JP2012102029A - Therapeutic agent for acetaminophen liver damage - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel pharmaceutical composition for treating or preventing acetaminophen liver damage.SOLUTION: The pharmaceutical composition for treating or preventing acetaminophen liver damage includes (E)-3-[p-(1H-imidazole-1-ylmethyl)phenyl]-2-propenoate (ozagrel) or its pharmacologically acceptable salt as an active ingredient. The ozagrel or its pharmacologically acceptable salt displays remarkable suppression effect on liver tissue damage such as suppression in increase of liver alanine aminotransferase (ALT) and increase of the total liver glutathione level when it is administrated before/after administrating acetaminophen.

Description

  The present invention relates to a pharmaceutical composition for the treatment or prevention of acetaminophen liver injury.

More specifically, the present invention provides the formula:
(E) -3- [p- (1H-imidazol-1-ylmethyl) phenyl] -2-propenoic acid [generic name: Ozagrel] or a pharmacologically acceptable salt thereof is effective The present invention relates to a pharmaceutical composition for treating or preventing acetaminophen liver injury, which is contained as a component.

  Acetaminophen (hereinafter sometimes referred to as “APAP”) is an aniline-based non-steroidal anti-inflammatory analgesic (NSAIDs), which is a safe and effective antipyretic analgesic when used in an appropriate amount. It is known to cause toxic liver injury by overdose such as drinking and suicide attempts. APAP administered was about 50% glucuronidated, about 30% sulfated and 5-10% cytochrome P450 (CYP) 2E1 to N-acetyl-p-benzoquinoneimine (NAPQI) in the liver And is further conjugated with glutathione and excreted in the urine. When NAPQI is produced and accumulated in large amounts in hepatocytes for some reason, liver injury is caused. For example, in elderly people who have reduced ability to sulphate or synthesize glutathione, or who have lower glutathione levels due to taking CYP2E1-inducing drugs or chronic drinking, liver damage is likely to develop and become severe. There is also a point of danger (eg, Non-Patent Document 1).

  For drug-induced liver injury, the principle of treatment is to discontinue the suspected drug promptly, and mild liver injury improves spontaneously. However, if transaminase elevation or jaundice persists, drug therapy is required. For liver injury due to APAP, intravenous infusion of N-acetylcysteine (NAC), a precursor, is effective for the purpose of supplementing liver glutathione within 10 hours after APAP. Since the drug is not marketed, oral or inhalation solution is initially administered at 140 mg / kg, then 70 mg / kg every 4 hours 17 times, total 18 times, orally or via nasogastric tube (See Non-Patent Document 1 or 2). Therefore, there are cases where sufficient effects may not always be obtained in patients who have been transported after elapse of APAP, and early development of new therapeutic agents that are easier to use is desired.

  Ozagrel or a pharmacologically acceptable salt thereof has thromboxane (TX) synthase inhibitory action, and bronchial asthma treatment agent as ozagrel hydrochloride hydrate, cerebral vasospasm after subarachnoid hemorrhage as ozagrel sodium and this It is used as a therapeutic agent for improving cerebral ischemic symptoms associated with cerebral ischemia and motor disorders associated with cerebral thrombosis (acute stage). In addition, the effectiveness of ozagrel has been reported in corneal diseases (see Patent Document 1), dry cough caused by an angiotensin converting enzyme inhibitor (see Patent Document 2), and the like.

The effect of ozagrel has been reported in an experimental liver injury model. Specifically, ozagrel inhibits transaminase GOT (AST) and GPT (ALT) elevation in a model of carbon tetrachloride-induced liver injury in which free radicals produced by liver drug-metabolizing enzymes are thought to cause hepatocyte degeneration. And it has been reported that histopathological improvement is shown (refer nonpatent literature 3). It has been reported that ozagrel exhibits inhibition of elevation of GOT (AST) and GPT (ALT) in bacterial lipopolysaccharide (LPS) and anti-basic liver protein (BLP) antibody-induced liver injury models (see Non-Patent Document 4). However, these liver injury models and APAP liver injury are clearly different from those of APAP liver injury in the causative drugs and toxic metabolites that cause liver injury. In addition, in the liver ischemia-reperfusion injury model, ozagrel has been reported to show an increase suppression of GPT (ALT) and an improvement in hepatic tissue blood flow, and the survival rate is improved (see Non-Patent Document 5).
However, none of the above references describes or suggests that ozagrel is useful for the treatment or prevention of APAP liver injury.

International Publication No. 2007/023877 Pamphlet WO 97/22362 pamphlet Ministry of Health, Labor and Welfare April 2008, Manual for Response to Severe Side Effects Disease Drug-induced liver injury p. 31-32 Today's Treatment Guidelines 2003, Medical School Publishing, p. 365 Hiroi Nagai and five others, Prostaglandins, 1989, 38, 4 p. 439-446 Hiroi Nagai and five others, Japan. J. Pharmacol., 1989, 51, p. 191-197 J Iida and 11 others, Transplant. Proc., 1999, 31, p. 1061-1062

  An object of the present invention is to provide a novel pharmaceutical composition for treating or preventing APAP liver injury.

  As a result of intensive studies to solve the above problems, the present inventors surprisingly suppressed the increase of liver transaminase and increased the amount of liver glutathione by administering ozagrel hydrochloride before or after APAP administration. For the first time, it has been found to show significant suppression of liver tissue injury, and the pharmaceutical composition containing ozagrel or a pharmacologically acceptable salt thereof is extremely effective for the treatment or prevention of APAP liver injury Knowledge was acquired and it came to make this invention.

That is, the present invention

[1] A pharmaceutical composition for treating or preventing acetaminophen liver injury, comprising ozagrel or a pharmacologically acceptable salt thereof as an active ingredient;

[2] The pharmaceutical composition according to the above [1], comprising ozagrel hydrochloride as an active ingredient;

[3] The pharmaceutical composition according to the above [1] or [2], wherein the dosage form is an injection;

  In the present invention, “acetaminophen liver injury” refers to drug-induced liver injury caused by administration of acetaminophen, for example, liver such as elevation of alanine transaminase (ALT), degeneration of hepatocyte tissue, or necrosis. Injuries and various symptoms resulting from them (jaundice, general malaise, etc.) can be mentioned.

  In the present invention, the prevention of APAP liver injury includes a person who has a high risk of developing or serious APAP liver injury (for example, an elderly person, a patient who continuously administers a drug that induces CYP2E1, a chronic drinker, etc. ) To prevent the onset or seriousness of APAP liver injury by administering in advance or simultaneously with APAP. Examples of the treatment of APAP liver injury include improving the symptoms of APAP liver injury or suppressing its severity by subsequent administration to a patient who has developed APAP liver injury.

  Ozagrel or a pharmacologically acceptable salt thereof, which is an active ingredient of the pharmaceutical composition of the present invention, can be easily produced by a known method (for example, Japanese Patent No. 1395666) or a method analogous thereto. Commercially available products (for example, APAC Pharmaceutical, LLC) can also be purchased.

  Ozagrel which is an active ingredient of the pharmaceutical composition of the present invention can be made into a pharmacologically acceptable salt. Such salts include acid addition salts with mineral acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, phosphoric acid, formic acid, acetic acid, methanesulfonic acid, benzenesulfonic acid, p-toluene. Acid addition with organic acids such as sulfonic acid, propionic acid, citric acid, succinic acid, tartaric acid, fumaric acid, butyric acid, oxalic acid, malonic acid, maleic acid, lactic acid, malic acid, carbonic acid, benzoic acid, glutamic acid, aspartic acid Salts, sodium salts, potassium salts, calcium salts, magnesium salts, zinc salts, salts with inorganic bases such as lithium salts, aluminum salts, N-methyl-D-glucamine, N, N′-dibenzylethylenediamine, 2-amino Ethanol, tris (hydroxymethyl) aminomethane, arginine, lysine, piperazine, choline, diethylamine, 4-phenyl-cyclohexyla An addition salt with an organic base such as min can be mentioned, and hydrochloride or sodium salt is preferred.

  In the present invention, the pharmacologically acceptable salt includes a solvate with a pharmacologically acceptable solvent such as water or ethanol, for example, monohydrate of ozagrel hydrochloride. .

  When the pharmaceutical composition of the present invention is used for actual prevention or treatment, the dose of ozagrel or a pharmacologically acceptable salt thereof, which is an active ingredient thereof, depends on the age, sex, weight, disease and degree of treatment of the patient. For example, in the case of oral administration per day for an adult, it can be administered in a single dose or divided into several doses in the range of generally about 100 to 800 mg, more preferably 200 to 400 mg. In the case of oral administration, it can be administered in a dose of about 10 to 160 mg, more preferably 20 to 80 mg, once or divided into several doses or continuously.

  When the pharmaceutical composition of the present invention is used for actual prevention or treatment, various dosage forms are used orally or parenterally depending on the usage, such as powders, fine granules, granules, tablets, Capsules, dry syrups, injections and the like can be mentioned. When oral administration is difficult depending on the patient's condition, the use of injections (continuous intravenous injection, etc.) is preferred.

  The pharmaceutical composition of the present invention is formulated by mixing with appropriate excipients, disintegrants, binders, lubricants and other pharmaceutical additives according to pharmacologically known methods according to the dosage form. can do.

For example, a tablet is a commercially available product, or an appropriate excipient, a disintegrant, a binder, a lubricant, etc. are added to the active ingredient, and tableted into a tablet according to a conventional method, and if necessary, A film-coated tablet, a sugar-coated tablet, an enteric-coated skin tablet, etc. can also be applied by appropriately coating. For example, the powder can be added to the active ingredient as necessary by adding an appropriate excipient, lubricant, etc., and mixed well to form a powder. For example, a capsule is prepared by adding an appropriate excipient, lubricant, etc. to an active ingredient and mixing well, or after granulating or finely granulating it according to a conventional method, filling it into an appropriate capsule and You can also Furthermore, in the case of such an orally administered preparation, an immediate release or sustained release preparation can be prepared depending on the prevention or treatment method.
For example, for injections, obtain commercially available products, or add appropriate diluents to the active ingredients, and if necessary, preservatives, isotonic agents, buffers, emulsifiers, dispersants, stabilization, dissolution Pharmaceutical additives such as adjuvants can be added to make an injection. Further, in the case of such injections, ampoule preparations, vial preparations, prefilled syringe preparations and the like can be used depending on the convenience of treatment.

  It has been found that the pharmaceutical composition of the present invention exhibits ALT elevation suppression, hepatic glutathione level increase, and remarkable suppression effect on hepatocyte damage. Therefore, the present invention can provide a pharmaceutical composition useful as a treatment or prevention of acetaminophen liver injury.

  The contents of the present invention will be described in more detail in the following examples, but the present invention is not limited to the contents.

  In the examples of the present specification, ozagrel hydrochloride monohydrate was used as ozagrel hydrochloride. In addition, the described quantity described the quantity converted as ozagrel hydrochloride.

Example 1
Ozagrel hydrochloride (administered 30 minutes later) suppresses ALT elevation in APAP liver injury (1) Preparation of APAP-induced liver injury model
C57BL / 6Njcl male mice (Specific pathogen free (SPF) grade, 8-10 weeks old, CLEA Japan, Inc.) were used in the experiment. APAP (550 mg / kg) was dissolved in phosphate buffered saline (PBS) by heating at about 60 ° C., and then intraperitoneally administered in a volume of 1 mL per 50 g of mouse body weight. The group not administered with APAP was defined as an untreated group.
(2) Measurement of alanine transaminase (ALT) As a blood biochemical marker of liver injury, the time course of serum ALT activity up to 24 hours after APAP administration and the effect of ozagrel hydrochloride on it were examined.
In the above model, 30 minutes after APAP administration, ozagrel hydrochloride (200 mg / kg) in saline (Saline) solution (Ozagrel administration group) or Saline (control group) at a drug volume of 0.6 mL per 50 g mouse body weight. It was administered intraperitoneally.
1, 2, 4, 8 and 24 hours after APAP administration, the mice were rapidly opened under ether inhalation anesthesia and blood was collected from the abdominal aorta, and serum ALT activity was measured by transaminase C II-Test Wako (Wako Pure Chemical Industries, Ltd.). Company). The absorbance of the reaction solution in this measurement was measured with a spectrophotometer (V-530, JASCO Corporation), and the ALT activity was calculated from a calibration curve obtained from a standard product.
(3) Results As shown in FIG. 1, a significant increase in ALT activity was observed after 8 and 24 hours in the control group compared to the untreated group. On the other hand, no increase in ALT activity was observed after any time in the ozagrel administration group. That is, it was confirmed that ozagrel hydrochloride has a remarkable inhibitory effect on ALT elevation in APAP liver injury.

Example 2
GSH increase effect of ozagrel hydrochloride (administered 30 minutes later) in APAP liver injury (1) Measurement of glutathione (GSH)
The time course of total GSH in liver tissue consumed for conjugation and detoxification of NAPQI, a toxic metabolite of APAP, and the effect of ozagrel hydrochloride on it were investigated.
To APAP-induced liver injury model mice prepared in the same manner as in Example 1, 30 minutes after APAP administration, ozagrel hydrochloride (200 mg / kg) physiological saline (Saline) solution (ozagrel administration group) or Saline (control group) The solution was intraperitoneally administered at a volume of 0.6 mL per 50 g of mouse body weight. At 0.5, 1, 2, 4, and 8 hours after administration of APAP, the mouse was rapidly laparotomized under ether inhalation anesthesia and the liver was removed. A part of the sample was used for GSH measurement, homogenized with Polytron PT 1600 (EKINEMATICA Inc.) in a 5% aqueous metaphosphoric acid solution, centrifuged, and the supernatant was used as a measurement sample. GSH was measured using BIOXYTECH GSH / GSSG-412 (OXIS International inc.). The absorbance of the reaction solution in this measurement was measured with a spectrophotometer (V-530, JASCO Corporation), the GSH concentration was determined, and the total GSH amount per 1 mg of wet weight of liver tissue was calculated.
(2) Results As shown in FIG. 2, in the control group, a significant decrease in GSH was observed from 30 minutes after APAP administration, and a significant decrease was observed up to 8 hours thereafter, compared with the untreated group. That is, the depletion of GSH, which is a feature of APAP liver injury, was confirmed. On the other hand, in the ozagrel administration group, GSH significantly decreased until 1 to 8 hours compared to the untreated group, but GSH after 2 to 8 hours showed a significantly high value compared to the control group. It was. That is, it was confirmed that ozagrel hydrochloride significantly increased GSH in APAP liver injury.

Example 3
Inhibitory effect of ozagrel hydrochloride (administered 30 minutes later) on APAP liver injury (1) Histopathological evaluation of the liver
The effect of ozagrel hydrochloride on hepatocellular injury was examined by histopathological evaluation of APAP-induced liver injury model mice.
To APAP-induced liver injury model mice prepared in the same manner as in Example 1, 30 minutes after APAP administration, ozagrel hydrochloride (200 mg / kg) physiological saline (Saline) solution (ozagrel administration group) or Saline (control group) The solution was intraperitoneally administered at a volume of 0.6 mL per 50 g of mouse body weight. At 8 and 24 hours after APAP administration, the mice were rapidly laparotomized under ether inhalation anesthesia and the liver was excised, and a part thereof was fixed with 10% neutral buffered formalin solution to prepare a pathological tissue specimen. The fixed tissue was embedded in paraffin by a conventional method, and about 3 μm book-cut specimens were prepared by a microtome, and then pathological analysis was performed by Hematoxylin-Eosin (HE) staining.
(2) Results In the control group, necrosis (arrow) of centrilobular hepatocytes characteristic of APAP liver injury was observed in all cases (FIG. 3). On the other hand, in the ozagrel-administered group, a finding that glycogen storage was decreased was observed, but hepatocyte necrosis as seen in the control group was not confirmed at all (FIG. 4).
The degree of hepatocellular injury was evaluated for each HE-stained pathological specimen (2 cases in each group) (Table 1). The symbol “-” in the table indicates no abnormality, “±” indicates a very slight change, “++” indicates a moderate change, and “++” indicates a severe change.
In APAP liver injury, hepatic tissue injury and necrosis include CYP2E1 in high concentrations in hepatocytes around the central vein of hepatic lobule, and low oxygen partial pressure and low glutathione concentration around the central vein of hepatic lobule Therefore, characteristically, NAPQI is expressed mainly around the central vein of the hepatic lobule where it tends to accumulate. Ozagrel hydrochloride was confirmed to remarkably suppress liver tissue injury characteristic of APAP liver injury.

Example 4
ALT elevation inhibitory effect of ozagrel hydrochloride (administered 30 minutes to 3 hours later) in APAP liver injury (1) Measurement of ALT The timing of administration on the therapeutic effect of ozagrel hydrochloride and N-acetylcysteine (NAC) on APAP liver injury The effects were compared.
APAP-induced hepatic injury model mice prepared in the same manner as in Example 1 were treated with a solution of osagrel hydrochloride (200 mg / kg) in Saline (ozagrel administration group), NAC (600 mg / kg) 0.5, 1, 2 and 3 hours after APAP administration. kg) of Saline solution (NAC administration group) or Saline (0.5 hours only, control group) was intraperitoneally administered at a drug volume of 0.6 mL per mouse body weight of 50 g. ALT activity was measured by the same method as in Example 1 24 hours after administration of APAP.
(2) Results As shown in FIG. 5, in both the ozagrel-administered group and the NAC-administered group, a significant ALT increase inhibitory effect was observed in the administration after 30 minutes to 2 hours compared to the control group. The ozagrel administration group showed a stronger inhibitory effect on ALT elevation than the NAC administration group. In addition, in the administration after 3 hours, the ALT elevation inhibitory effect was not obtained in any group.

Example 5
(1) Comparison of survival rate in APAP liver injury
APAP (330 mg / kg) was administered in the same manner as in Example 1 using Crlj: CD1 (ICR) male mice (SPF grade, 7-8 weeks old, Charles River Japan, Inc.) and induced APAP A liver injury model was prepared. 30 minutes after APAP administration, Saline solution of ozagrel hydrochloride (200 mg / kg) (ozagrel administration group), NAC (600 mg / kg) of Saline solution (NAC administration group) or Saline (control group), mouse weight 50 g The solution was intraperitoneally administered at a volume of 0.6 mL per solution. Observations were made up to 72 hours after APAP administration.
(2) Results As shown in FIG. 6, all cases died within 12 hours in the control group. In the NAC group, deaths were observed after 40 hours. On the other hand, in the ozagrel administration group, the survival rate after 72 hours was 100%.

Example 6
In order to investigate the production of TXA ₂ in hepatic tissue effect on thromboxane (TX) Production (1) TXB _{2, 2,} of 3-Dinor measuring liver tissue, TXB _2, 2 is stable metabolite of TXA _2, The concentration of 3-Dinor in liver tissue was measured.
To APAP-induced liver injury model mice prepared in the same manner as in Example 1, 30 minutes after administration of APAP, Saline solution (ozagrel administration group) or Saline (control group) of ozagrel hydrochloride (200 mg / kg) was added to a mouse weight of 50 g. The solution was intraperitoneally administered at a volume of 0.6 mL per solution.
Three hours after administration of APAP, the mouse was rapidly opened under ether inhalation anesthesia, and the liver was removed. A portion of the liver and TXB _2, 2, for the determination of 3-Dinor, 0.1M PBS for 5 volumes of extraction buffer (15% (v / v) methanol and 1% (w / v) indomethacin (Sigma) Solution, pH 7.5), and homogenized with Polytron PT 1600 (EKINEMATICA Inc.) and then centrifuged, and the supernatant was used as a measurement sample. The TXB ₂ , 2, 3-Dinor, EIA Kit (Cayman chemical) was used to measure the TXB ₂ , 2, 3-Dinor concentration in the measurement sample. The absorbance of the reaction solution was measured using a plate reader (V-530, JASCO Corporation). From the calibration curve obtained from the standard product, the amount of TXB ₂ , 2, 3-Dinor per 1 mg wet weight of liver tissue was measured. Calculated.
(2) Results As a result, TXB ₂ , 2, 3-Dinor concentrations were 32.6 ± 0.789, 27.5 ± 14.3 and 29.1 ± 6.42 (pg / mg tissue weight) in the untreated group, the control group and the ozagrel administration group, respectively. There was no significant difference in all groups.

Example 7
Effect on human and mouse CYP2E1 activity (1) Measurement of CYP2E1 activity Using Vivid (registered trademark) CYP2E1 Blue Screening Kit (Invitrogen) composed of microsomes prepared from insect cells expressing human CYP2E1 and rabbit NADPH-P450 reductase The presence or absence of human CYP2E1 inhibitory activity of ozagrel hydrochloride was examined.
In addition, using microsomes collected from C57BL / 6Njcl male mouse liver, CYP2E1 has high specificity according to the method described in the method of John W. Allis et al. (Analytical Biochemistry 219, p. 49-52 (1994)). By determining the amount of p-nitrocatechol (PNC) produced from the substrate p-nitrophenol (PNP), the effect of ozagrel hydrochloride addition (0.001-1 mg / mL) on mouse CYP2E1 activity was examined. The PNP and PNC concentrations were measured using the Ultratrospec 3300 (GE Healthcare UK Ltd.) based on a standard product purchased from Sigma-Aldrich, and calculated as PNC production (nmol / mg / mg protein). did.
(2) Results In both human and mouse, 0.001-1 mg / mL ozagrel hydrochloride showed no significant inhibitory action on CYP2E1.

Example 8
Effect of Ozagrel hydrochloride (pre-administration) on GSH increase in APAP liver injury (1) Measurement of GSH In the APAP-induced liver injury model mouse described in Example 1, ozagrel hydrochloride (200) was administered 30 minutes before or 30 minutes after APAP administration. mg / kg) Saline solution (pre-administration group and post-ozagrel administration group, respectively) or Saline (pre-administration control group and post-administration control group, respectively) was administered intraperitoneally at a drug volume of 0.6 mL per 50 g of mouse body weight. .
One hour after APAP administration, the mouse was rapidly opened under ether inhalation anesthesia and the liver was removed, and the total GSH was measured in the same manner as described in Example 2.
(2) Results As shown in FIG. 7, the APAP administration showed a significant decrease in GSH (P <0.01) in all administration groups compared to the untreated group, but in the pre-ozagrel administration group, Compared to both the pre-administration control group and the post-ozagrel administration group, the amount of GSH was significantly higher at P <0.01 and P <0.05, respectively.

Example 9
NAPQI-induced in vitro hepatocyte injury inhibitory effect (1) Cells used
Rat liver epithelial cells RLC-16 (RIKEN BioResource Center) is added to Minimum essential medium (MEM) medium (Sigma), then 10% fetal bovine serum (FBS) (Biowest), 100 IU / mL penicillin (Invitrogen), 100 μg / mL streptomycin (Invitrogen) and 0.1 mM non-Essential Amino Acids Solution (Invitrogen) were added and cultured, and subcultured once every 3 to 4 days.
(2) Cell viability measurement
RLC-16 cells were seeded on a 96-well plate at 1 × 10 ⁴ cells / well, cultured for 24 hours to allow the cells to engraft, and then the culture solution was removed. A dimethylsulfoxide solution of 0.25 mM NAPQI (Sigma-Aldrich) was added to each medium (1, 10, 100 μM) of ozagrel hydrochloride PBS solution, 1 mM NAC PBS solution or PBS (control group). . After 24 hours of culture, the WST-1 assay was performed using a cell counting kit (DOJINDO). After 4 hours, the absorbance at 450 nm was measured with a microplate reader (Immuno mini NJ-2300, Nalge Nunc International KK). Based on the above, the number of viable cells was determined, and the survival rate (%) of each group was determined with the value of NAPQI-free cells as 100.
(3) Results As shown in FIG. 8, a significant decrease in cell viability was observed by exposure to NAPQI (0.25 mM). In contrast, the addition of ozagrel hydrochloride (100 μM) or NAC (1 mM) showed a significantly higher cell viability than the control group.

  In addition, the statistical process of each experimental result in this specification was performed using GraphPad Prism ver.3.00 (GraphPad Software). The significant difference test of each group compared between groups by the multiple comparison test with respect to the experimental result. In other words, the Bartlett test is used to test the homogeneity of the variance. If the variance is uniform, one-way analysis of variance (one-way ANOVA) is performed. If the variance is not uniform, the Kruskal-Walis test is performed. Only when a significant difference was observed in these, a significant difference test between groups was performed by Tukey's Multiple Comparison Test. Survival curves were analyzed by the Kaplan-Meier method, and a significant difference test was performed between groups by log-rank test. Based on the above test, it was determined to be significant when the risk rate was less than 5%.

  As described above, ozagrel hydrochloride exhibited extremely excellent therapeutic effects on APAP liver injury such as suppression of ALT elevation, increase in liver GSH (inhibition of decrease), and suppression of liver tissue injury. In addition, it is effective at administration timing before APAP administration, and has a wider tolerance in administration timing, such as better ALT elevation suppression effect at various administration timing compared to NAC which is an existing drug It was suggested that

The pharmaceutical composition of the present invention is useful for the treatment or prevention of APAP liver injury.

FIG. 1 is a graph showing the time course of ALT activity in APAP-induced liver injury model mice. The vertical axis represents serum ALT activity (IU / L), and the horizontal axis represents elapsed time (hours) after APAP administration. From the left of the figure, 0 (no treatment group), 1, 2, 4, 8, and 24 hours after the mean value and standard error of each of the control group (black) and the ozagrel-treated group (white) (4-12 for each group) Example). In the figure, “**” indicates that there is a significant difference at P <0.01 compared to the untreated group.

FIG. 2 is a graph showing changes over time in the total amount of GSH in liver tissue in APAP-induced liver injury model mice. The vertical axis represents the total amount of GSH in the liver (nmol / mg tissue weight), and the horizontal axis represents the elapsed time (hours) after APAP administration. From the left of the figure, mean values and standard errors after 0.5, 1, 2, 4, and 8 hours of 0 (no treatment group), control group (black), and ozagrel administration group (white) (each group 4-6) Example). In the figure, “*” and “**” are significant differences at P <0.05 and P <0.01 compared to the control group, respectively, “†” are significant differences at P <0.01 compared to the untreated group Represent that there is.

FIG. 3 shows an example of the result of HE staining of liver tissue 8 hours after APAP administration (control group). The arrow in the figure indicates a portion where centrilobular hepatocyte necrosis is observed.

FIG. 4 shows an example of the result of HE staining of liver tissue 8 hours after APAP administration (Ozagrel administration group).

FIG. 5 is a graph showing the influence of drug administration timing on ALT activity 24 hours after APAP administration. The vertical axis represents ALT activity (IU / L), and the horizontal axis represents the elapsed time (hours) after administration of APAP administered with ozagrel hydrochloride or NAC. From the left of the figure, mean values and standard errors in the non-treated group (horizontal stripes), the control group (black), the ozagrel-treated group (white), and the NAC-treated group (grey) after 0.5, 1, 2, and 3 hours (3 to 6 cases in each group) are shown.

FIG. 6 is a graph showing the transition of survival rate in APAP-induced liver injury model mice. The vertical axis shows the survival rate (%) (5 to 11 cases in each group), and the horizontal axis shows the elapsed time (hours) after APAP administration. In the graph, the x mark indicates the ozagrel administration group, the ● mark indicates the NAC administration group, and the ■ mark indicates the control group. In the figure, “***” indicates that there is a significant difference at P <0.001 compared to the control group.

FIG. 7 is a graph showing the total amount of GSH in liver tissue 1 hour after administration of APAP in APAP-induced liver injury model mice. The vertical axis represents the total amount of GSH in the liver (nmol / mg tissue weight), and the horizontal axis represents the administration group. From the left of the figure, the mean value and standard error of the untreated group (horizontal stripes), the post-administration control group (black), the post-ozagrel administration group (white), the pre-administration control group (hatched line), and the pre-ozagrel administration group (gray) 3-4 cases in each group) are shown.

FIG. 8 is a graph showing cell viability in in vitro NAPQI-induced hepatocyte injury. The vertical axis represents the cell viability (%), and the horizontal axis represents the group. From the left of the figure, mean values and standard deviations (4 to 9 cases for each group) of the control group (black), ozagrel hydrochloride 1, 10 and 100 μM (shaded), and the NAC administration group (grey) are shown. In the figure, “***” indicates that there is a significant difference at P <0.001 compared to the control group.

Claims (3)

A pharmaceutical composition for treating or preventing acetaminophen liver injury comprising ozagrel or a pharmacologically acceptable salt thereof as an active ingredient. The pharmaceutical composition according to claim 1, comprising ozagrel hydrochloride as an active ingredient. The pharmaceutical composition according to claim 1 or 2, wherein the dosage form is an injection.