containing TNP (N2- (m-Tr if kiorobenzyl), N6-(-nitr Anlagenzy 1) pur i ne) or a pharmaceutically acceptable salt thereof as an active ingredient.
  • TNP known as 5-anosi as an inhibitor of inosi tol pyrophosphate
  • GSH glutathione
  • JNK phosphorylated JNK
  • TNP N2- (m-Tr i f luorobenzyl), N6- (p-n i trobenzyl) pur ine).
  • FIG. 2 shows acetaminophen and hepatocytes differentiated from human embryonic stem cells.
  • Figure 3 is a graph showing the analysis of apoptosis according to acetaminophen and TNP treatment of mouse-derived hepatocytes.
  • Figure 4 is a graph showing the analysis of apoptosis according to acetaminophen and TNP treatment of the human liver cancer cell line (HepG2).
  • FIG. 5 is an enlarged view of the photograph of FIG. 4 by 200 times.
  • Figure 6 is a graph showing the analysis of the intracellular glutathione concentration according to acetaminophen and TNP treatment of mouse-derived hepatocytes.
  • Figure 7 is a diagram showing the analysis of JNK phosphorylation according to acetaminophen and TNP treatment of mouse-derived hepatocytes.
  • FIG. 8 is a diagram showing the analysis of JNK phosphorylation according to acetaminophen and TNP treatment of human liver cancer cell line.
  • FIG. 9 is a diagram showing the survival rate analysis of acetaminophen and TNP treatment in a mouse model.
  • FIG. 10 is a diagram showing histopathological analysis of several TNPs of acet 4-Menofene in 1 ⁇ of mouse hair.
  • FIG. 11 is a diagram showing blood analysis according to acetaminophen and TNP treatment in a mouse model.
  • FIG. 12 is a diagram showing a JNK phosphorylation analysis according to acetaminophen and TNP treatment in a mouse model.
  • the present invention is a pharmaceutical composition for the prevention and treatment of liver toxicity containing TNP (N2- (m-Tr if luorobenzyl), N6— (p-ni t robenzyl) pur ine) or a pharmaceutically acceptable salt thereof as an active ingredient.
  • TNP N2- (m-Tr if luorobenzyl), N6— (p-ni t robenzyl) pur ine
  • a pharmaceutically acceptable salt thereof as an active ingredient.
  • the TNP preferably has a structure of Formula 1, but is not limited thereto.
  • the TNP inhibits acetaminophen-derived cell death, and in hepatocytes .
  • Glutathione increases (glutathione, GSH), and preferably inhibits the increase in the stress response caused by acetaminophen.
  • GSH glutthione
  • the present inventors confirmed the change according to TNP treatment of cell death induced by acetoaminophen in human embryonic stem cell-derived hepatocytes, mouse-derived hepatocytes, and human liver cancer cell lines (HepG2). It was confirmed that hepatic cell death induced by TNP treatment was significantly inhibited (see FIGS.
  • TNP known as an inhibitor of inosi tol pyrophosphate of the present invention inhibits apoptosis by acetaminophen in human embryonic stem cell-derived hepatocytes and mouse-derived hepatocytes and human liver cancer cell lines, It increased the concentration of reduced glutathione (GSH) in hepatocytes and confirmed that it inhibits phosphorylated JNK, an increased stress response by acetaminophen, and the same acetaminophen-induced hepatotoxicity in animal models.
  • GSH reduced glutathione
  • the present invention is not only the TNP represented by the formula (1), Pharmaceutically acceptable salts thereof, possible solvates, hydrates, d) It includes both miche, or stereoisomer thereof.
  • the TNP represented by Formula 1 of the present invention may be used in the form of a pharmaceutically acceptable salt, and as the salt, an acid addition salt formed by a pharmaceutically acceptable free acid is useful.
  • Acid addition salts include inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid, aliphatic mono and dicarboxylates, phenyl-substituted alkanoates, hydroxy alkanoates and alkanes.
  • non-toxic organic acids such as dioates, aliphatic acids, aliphatic and aromatic sulfonic acids.
  • These pharmaceutically toxic salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate phosphate, monohydrogen phosphate, dihydrogen phosphate metaphosphate, pyrophosphate chloride, bromide and iodide fluoride.
  • the acid minor salts according to the present invention can be dissolved in conventional methods, for example, by dissolving the TNP represented by the formula (1) in an excess of aqueous acid solution, and dissolving the salts with a miscible organic solvent such as methane, hyehanol, acetone or It can be prepared by precipitation with acetonitrile. In addition, the mixture may be prepared by evaporating a solvent or excess acid to dry, or by suction filtration of the precipitated salt. Bases can also be used to make pharmaceutically acceptable metal salts.
  • Alkali metal or alkaline earth metal salts are obtained, for example, by dissolving the compound in an excess of alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the compound salt at cost, and evaporating and drying the filtrate. At this time, it is pharmaceutically suitable to prepare sodium, potassium or calcium salt as the metal salt.
  • Corresponding silver salts are also obtained by reacting an alkali or alkaline earth metal salt with a suitable negative salt (eg silver nitrate). When formulating the composition, it is prepared using diluents or excipients such as commonly used layering agents, extenders, binders, wetting agents, disintegrating agents, surfactants.
  • Solid preparations for oral administration include tablets, patients, powders, granules, capsules, troches and the like, and such solid preparations contain at least one excipient such as starch in one or more TNPs represented by Formula 1 of the present invention.
  • a excipient such as starch in one or more TNPs represented by Formula 1 of the present invention.
  • Calcium carbonate It is prepared by mixing sucrose or lactose or gelatin.
  • lubricants such as magnesium styrate talc are also used.
  • Liquid preparations for oral administration include suspensions, solutions, emulsions or syrups, and include various excipients such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin.
  • Sieves for parenteral administration include thermophilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, suppositories, and the like.
  • Non-aqueous solvents and suspending solvents may be used propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as etholeate.
  • w epsol, macrogol, tween 61, cacao butter, laurin butter, glycerol, gelatin and the like can be used as the base of the suppository.
  • the composition according to the invention is administered in a pharmaceutically effective amount.
  • compositions of the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be single or multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect in a minimum amount without side effects, which can be easily determined by those skilled in the art.
  • the effective amount of the compound according to the present invention may vary depending on the age, sex and weight of the patient, generally between 0.01 mg and 100 mg, preferably 0.5 mg and 10 mg per kg of body weight daily or It can be administered every other day or divided into one to three times a day.
  • the severity of obesity, sex, weight and age The dosage may be increased or decreased depending on the above or the like, and the above dosage does not limit the scope of the present invention by any method.
  • the present invention provides a health functional food for preventing and improving liver toxicity containing TNP or a pharmaceutically acceptable salt thereof as an active ingredient.
  • the TNP preferably has a structure of Chemical Formula 1, but is not limited thereto.
  • the TNP inhibits acetoaminophen-derived cell death, increases glutathione (GSH) in hepatocytes, and preferably suppresses an increase in stress response caused by acetaminophen, but is not limited thereto.
  • GSH glutathione
  • TNP known as an inhibitor of inosi tol pyrophosphate of the present invention inhibits apoptosis caused by acetaminophen in human embryonic stem cell-derived hepatocytes, mouse-derived hepatocytes, and human hepatocarcinoma cell lines
  • Reduced glutathione increased the concentration of (glutathione, GSH) and confirmed that it inhibits phosphorylated JNK, an increased stress response by acetaminophen, and the same acetaminophen-induced hepatotoxicity in animal models.
  • GSH glutathione
  • TNP or a pharmaceutically acceptable salt thereof can be usefully used as a health functional food for the prevention and improvement of liver toxicity.
  • the TNP of the present invention may be prepared by various methods known in the food science or pharmaceutical fields, and may be used by itself or as a food acceptable carrier, excipient, diluent, and the like. It can be prepared in any food form that is compatible and orally available. Preferably in the form of beverages, pills, granules, tablets or capsules.
  • the health functional food of the present invention may further include ingredients that are commonly added during food production and are food acceptable.
  • ingredients that are commonly added during food production and are food acceptable.
  • citric acid liquid fructose
  • sugar sugar
  • glucose glucose
  • acetic acid it may further include one or more components in malic acid, juice, and the like.
  • the amount that can be included as an active ingredient of the health functional food according to the present invention can be appropriately selected according to the age, sex, weight, condition, symptoms of the disease of the person who wants to prevent and improve liver toxicity, preferably 1 day for adults It is good to include about 0.01 g to 10.0 g, and by ingesting a health functional food having such a content, the effect of preventing and improving liver toxicity can be obtained.
  • the present invention provides a pharmaceutical composition for preventing and treating liver disease, which contains TNP or a pharmaceutically acceptable salt thereof as an active ingredient.
  • the present invention provides a health functional food for preventing and improving liver disease containing TNP or a weakly acceptable salt thereof as an active ingredient.
  • the TNP preferably has a structure of Formula 1, but is not limited thereto.
  • the TNP inhibits acetaminophen-derived cell death, increases glutathione (GSH) in hepatocytes, and preferably inhibits an increase in stress response caused by ace Baminophen, but is not limited thereto.
  • GSH glutathione
  • the acetaminophen derived liver disease includes acute liver failure (fulminant hepat ic fai lure), liver necrosis (l iver necrosis), neurotoxin (nephrotoxici ty) and. . liver It is preferably any one selected from the group consisting of cirrhosis, but not limited thereto.
  • TNP known as an inhibitor of 5-inosine pyrophosphate of the present invention inhibits apoptosis by acetaminophen in human embryonic stem cell-derived hepatocytes, mouse-derived hepatocytes, and human hepatocarcinoma cell lines, and is reduced in hepatocytes.
  • Glutathione increased the concentration of (glutathione, GSH) and was found to inhibit phosphorylated JNK, an increased strain i response by acetaminophen, and to protect the same acetaminophen-induced hepatotoxicity in animal models.
  • the TNP or a pharmaceutically acceptable salt thereof can be usefully used as a pharmaceutical composition for preventing and treating liver disease, and as a dietary supplement.
  • the present invention will be described in detail by Examples and Experimental Examples.
  • hESC human embryonic stem cells
  • the hESC cell line was a CHA-hESC cell line and cultured for 3 days in a confluent dotok in a controlled wfl ⁇ (conditioned medium) in a feederfree system. After incubation, the hESC cells were treated with 50 ng / m £ activin MActivin A; Differentiation was induced by incubation for 5 days in RPMI-1640 (Hyc lone, USA) medium containing Peprotech Inc., USA. The differentiated cells contained 30 ng / mi fibroblast growth factor 4 (Peprotech) and 20 ng / mi bone morphogenetic protein 2 (BMP2; Peprotech).
  • HCM hepatocyte culture medium
  • HGF hepatocyte growth factor
  • Matured hepatocytes were obtained by inducing the maturation of hepatocytes while incubating for 5 days in hepatocyte culture medium to which R & D Systems Inc., USA) and 0.1 ⁇ M dexamethasone (Sigma-Aldrich, USA) were added.
  • Example-2 Cultivation of Mouse-Derived Hepatocytes
  • Mouse-derived hepatocyte culture was performed as follows using the collagenase perfusion method.
  • the filtrate was centrifuged at 100 X g for 3 minutes, and then the supernatant was discarded and the precipitated hepatocytes were suspended in HBSSCHanks' Balanced salt solution, WelGene) buffer and centrifuged.
  • the precipitated hepatocytes were centrifuged in Percol solution (Autoclaved percolKsigma), 10 X PBS, 1M HEPES PH7.4 for 10 minutes at 2400 rpm, and then the supernatant was discarded and the precipitated hepatocytes were HBSSCHanks' Balanced salt solution, WelGene. Suspended in buffer and centrifuged again at 100 X g.
  • Precipitated hepatocytes were suspended in a culture solution to obtain a cell suspension, and then adjusted to a concentration of hepatocytes of 1 X 10 cells / ml, and then transplanted into a culture vessel previously coated with gelatin.
  • Culture medium was 10% FBS, 10 nM dexa, 10 nM insulin, Containing penicillin / streptomycin (penicillin / streptomycin LOO g / ml)
  • DMEM medium (bic est) was used and cultured by supplying a mixed gas of C025> in a 37 0 C incubator maintained at a constant temperature and humidity.
  • HepG2 Human Human Cancer Cell Line
  • HepG2 cells which are human liver cancer cells, were divided into 6-well plates pre-coated with gelatin at a cell concentration of 1 ⁇ 10 cell / n and incubated for 24 hours for cell attachment.
  • the culture medium was a DMEM bovine added with 10% FBS, 10 nM dexa, 10 nM insulin, and penicillin / s: ⁇ ⁇ mycin a3 ⁇ 40 irl r, and C025% of the mixed gas in a 37 0 C incubator maintained at a constant temperature and humidity. Supplied and incubated.
  • hepatocytes were divided into 6-well plates at a concentration of 9 OT per well. 1000 mg / ⁇ DMEM—low glucose culture (Dulbecco's Modified Eagle's Medium, Wei gene No. 001-11) was replaced and incubated for 2 hours. Acetaminophen 25 mM single treatment group, acetaminophen 25 mM, TNP mixed group at a concentration of 100 ⁇
  • mice-derived hepatocytes were dispensed at a concentration of 100% per well in a 6-well plate, and cell viability results were obtained in the same manner as in ⁇ Experimental Example 1>.
  • all experimental values were expressed as mean ⁇ standard error (mean ⁇ SD), and statistical analysis was performed by Student's t-test, and the maximum significance limit was set to p ⁇ 0.05.
  • HepG2 human liver cancer cell line was dispensed at a concentration of 90% per well in a 6-well plate, and cell viability results were obtained in the same manner as in ⁇ Experimental Example 1>.
  • all experimental values were expressed as mean ⁇ standard error (mean ⁇ SD), and statistical analysis was performed by Student's t_test, and the maximum significance limit was set to p ⁇ 0.05.
  • mice-derived hepatocytes treated with acetaminophen and TNP were collected in the same manner as in ⁇ Experimental Example 2>, and then cell extracts were prepared for protein analysis, followed by SDS ⁇ PAGE analysis and Western blotting.
  • Cells were 10 mi SDS buffer solution (50 mM Tris-HCl, 1% IGEPAL CA-630, 0.25% deoxycholic acid, 150) containing a protease inhibitor cocktai 1 (Complete Mini, Roche, Germany).
  • Electrophoretic proteins were transferred to Immobilon-P transfer membranes (Mill ipore Corporate, USA) using an electroblotting device, and the membrane was then replaced with 0.1% Tween-20 and 10%. After blocking for 1 hour in TBS (Tris-buffered saline) containing% formula, phosphorylated JNK Cell Signaling) and whole JNK Cell Signaling) antibodies were added and reacted overnight.
  • TBS Tris-buffered saline
  • HRP horseradish peroxidase
  • the protein bands were Western blotting luminol reagent (luminol) reagent) (Santa Cruz Biotechnology) Activity was analyzed using Image J software (version 1.37, NIH, USA).
  • TNP acetaminophen-induced hepatotoxicity
  • mice 500 mg / kg acetaminophen (Sigma) was dissolved in water and orally administered to mice using male C57BL / 6 mice aged 8 to 10 weeks, and then 10 mg / kg TNP (Caibiocham) to olive oil (Sigma). After dissolving, mice were injected intraperitoneally to analyze the survival rate. Statistical analysis was performed using Kaplan-Meier survival analysi s, and the maximum limit of significance was set to ⁇ .05.
  • TNP treatment confirmed the protective effect of the remarkable individual survival rate against acetaminophen-induced hepatotoxicity (Fig. 9).
  • livers were extracted 6 hours after TNP injection in male mice injected with acetaminophen and TNP to determine histopathological analysis, blood analysis (AST, ALT) and stress signaling activity (JNK). Analyzed. Hematological analysis of blood samples obtained by orbital blood collection of mice was performed by centrifugation at 13000 rpm for 10 minutes for analysis of AST and ALT (Idexx VetTest8008 chemi stry analyzer) with serum 100 ⁇ and extracted to analyze stress signaling activity.
  • IM tr is— cl (PH7.4), IM Nacl, 0.5M EDTA, NP-40, 10% sodium deoxycholate, 10% SDS), digested with homogenizer and centrifuged. after separation by collecting the supernatant were performed SDS-PAGE analysis, and Western blotting. Phosphorylated JNK and total JNK amount were measured by Western blotting in the same manner as in ⁇ Example 5>.
  • FIG. 10 histological analysis of liver tissue by acetaminophen treatment showed a very pathological phenotype of necrosis in the liver of mice treated with TNP (Fig. 10).
  • FIG. 11 it was confirmed that AST and ALT levels of hepatotoxicity serum were significantly reduced in mice treated with TNP (FIG. 11).
  • FIG. 12 stress reaction by acetaminophen treatment was shown. The increase of phosphorylation induced an increase in phosphorylated JNK signal, but it was confirmed that the phosphorylated JNK increase was significantly inhibited by TNP co-treatment (FIG. 12).

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Abstract

La présente invention concerne une composition pour la prévention et le traitement de l'hépatotoxicité induite par acétaminophène (AP), contenant du TNP (N2-(m-trifluorobenzyl) et N6-(p-nitrobenzyl) purine) en tant qu'ingrédients actifs. Il est confirmé que le TNP, qui est connu en tant qu'inhibiteur de pyrophosphate 5-inositol, inhibe la mort cellulaire induite par acétaminophène dans des hépatocytes dérivés de cellules souches d'embryon humain, d'hépatocytes dérivés de la souris et la lignée cellulaire d'hépatome humain, augmente la concentration de glutathione réduite (GSH) dans les hépatocytes, et inhibe le JNK phosphorylé, qui est une réponse au stress augmenté par l'acétaminophène. Il est également confirmé que la composition a le même effet de protection contre l'hépatotoxicité induite par acétaminophène que dans le modèle animal, et, de ce fait, le TNP peut être utilisé en tant que principe actif dans la composition pour la prévention et le traitement de l'hépatotoxicité induite par acétaminophène.
PCT/KR2015/006876 2015-02-13 2015-07-03 Composition pour la prévention et le traitement de l'hépatotoxicité induite par acétaminophène, contenant du tnp (n2- (m-trifluorobenzyl) et n6-(p-nitrobenzyl) en tant qu'ingrédients actifs Ceased WO2016129760A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
US15/032,295 US9636343B2 (en) 2015-02-13 2015-07-03 Composition for preventing and treating acetaminophen inducing hepatotoxicity containing TNP(N2-(m-Trifluorobenzyl), N6-(p-nitrobenzyl)purine) as an effective ingredient
JP2016539060A JP6145223B2 (ja) 2015-02-13 2015-07-03 有効成分としてＴＮＰ（Ｎ２−（ｍ−トリフルオロベンジル），Ｎ６−（ｐ−ニトロベンジル）プリン）を含む、アセトアミノフェンに起因する肝毒性の予防及び治療のための組成物

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
KR20150022315 2015-02-13
KR10-2015-0022315 2015-02-13
KR10-2015-0088220 2015-06-22
KR1020150088220A KR101666605B1 (ko) 2015-02-13 2015-06-22 TNP(N2-(m-Trifluorobenzyl), N6-(p-nitrobenzyl)purine)를 유효성분으로 함유하는 아세트아미노펜 유래 간 독성 예방 및 치료용 조성물

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