WO2023210716A1 - Liver fibrosis therapeutic regimen for cbp/catenin inhibitor - Google Patents

Abstract

[Problem] To provide a pharmaceutical composition useful for the prevention or treatment of liver disease, in particular, liver fibrosis or liver cirrhosis. [Solution] Provided is a pharmaceutical composition that is for the prevention and/or treatment of liver disease and that contains 4-({(6S,9S,9aS)-1-(benzylcarbamoyl)-2,9-dimethyl-4,7-dioxo-8-[(quinoline-8-yl)methyl]octahydro-2H-pyrazino[2,1-c][1,2,4]triazine-6-yl}methyl)phenyl dihydrogen phosphate or a pharmaceutically acceptable salt thereof. The pharmaceutical composition is characterized in that 100 to 400 mg/m² of the 4-({(6S,9S,9aS)-1-(benzylcarbamoyl)-2,9-dimethyl-4,7-dioxo-8-[(quinoline-8-yl)methyl]octahydro-2H-pyrazino[2,1-c][1,2,4]triazine-6-yl}methyl)phenyl dihydrogen phosphate or a pharmaceutically acceptable salt thereof is administered intravenously to a human once or twice a week for 2 to 5 hours each time, and the administration is continued for 2 to 6 months.

Description

CBP/catenin inhibitor liver fibrosis treatment regimen

The present invention relates to a pharmaceutical composition for the prevention and treatment of liver diseases, particularly liver diseases such as liver fibrosis and cirrhosis.

Chronic organ damage causes fibrosis of affected tissues such as the liver, heart, kidneys, and lungs. In the liver, fibrosis is the first step toward the development of cirrhosis, which causes liver dysfunction, esophageal varices, and hepatocellular carcinoma, and is responsible for more than 1 million deaths annually worldwide. Causes of liver cirrhosis include hepatic steatosis due to excessive drinking and overeating, and hepatitis due to viral infection, and each is treated with alcohol abstinence, diet therapy, and antiviral drugs. In addition, chronic cholestasis and subsequent liver damage and cirrhosis can be caused by certain drugs, primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC), familial intrahepatic cholangitis, Alagille syndrome, It may be caused by certain diseases such as gestational intrahepatic cholangitis. PBC and PSC are chronic progressive biliary liver diseases caused by autoimmune diseases of unknown cause, and currently there is no effective treatment other than liver transplantation. The prevalence of PBC is 140 cases/million people, and the prevalence of PSC is 0-317 cases/million people. Bile acidic liver damage is the result of destruction of hepatic parenchymal cells due to retention of hydrophobic bile acids (BA). Initial hepatocyte damage induces a subsequent inflammatory response, which exacerbates hepatocyte and intralobar bile duct damage, leading to fibrosis and ultimately liver failure due to cirrhosis. Although ursodeoxycholic acid and bezafibrate are recognized as therapeutic drugs for PBC, antifibrotic drugs for liver cirrhosis associated with PBC and PSC have not yet been put into practical use (Non-patent Documents 1 and 2). BA receptors such as FXR, its transporter system, and fibroblast growth factor 19 are attracting attention as therapeutic targets for cholestatic liver disease and liver fibrosis (Non-patent Documents 3 and 4). However, there are currently no therapeutic agents that reduce the amount of BA itself.

Studies have previously reported that abnormalities in Wnt/β-catenin signaling are involved in fibrosis (Non-Patent Documents 5 to 7). It has been reported that β-catenin deletion increased cholestatic liver damage and fibrosis in Mdr2-KO mice, but suppressed cholestatic liver damage and fibrosis in bile duct ligation mice ( Non-Patent Documents 8, 9).

4-({(6S,9S,9aS)-1-(benzylcarbamoyl)-2,9-dimethyl-4,7-dioxo -8-[(quinolin-8-yl)methyl]octahydro-2H-pyrazino[2,1-c][1,2,4]triazin-6-yl}methyl)phenyl dihydrogen phosphate (foscenvivint) is known. It has been reported that this compound is useful as a prophylactic/therapeutic agent for liver fibrosis (Patent Document 1). Also. The compound has the effect of improving liver function, particularly liver glucose metabolism function, electron transport system function, and liver synthesis ability, and is effective as a liver function improving agent (Patent Document 2).

Patent No. 6303112 WO2020/122022

The present invention aims to provide a pharmaceutical composition useful for preventing or treating liver diseases.

The following formula:

The compound represented by 4-({(6S,9S,9aS)-1-(benzylcarbamoyl)-2,9-dimethyl-4,7-dioxo-8-[(quinolin-8-yl)methyl]octahydro -2H-pyrazino[2,1-c][1,2,4]triazin-6-yl}methyl)phenyl dihydrogen phosphate (foscenvivint, (also known as foscenvivint, PRI-724, OP-724)) hereinafter referred to as "the present disclosure" (also referred to as "compound") is a small molecule therapeutic agent associated with the inhibition of intracellular protein-protein interactions in the Wnt pathway, which is essential for the regulation of cancer stem cells (CSCs).
Following an investigator-initiated domestic trial of foscenvivint in Japanese patients with liver cirrhosis caused by hepatitis B virus or hepatitis C virus, the present inventor conducted an investigator-initiated domestic trial of foscenvivint in patients with advanced PBC. We have started an investigator-initiated domestic clinical trial targeting patients with liver cirrhosis caused by co-infection with hemophilia-associated human immunodeficiency virus and hepatitis C virus (hereinafter referred to as hemophilia HIV/HCV co-infection), and in the process, we have completed this clinical trial. The inventors have diligently studied and optimized the dosage and evaluation index of the disclosed compound, and have completed the present invention.
The present disclosure includes the following features.

[1] 100 to 400 mg/m ² of 4-({(6S,9S,9aS)-1-(benzylcarbamoyl)-2,9-dimethyl-4,7-dioxo-8-[(quinolin-8-yl) ) methyl]octahydro-2H-pyrazino[2,1-c][1,2,4]triazin-6-yl}methyl)phenyl dihydrogen phosphate, or a pharmaceutically acceptable salt thereof, is administered once a week to humans. 4-({(6S,9S,9aS )-1-(benzylcarbamoyl)-2,9-dimethyl-4,7-dioxo-8-[(quinolin-8-yl)methyl]octahydro-2H-pyrazino[2,1-c][1,2, 4] A pharmaceutical composition for the prevention and/or treatment of liver diseases containing triazin-6-yl}methyl)phenyl dihydrogen phosphate or a pharmaceutically acceptable salt thereof.
[2] 280 mg/m ² of 4-({(6S,9S,9aS)-1-(benzylcarbamoyl)-2,9-dimethyl-4,7-dioxo-8-[(quinolin-8-yl)methyl ] Octahydro-2H-pyrazino[2,1-c][1,2,4]triazin-6-yl}methyl)phenyl dihydrogen phosphate, or a pharmaceutically acceptable salt thereof, is administered twice weekly to humans. The pharmaceutical composition according to [1] above, which is administered intravenously over 4 hours each time, and is used so that the administration is continued for 12 weeks.
[3] The pharmaceutical composition according to [1] or [2] above, wherein the liver disease is accompanied by liver fibrosis or cirrhosis.
[4] The pharmaceutical composition according to [3] above, wherein the liver fibrosis or cirrhosis is a disease caused by hepatitis B or C, or primary biliary cholangitis (PBC).
[5] The pharmaceutical composition according to [3], wherein the liver fibrosis or cirrhosis is a disease caused by hemophilia, HIV/HCV co-infection, or non-alcoholic steatohepatitis.
[6] The pharmaceutical composition according to [3], wherein the liver disease is classified as A, B or C according to the Child-Pugh score.
[7] The pharmaceutical composition according to any one of [1] to [6] above, which is for maintaining or improving liver tissue hardness.
[8] The pharmaceutical composition according to [7] above, wherein maintenance or improvement is evaluated during drug administration or at the end of drug administration compared to before the start of drug administration.
[9] The pharmaceutical composition according to [7] above, wherein maintenance or improvement is evaluated 6 to 12 months after the end of drug administration, compared to the time point at which drug administration ended.
[10] The pharmaceutical composition according to [7] above, wherein the liver tissue hardness is indicated by a Fibroscan measurement value.
[11] The pharmaceutical composition according to [7], wherein the liver tissue hardness is indicated by an MR elastography measurement value.
[12] The pharmaceutical composition according to any one of [1] to [6] above, which is for maintaining or improving a liver reserve index.
[13] The pharmaceutical composition according to [12] above, wherein maintenance or improvement is evaluated during drug administration or at the end of drug administration compared to before the start of drug administration.
[14] The pharmaceutical composition according to [12] above, wherein maintenance or improvement is evaluated 6 to 12 months after the end of drug administration compared to the time point at which drug administration ended.
[15] The pharmaceutical composition according to [12] above, wherein the hepatic reserve index is represented by serum albumin concentration, serum bilirubin concentration, prothrombin activity value, or ALBI score.
[16] The pharmaceutical composition according to [12], wherein the hepatic reserve index is indicated by serum bilirubin concentration, prothrombin activity value, or ALBI score.
[17] The pharmaceutical composition according to [12] above, wherein the hepatic reserve index is indicated by a change in Child Pugh classification (A, B, C) or a change in score.

[18] The pharmaceutical composition according to any one of [1] to [6] above, which is for maintaining or improving a cholestasis index.
[19] The pharmaceutical composition according to [18] above, wherein maintenance or improvement is evaluated 6 to 12 months after the end of drug administration compared to the time point at which drug administration ended.
[20] The pharmaceutical composition according to [18] above, wherein the cholestasis index is indicated by serum total bile acid concentration.
[21] 4-({(6S,9S,9aS)-1-(benzylcarbamoyl)-2,9-dimethyl-4,7-dioxo-8-[(quinolin-8-yl)methyl]octahydro-2H- A method for assisting in evaluating the therapeutic effect of pyrazino[2,1-c][1,2,4]triazin-6-yl}methyl)phenyl dihydrogen phosphate, or a pharmaceutically acceptable salt thereof, on liver diseases, the method comprising: , liver tissue hardness, liver reserve index, and cholestasis index.
[22] The method according to [21] above, wherein the measurement is performed 6 to 12 months after the end of drug administration, in comparison with the time point at which drug administration ended.
[23] The method according to [21] or [22] above, wherein the liver tissue hardness is indicated by a fibroscan measurement value.
[24] The method according to [21] or [22] above, wherein the hepatic reserve index is indicated by serum albumin concentration.
[25] The method according to [21] or [22] above, wherein the cholestasis index is indicated by serum total bile acid concentration.
[26] 100 to 400 mg/m ² of 4-({(6S,9S,9aS)-1-(benzylcarbamoyl)-2,9-dimethyl-4,7-dioxo-8-[(quinolin-8-yl) ) methyl]octahydro-2H-pyrazino[2,1-c][1,2,4]triazin-6-yl}methyl)phenyl dihydrogen phosphate, or a pharmaceutically acceptable salt thereof, is administered once a week to humans. A method for preventing and/or treating liver diseases, which is administered intravenously once or twice over 2 to 5 hours each time, and the administration is continued for 2 to 6 months.
[27] 280 mg/m ² of 4-({(6S,9S,9aS)-1-(benzylcarbamoyl)-2,9-dimethyl-4,7-dioxo-8-[(quinolin-8-yl)methyl ] Octahydro-2H-pyrazino[2,1-c][1,2,4]triazin-6-yl}methyl)phenyl dihydrogen phosphate, or a pharmaceutically acceptable salt thereof, is administered twice weekly to humans. The method for preventing and/or treating liver diseases according to [26] above, wherein the method is administered intravenously over 4 hours each time, and the administration is continued for 12 weeks.
[28] 4-({(6S,9S,9aS)-1-(benzylcarbamoyl)-2,9-dimethyl-4,7-dioxo- 8-[(quinolin-8-yl)methyl]octahydro-2H-pyrazino[2,1-c][1,2,4]triazin-6-yl}methyl)phenyl dihydrogen phosphate, or a pharmaceutically acceptable thereof The use of salt,
100 to 400 mg/m ² of 4-({(6S,9S,9aS)-1-(benzylcarbamoyl)-2,9-dimethyl-4,7-dioxo-8-[(quinolin-8-yl)methyl] Octahydro-2H-pyrazino[2,1-c][1,2,4]triazin-6-yl}methyl)phenyl dihydrogen phosphate, or a pharmaceutically acceptable salt thereof, is administered to humans once or twice a week. Use characterized in that it is administered intravenously over a period of 2 to 5 hours each time, and the administration is continued for 2 to 6 months.
[29] 280 mg/m ² of 4-({(6S,9S,9aS)-1-(benzylcarbamoyl)-2,9-dimethyl-4,7-dioxo-8-[(quinolin-8-yl)methyl ] Octahydro-2H-pyrazino[2,1-c][1,2,4]triazin-6-yl}methyl)phenyl dihydrogen phosphate, or a pharmaceutically acceptable salt thereof, is administered twice weekly to humans. The use according to [28] above, wherein the use is administered intravenously over 4 hours each time, and the administration is continued for 12 weeks.

4-({(6S,9S,9aS)-1-(benzylcarbamoyl)-2,9-dimethyl-4,7-dioxo-8-[(quinolin-8-yl)methyl]octahydro-2H-pyrazino[2 ,1-c][1,2,4]triazin-6-yl}methyl)phenyl dihydrogen phosphate or a pharmaceutically acceptable salt thereof is administered at a dosage of 100 to 400 mg/m ² (e.g. , 280 mg/m ² ) is administered intravenously once or twice a week (e.g., twice a week) over 2 to 5 hours (e.g., 4 hours) each time, and the administration is continued for 2 to 6 months ( For example, when continued for 12 weeks), it shows an excellent therapeutic effect on liver cirrhosis (liver fibrosis).

FIG. 1 is a diagram showing that the compound of the present disclosure has the effect of suppressing liver fibrosis in Mdr2-KO mice. Mdr2-KO mice (MDR2KO, male, 8-10 weeks old) and FVB background control mice were administered compounds of the present disclosure (20 mg/kg, three times a week) for 10 weeks. Mice that were not administered were used as controls. A shows the scheme of the treatment protocol. B shows the results of Sirius red staining (scale bar, 500 μm, 1 mm, and 500 μm from the upper figure, respectively). FIG. 2 is a diagram showing that the compound of the present disclosure has the effect of suppressing liver fibrosis in BDL mice. Wild type C57BL/6 mice (male, 8-10 weeks old) underwent BDL or sham surgery and were treated with compounds of the present disclosure (20 mg/kg, three times a week) or PBS. Animals were euthanized 14 days after surgery. A shows the scheme of the treatment protocol. B shows the results of hematoxylin and eosin staining, Sirius red staining, and CK19 immunohistochemical staining (scale bars, 100 μm, 500 μm, and 250 μm, respectively, from the upper figure). FIG. 3 is a diagram showing that the compound of the present disclosure has the effect of suppressing liver fibrosis in DDC mice. Wild type C57BL/6 mice (male, 8 weeks old) were fed a 0.1% DDC diet for 18 days and administered a compound of the present disclosure (20 mg/kg, three times a week) or PBS. The results of hematoxylin and eosin staining and Sirius red staining (scale bar, 250 μm and 500 μm from the above figure, respectively) are shown. FIG. 4 shows that compounds of the present disclosure reduce bile acid (BA) synthesis in Mdr2-KO mice. Mdr2-KO mice (8-10 weeks old) and FVB background control mice were administered compounds of the present disclosure (20 mg/kg, three times a week) for 10 weeks. Mice that were not administered were used as controls. Liver and serum TBA, CA, and CDCA levels (n=10 for each group) are shown. Results shown represent the mean±SD of at least three independent experiments. **p<0.01; ***p<0.0001, Student's t-test. FIG. 5 shows that compounds of the present disclosure reduce bile acid (BA) synthesis in BDL mice. Wild type C57BL/6 (male, 8-10 weeks old) mice underwent BDL or sham surgery and were treated with compounds of the present disclosure (20 mg/kg, 3 times a week) or PBS. Liver TBA, CA, and CDCA levels (n=6-9 per group) are shown. Results shown represent the mean±SD of at least three independent experiments. *p<0.05; **p<0.01; ***p<0.0001, one-way ANOVA. FIG. 6 shows that compounds of the present disclosure reduce bile acid (BA) synthesis in DDC mice. Wild type C57BL/6 (male, 8 weeks old) mice were fed a 0.1% DDC diet for 18 days and administered a compound of the present disclosure (20 mg/kg, three times a week) or PBS. Liver and serum TBA, CA, and CDCA levels (n=6 for each group) are shown. Results shown represent the mean±SD of at least three independent experiments. **p<0.01, unpaired Student's t-test. FIG. 7 is a diagram showing that the compound of the present disclosure improves liver dysfunction in Mdr2-KO mice. Male Mdr2-KO mice and FVB background control mice were administered compounds of the present disclosure (20 mg/kg, three times a week) for 10 weeks. Mice that were not administered were used as controls. Serum ALT, ALP, and T. Bil levels (n=5 to 7 for each group) are shown. Results shown represent the mean±SD of at least three independent experiments. **p<0.01; ***p<0.005; ***p<0.0001, one-way ANOVA. FIG. 8 is a diagram showing that the compound of the present disclosure improves liver dysfunction in BDL mice. Wild type C57BL/6 (male, 8-10 weeks old) mice underwent BDL or sham surgery and were treated with compounds of the present disclosure (20 mg/kg, 3 times a week) or PBS. Serum ALT, ALP, and T. Bil level (n=6 to 9 for each group) is shown. Results shown are representative of at least three independent experiments. Data represent mean ± SD. **p<0.01; ***p<0.005, one-way ANOVA. FIG. 9 is a diagram showing that the compound of the present disclosure improves liver dysfunction in DDC mice. Wild type C57BL/6 (male, 8 weeks old) mice were fed a 0.1% DDC diet for 18 days and administered a compound of the present disclosure (20 mg/kg, three times a week) or PBS. Serum ALT, ALP, and T. Bil level (n=6 for each group) is shown. Results shown represent the mean±SD of at least three independent experiments. **p<0.01, unpaired Student's t-test. FIG. 10 is a diagram showing that the compounds of the present disclosure improve liver fibrosis in Mdr2-KO mice. Male Mdr2-KO mice and FVB background control mice were administered compounds of the present disclosure (20 mg/kg, three times a week) for 10 weeks. Mice that were not administered were used as controls. Shows the mRNA expression levels of certain genes (Col1a1, Col1a2) in the liver determined by RT-qPCR. Results shown represent the mean±SD of at least three independent experiments. *p<0.05; **p<0.01, one-way ANOVA. FIG. 11 is a diagram showing that the compound of the present disclosure improves liver fibrosis in DDC mice. Wild type C57BL/6 (male, 8 weeks old) mice were fed a 0.1% DDC diet for 18 days and administered a compound of the present disclosure (20 mg/kg, three times a week) or PBS. Shows the mRNA expression levels of certain genes (Col1a1, Col1a2, Col3a1) in the liver determined by RT-qPCR. Results shown represent the mean±SD of at least three independent experiments. **p<0.01, unpaired Student's t-test. FIG. 12 is based on the results of a Phase I clinical trial of compounds of the present disclosure in patients with advanced PBC (n=5). Figure 2 shows changes in serum TBA concentration before (baseline), during (after 4 weeks, after 8 weeks) and after 12 weeks (end of administration) of a compound of the present disclosure. Data represent mean ± SD. paired student's t-test. FIG. 13 is based on the results of a clinical Phase I/IIa study of compounds of the present disclosure in patients with liver cirrhosis. Figure 2 shows changes in liver tissue stiffness before administration of a compound of the present disclosure (baseline) and after 12 weeks (end of administration). paired t-test. Fibroscan was used to measure liver tissue hardness. FIG. 14 is based on the results of a clinical Phase IIa study of compounds of the present disclosure in patients with liver cirrhosis. Figure 2 shows changes in serum albumin concentration before (baseline), during (3 weeks, 6 weeks, 9 weeks after) and 12 weeks (end of administration) administration of a compound of the present disclosure (A). Changes in serum albumin concentration before compound administration (baseline) and 9 months after the start of medication are shown (B). FIG. 15 is based on the results of a clinical trial conducted on patients with liver cirrhosis using the dosing schedule of Protocol A. Figure 3 shows the change in FIB-4 index before administration of a compound of the present disclosure (baseline) and after 12 weeks (end of administration). *p<0.05 The figure on the left shows each patient (KOM-6, 7, 9, 10, 12, 13, 15, 17, 22, 28, 26, 30, 32) at the start of medication (each bar graph on the left) and at the end of medication (12 Fibroscan measurements are shown after 1 week) (center of each bar graph) and 9 months after the start of medication (right side of each bar graph). The figure on the right shows changes in liver tissue hardness at the start of medication and 9 months after the start of medication. **p<0.01; paired t-test. FIG. 17-1 is a diagram showing the effect of the compound of the present disclosure on improving liver function. Figure 17-2 shows a case with Child-Pugh (CP) classification B. FIG. 18-1 is a diagram showing the effect of the compound of the present disclosure on improving liver function. Figure 18-2 shows a case with Child-Pugh (CP) classification B. FIG. 19-1 is a diagram showing the effect of the compound of the present disclosure on improving liver function. Figure 19-2 shows a case with Child-Pugh (CP) classification B. Figure 20 is based on the results of a Phase I clinical trial of compounds of the present disclosure in hemophilic HIV/HCV coinfected cirrhotic patients. FIG. 2 is a diagram showing the effect of improving liver fibrosis. FIG. 21 is a diagram showing the effect of the compound of the present disclosure on improving liver fibrosis.

In some embodiments, the present disclosure provides 4-({(6S,9S,9aS)-1-(benzylcarbamoyl)-2,9-dimethyl-4,7-dioxo-8-[(quinoline-8- yl)methyl]octahydro-2H-pyrazino[2,1-c][1,2,4]triazin-6-yl}methyl)phenyl dihydrogen phosphate (foscenvivint), a compound of the present disclosure or PRI- 724 or OP-724) or a pharmaceutically acceptable salt thereof as an active ingredient, for the prevention and/or treatment of liver diseases (hereinafter also referred to as "the pharmaceutical composition of the present disclosure") The pharmaceutical composition of the present disclosure provides foscenvivint at a dose of 100 to 400 mg/m ² (e.g., 280 mg/m ² ) once or twice a week (e.g. , twice a week), administered intravenously over 2 to 5 hours (e.g., 4 hours) each time, and the administration is continued for 2 to 6 months (e.g., 12 weeks) to improve liver cirrhosis ( Shows therapeutic effect on liver fibrosis.
This will be explained in detail below.

In this disclosure, "liver disease" refers to any disease in the liver. In some embodiments, the liver disease targeted by the present invention may be any liver disease, but may be a disease accompanied by liver fibrosis or even more advanced symptoms of liver cirrhosis in some cases. Liver fibrosis and cirrhosis occur during the repair process of liver cells that have become necrotic due to various liver disorders such as viral hepatitis, alcoholic liver injury, autoimmune liver injury, and metabolic liver injury. This is a state in which the production of fibrous tissues called extracellular matrix is promoted, and these fibrous tissues gradually accumulate as inflammation progresses. Diseases that cause liver fibrosis and cirrhosis are not particularly limited, but include viral hepatitis (e.g., hepatitis B, hepatitis C), alcoholic liver damage, autoimmune liver damage (e.g., primary biliary cholangitis), Liver disorders include drug-induced liver injury, metabolic liver injury (eg, fatty liver, non-alcoholic steatohepatitis, hemochromatosis). In some embodiments, liver diseases to be prevented or treated with the pharmaceutical composition of the present disclosure include those accompanied by liver fibrosis or cirrhosis. In some embodiments, hepatitis B or C with primary biliary cholangitis, hemophilia, HIV/HCV co-infection, or liver fibrosis or cirrhosis due to non-alcoholic steatohepatitis. Can be mentioned.

In the present disclosure, prevention or treatment of liver disease means improvement and maintenance of liver function in some embodiments.

In the present disclosure, "liver function" means all functions possessed by the liver, and is not particularly limited. In some embodiments, the functions of the liver include blood storage (e.g., regulation of circulating volume); processing of hemoglobin (e.g., processing and excretion of hemoglobin); production of bile and bile pigments and enterohepatic circulation; Functions in the blood and circulation such as synthesis of phase proteins, albumin, blood clotting factors, steroid binding proteins, other hormone binding proteins, etc. [liver synthesis capacity]; nutrients and vitamins (glucose and/or other sugars, amino acids, lipids) and/or metabolic functions of nutrients, such as the metabolism of fatty acids, cholesterol, lipoproteins, fat-soluble vitamins, and water-soluble vitamins; various substances (toxins, steroids such as estrogen and androsterone, and other Detoxification or decomposition functions such as inactivation of hormones, etc.); and immune functions. Liver mitochondria also contribute to energy production in the liver through their electron transport chain [electron transport chain function] (Sherlock's Diseases of the Liver & Biliary System, 13th ed. S.Dooley, A.S.F.Lok, G.Garcia-Tsao Other authors, published June 2018, WILEY-BLACKWELL, Inc.).

In the present disclosure, "improvement/maintenance" of liver function refers to improving each of the liver functions described above and preventing or suppressing (maintaining) decline in liver function due to liver dysfunction due to aging and disease. The term also includes therapeutic effects on liver fibrosis and cirrhosis, which cause liver dysfunction. The effect of "improving/maintaining" liver function can also be translated into a therapeutic effect on liver diseases. In some embodiments, "improvement/maintenance" of liver function includes, but is not particularly limited to, improvement/maintenance of liver reserve capacity (hepatic synthetic ability), improvement/maintenance of bile production and enterohepatic circulation. .

In some embodiments, improvement and maintenance of liver reserve capacity (hepatic synthetic capacity) is evaluated by a method that measures the amount and function of proteins synthesized in the liver. In some embodiments, it can be assessed by measuring serum albumin concentration as an indicator of hepatic reserve. In some embodiments, serum albumin concentration can be measured by spectrophotometry (eg, Lowry method), electrophoresis-dye densitometry, high performance liquid chromatography-ultraviolet absorption detection, and the like. In some embodiments, it can also be evaluated by measuring the amount of serum alanine aminotransferase (ALT), alkaline phosphatase (ALP), cholinesterase, or cholesterol. In some embodiments, other liver-synthesized proteins include prothrombin, and methods of assessing its function include measuring prothrombin time. When liver function is impaired, bile secretion decreases and absorption of fat-soluble vitamin K is inhibited. Among the coagulation factors, II, VII, IX and X are vitamin K dependent and decrease faster than other factors synthesized in the liver. Among these factors, factor VII has the shortest half-life, so when the liver synthesis ability decreases, the prothrombin time in which factor VII is involved is acutely prolonged. Furthermore, although factor VII is an extrinsic coagulation factor, over time, the activated partial thromboplastin time involving endogenous coagulation factors also increases.

In some embodiments, improvement and maintenance of bile and bile pigment production and enterohepatic circulation can be evaluated by measuring total bile acid concentration in serum using cholestasis as an indicator. Bile is produced by liver cells and excreted into the duodenum through the biliary system, but a condition in which the flow of bile is obstructed somewhere along this route is called cholestasis. Bile components remain in the liver and bile ducts and even leak into the blood. Bilirubin is a yellow pigment produced when hemoglobin in old red blood cells breaks down. Bilirubin is carried through the bloodstream to the liver, where it is processed and excreted into the bile. Bilirubin before it is processed by the liver is called "indirect bilirubin", and bilirubin that is processed and enters the bile is called "direct bilirubin", and the two together are called "total bilirubin".

In some embodiments, the Child-Pugh score can be used to assess liver reserve.

The Child-Pugh score (classification) is a functional evaluation method for liver cirrhosis based on five items: (1) hepatic encephalopathy, (2) ascites, (3) serum bilirubin level, (4) serum albumin level, and (5) prothrombin activity. Yes (Table 1). Based on the total score of each item, the evaluation is divided into three levels: class A (5 to 6 points), class B (7 to 9 points), and class C (10 to 15 points).

In some embodiments, the liver disease is classified as A, B, or C according to the Child-Pugh score.

In some embodiments, the liver disease is at least 5, or at least 6, or at least 7, or at least 8, or at least 9, or at least 10, or at least 11, or at least 12, or at least 13, or at least 14, Or classified according to the total score according to the Child-Pugh classification of 15.

In some embodiments, hepatic reserve can be assessed by improvement in Child-Pugh classification grade (A, B, C).

In some embodiments, hepatic reserve can also be evaluated by the amount of change in the Child-Pugh score (5 to 15 points). It can also be evaluated by the amount of change in the scores (1 to 3 points) of each evaluation item (hepatic encephalopathy, ascites, serum bilirubin level, serum albumin level, prothrombin activity) that make up the Child-Pugh score.

In some embodiments, the ALBI grade can be used to assess liver reserve.

ALBI grade (Albumin-bilirubin grade) is a liver reserve evaluation method that evaluates liver reserve into three levels based on the ALBI score calculated only from albumin and bilirubin values. [(0.66×log ₁₀ bilirubin (μmol/L)) + (-0.085×albumin (g/L)): grade 1:2:3 = ≦-2.60: ＞-2.60 to ≦-1.39: ＞-1.39.

In some embodiments, the (MELD) score can be used to assess liver reserve.

The Model for end-stage liver disease (MELD) score is used to predict the short-term prognosis of patients with decompensated liver cirrhosis and to determine suitability for liver transplantation. (MELD) score is calculated from three items: (1) serum bilirubin, (2) prothrobin time-international normalized ratio (PT-INR), and (3) serum creatinine value.

serum bilirubin
[Reference value] 0.3 to 1.2 (mg/dL)
In the reticuloendothelial system, hemoglobin in old red blood cells is degraded, heme is cleaved, and unconjugated (indirect) bilirubin is produced. Unconjugated bilirubin binds to albumin, is transported to the liver, and is taken up by hepatocytes from the sinusoidal side. The taken up bilirubin binds to ligandin, is transported to the endoplasmic reticulum, undergoes glucuronidation by bilirubin UDP-glucuronosyl transfer (BUGT), is transported to the bile canaliculi side, and is removed from the bile canaliculi by multidrug resistance protein 2 (MRP2). via bile duct It is excreted into the duodenum. Hyperbilirubinemia occurs due to abnormalities in this bilirubin metabolism and excretion pathway. Table 2 shows diseases in which bilirubin increases.

Prothrobin time (PT)
[Reference value] PT: 80-130 (%)
Prothrobin time (PT) reflects the overall coagulation activity of extrinsic coagulation factors VII, X, V, and II factors (prothrobin) and fibrinogen. (PT) is the actual measured value (seconds). % activity relative to normal plasma. PT is a more sensitive indicator of liver reserve capacity (protein synthesis ability) than albumin, and its value is lower than that of severe liver failure, vitamin K deficiency, and oral warfarin administration.

In some embodiments, the therapeutic effect on liver fibrosis and liver cirrhosis can be evaluated by observing the state of fibrosis and defibrosis in the liver parenchymal region. In some embodiments, by tissue staining using histopathological techniques (eg, Sirius Red staining). Sirius red staining is a staining for evaluating fibrosis, and stains collagen fibers red. In some embodiments, the liver tissue stiffness can also be evaluated by measuring the stiffness of the liver tissue, and in some embodiments, the method includes Improvement and maintenance of hardness can be evaluated. A fibroscan test is a test that measures the hardness of the liver and the amount of fat in the liver tissue by applying a special "probe" to the surface of the body and measuring the vibrations and ultrasound transmitted by the probe. The basic principle of MR elastography (MRE) is to convert the vibrational phase of shear elastic waves generated in the liver into the rotational phase of protons using a heating device that generates extracorporeal vibrations, and detect this phase difference using an MRI phase image. , the shear modulus is obtained. The velocity (V) of an elastic wave propagating within the liver is expressed by the formula V ² =μ/ρ using the shear modulus (μ) and density (ρ) of the substance.

In some embodiments, the therapeutic effect can also be confirmed by evaluating liver fibrosis by measuring the expression level of collagen genes.

In some embodiments, the FIB-4 index can be measured to evaluate the degree of progression of liver fibrosis.

The FIB-4 index is a scoring system that combines blood test data to evaluate the degree of progression of liver fibrosis. This test method calculates the degree of progression of fibrosis by combining four blood test items: AST value, ALT value, platelet count, and age. This is a score that predicts liver fibrosis in hepatitis C. Recently, it has been used for other liver diseases such as NASH, and it is important to use FIB-4 index as an indicator in fatty liver disease to detect advanced cases of liver fibrosis early in the follow-up of NAFLD patients. It is believed that.

In some embodiments, APRI can be measured to evaluate the degree of progression of liver fibrosis.

APRI (app = aspartate aminotransferase to platelet ratio index) evaluates the degree of fibrosis by combining AST, ALT, and platelet count. This is a score that predicts liver fibrosis in hepatitis C.

In some embodiments, the effect of "improving/maintaining" liver function, that is, the "therapeutic effect on liver disease" can be evaluated based on each of the above-mentioned indicators. In some embodiments, each of the indicators described above can be used to assist in evaluating the effectiveness. In some embodiments, by measuring each index, the optimal dosage regimen for treatment can be determined. In some embodiments, the evaluation indicators include liver tissue stiffness (e.g., fibroscan measurements), liver reserve indicators (e.g., serum albumin levels), and cholestatic indicators (e.g., serum total bile acid levels). At least one, two, or all three can be used.

The above evaluation indicators are usually measured before starting drug administration, during the drug administration period, and at the end of drug administration, and are used to evaluate the effect of the drug on improving and maintaining liver function (therapeutic effect on liver disease). I can do it. Furthermore, by measuring after a certain period of time (e.g. 6 to 12 months) after the end of drug administration, it is possible to determine the durability of the drug's further improvement in liver function and maintenance effect (therapeutic effect on liver disease). can be evaluated.

In some embodiments, the pharmaceutical compositions of the present disclosure contain 4-({(6S,9S,9aS)-1-(benzylcarbamoyl)-2,9-dimethyl-4,7-dioxo-8-[( quinolin-8-yl)methyl]octahydro-2H-pyrazino[2,1-c][1,2,4]triazin-6-yl}methyl)phenyl dihydrogen phosphate, or a pharmaceutically acceptable salt thereof . In some embodiments, the pharmaceutical compositions of the present disclosure include 4-({(6S,9S,9aS)-1-(benzylcarbamoyl)-2,9-dimethyl-4,7-dioxo- 8-[(quinolin-8-yl)methyl]octahydro-2H-pyrazino[2,1-c][1,2,4]triazin-6-yl}methyl)phenyl dihydrogen phosphate, or pharmaceutically acceptable thereof Contains salt obtained.

In this disclosure, "pharmaceutically acceptable" means useful in preparing pharmaceutical compositions that are generally safe, non-toxic, and not biologically or otherwise undesirable. , and is acceptable for human as well as veterinary uses.

In the present disclosure, the term "pharmaceutically acceptable salt" refers to a salt of the compound of the present invention that is pharmaceutically acceptable and has the desired pharmacological activity, as defined above. In some embodiments, such salts include inorganic acids, such as, for example, hydrochloric, hydrobromic, sulfuric, nitric, and phosphoric acids; or, for example, acetic, propionic, hexanoic, heptanoic, cyclopentane. Propionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, o-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid , methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, p-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, p-toluenesulfonic acid, camphorsulfonic acid, 4-Methylbicyclo[2.2.2]oct-2-ene-1-carboxylic acid, glucoheptonic acid, 4,4'-methylenebis(3-hydroxy-2-ene-1-carboxylic acid), 3-phenylpropion acid addition salts formed with organic acids such as trimethylacetic acid, tert-butylacetic acid, lauryl sulfate, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid and muconic acid. In some embodiments, pharmaceutically acceptable salts also include base addition salts that may be formed when acidic protons present are capable of reacting with an inorganic or organic base. In some embodiments, acceptable inorganic bases include sodium hydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide, and calcium hydroxide. In some embodiments, acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like.

In some embodiments, the pharmaceutical compositions of the present disclosure may be in a dosage form that is used orally or parenterally. These dosage forms can be formulated by those skilled in the art by incorporating appropriate combinations of pharmaceutically acceptable carriers and excipients into unit dosage forms as required by generally accepted pharmaceutical practice. can. In some embodiments, the pharmaceutical compositions of the present disclosure can be manufactured according to known methods, such as those described in the Japanese Pharmacopoeia or the United States Pharmacopeia (USP).
In some embodiments, methods of administering the pharmaceutical compositions of the present disclosure include intravenous administration, continuous intravenous administration, and the like.

In some embodiments, the pharmaceutical composition of the present disclosure is administered to humans as a pharmaceutical preparation (eg, injection, drip) prepared by a conventional method. In some embodiments, the administration is at a dose of 100 to 400 mg/m ² of active ingredient.

In some embodiments, the administration is at a dose of 200 to 300 mg/m ² of active ingredient.

In some embodiments, the administration is in an amount of active ingredient of 200 mg/m ² , 210 mg/m ² , 220 mg/m ² , 230 mg/m ² , 240 mg/m ² , 250 mg/m ² , 260 mg / ^m2 , 270mg/ ^m2 , 280mg/ ^m2 , 290mg/ ^m2 , 300mg/ ^m2 .

In some embodiments, the administration is at a dose of 280 mg/m ² of active ingredient.

In some embodiments, the administration is administered intravenously over one or more hours each time at least once a week.

In some embodiments, the administration is administered intravenously once or twice a week over a period of 2 to 5 hours each time. In some embodiments, the administration is administered intravenously twice a week over a 4 hour period each time.

In some embodiments, the administration is continued for one month.

In some embodiments, the administration is continued for 2 to 6 months. In some embodiments, the administration is used to continue for 12 weeks.

In some embodiments, the pharmaceutical composition of the present disclosure is administered to humans once or twice a week for 2 to 5 hours each time as a conventionally formulated pharmaceutical preparation (e.g., injection, infusion). The drug is administered intravenously over a period of time, and the administration is continued over a period of 2 to 6 months. In some embodiments, the pharmaceutical composition of the present disclosure is administered intravenously to humans twice a week for 4 hours each time as a pharmaceutical formulation (e.g., injection, infusion) formulated in a conventional manner. It is used such that the administration is continued for 12 weeks.

The present invention will be explained in more detail with reference to Examples below, but these are not intended to limit the scope of the present invention.

All experiments were performed in accordance with the National Academy of Sciences' Guide for the Care and Use of Laboratory Animals. The research protocol was approved by the Tokyo Metropolitan Komagome Hospital Research Committee. All analyzes using human samples were approved by the Tokyo Metropolitan Komagome Hospital Ethics Committee. This study complies with the principles of the Declaration of Helsinki.
All experimental animals (mice) were maintained in ventilated cages under a 12 h/12 h light/dark cycle with free access to enrichment, water, and food.

(List of abbreviations)
ALP: alkaline phosphatase
ALT: alanine aminotransferase
BA: bile acid
BDL: bile duct ligation
CA: cholic acid
CDCA: chenodeoxycholic acid
CK19: cytokeratin-19 (cytokeratin 19)
FXR: farnesoid X receptor
MDR2: multidrug resistance-associated protein 2
PBC: primary biliary cholangitis
PSC: primary sclerosing cholangitis
T.Bil: total bilirubin
TBA: total bile acid

(Materials and methods)
1. Mdr2-KO Mouse Model Mdr2-KO (FVB.129P2-Abcb4tm1Bor/J, #002539) mice were purchased from Jackson Laboratory (Bar Harbor, ME, USA) and wild-type littermates on FVB/NJ background were used as controls. . 8-10 week old male Mdr2-KO mice and littermate controls were treated weekly for 10 weeks with 20 mg/kg of a compound of the present disclosure (foscenvivint; Prism BioLab, Tokyo, Japan) dissolved in PBS or PBS as a control. Three intraperitoneal injections were given.

2. BDL Mouse Model Male wild-type (C57BL/6J) mice aged 8 to 10 weeks were obtained from Japan SLC (Shizuoka, Japan) and used as previously reported (Osawa Y, et al. Inhibition of Cyclic Adenosine Monophosphate (cAMP)-response Element. -binding Protein (CREB)-binding Protein (CBP)/beta-Catenin Reduces Liver Fibrosis in Mice. eBioMedicine 2, 1751-1758 (2015).) The common bile duct above the pancreas was incised and BDL was performed. The mice received 20 mg/kg of a compound of the present disclosure dissolved in PBS intraperitoneally three times a week.

3. DDC Mouse Model Eight-week-old C57BL/6J wild-type male mice were fed 0.1% DDC concentrate diet (Sigma-Aldrich, St. Louis, MO, USA) for 18 days.

4. Bilirubin Serum bilirubin was measured using the QuantiChrom Bilirubin Assay Kit (BioAssay Systems, Hayward, CA, USA).

5. BA analysis Serum and liver TBA, CA, and CDCA concentrations were determined using the Total Bile Acid Assay Kit, Cholic Acid ELISA Kit, and Chenodeoxycholic Acid ELISA Kit (Cell Biolabs, San Diego, CA, USA), respectively, according to the manufacturer's instructions. Analyzed.

6. RT-qPCR
For RNA extraction, DNA removal, and reverse transcription from liver tissue and cultured cells, RNeasy and DNase Kits (Qiagen, Valencia, CA, USA) and High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) were used. was used. qPCR was performed in triplicate on a LightCycler 480 (Roche Applied Science, Mannheim, Germany) using probe and primer sets purchased from Thermo Fisher Scientific and TaqPath qPCR Master Mix, CG (Applied Biosystems). The expression level of the target gene was normalized to the expression level of GAPDH in each sample.
(mouse primer set)

7. Histological analysis Mouse liver tissues were fixed in 10% formalin, sectioned, and stained with hematoxylin and eosin (HE). Collagen deposits were stained with Sirius Red (saturated picric acid plus 0.1% Direct Red 80 and 0.1% Fast Green FCF). In addition, samples were immunohistochemically stained using an antibody against cytokeratin 19 (Abcam, Cambridge, UK). To quantify the Sirius Red positive area, HistoQuant software (3DHISTECH, Budapest, Hungary) was used.
(Hematoxylin and eosin (HE) staining)
HE staining is a staining method that allows the cell nucleus and cytoplasm to be dyed separately to observe the morphology of tissues. HE staining makes it possible to grasp the overall picture of cells and tissue structures.
Sections are cut from paraffin blocks of liver tissue prefixed with Bouin solution and stained with Lillie-Mayer's Hematoxylin (Muto Chemical Co., Ltd., Japan) and eosin solution (Fujifilm Wako Pure Chemical Industries, Ltd., Japan).
(Sirius red staining)
To visualize collagen deposition, Bouin-fixed liver sections are stained using picrosirius red solution (Waldeck GmbH & Co., Germany). For quantitative analysis, a digital camera (DFC280, Leica, Germany) was used to capture Sirius Red-stained sections near the central vein at 200x magnification, and positive areas were identified in 5 fields per section using ImageJ. Measure using software (ImageJ, National Institute of Health, USA).

8. Fib-4 Index Measurement The Fib-4 Index is a score that predicts liver fibrosis, and is calculated by combining four items: AST, ALT, platelet count, and age. It is possible to evaluate the degree of progress.

9. Fibroscan test As a non-invasive testing method, Fibroscan is a non-invasive testing method based on the principle of transient elastography, which measures the propagation velocity of pulsed vibration waves in tissues in terms of elasticity (kPa) using ultrasonic image analysis. FibroScan manufactured by ECHOSENS is used.

10. Serum Albumin Concentration Serum albumin is a protein produced in the liver, and its amount is known to decrease as liver function declines.
Serum albumin can be measured according to a conventional method. For example, the amount of albumin in a sample is determined by using a reagent that generates a pigment by binding with albumin and measuring the pigment spectroscopically. Examples of such dyes include bromocresol green, which reacts with albumin at around pH 4.0 to produce a blue albumin-binding dye.
A commercially available measurement reagent (Shikaliquid ALB, Kanto Kagaku Co., Ltd.) can also be used. Mix the reagent with the sample and measure the absorbance of the bound dye produced.

Example 1
Preclinical test (evaluation in PBC model mice) 1 (hepatic fibrosis improving effect)
As nonclinical test models of primary biliary cholangitis (PBC), we have used bile duct ligation model mice (BDL mice), which induce liver fibrosis due to cholestasis, MDR2 knockout mice (Mdr2-KO mice), and bile duct injury model mice ( The hepatic fibrosis-improving effect of the compound of the present disclosure was investigated using DDC mice).
MDR2KO mice and BDL mice were treated with compounds of the present disclosure according to the protocols in Figures 1A and 2A, respectively. DDC mice were wild-type C57BL/6 male 8-week-old mice fed with 0.1% DCC feed for 18 days. During that time, compounds of the present disclosure (20 mg/kg three times per week) or PBS were administered intraperitoneally. After the treatment, the liver was collected and the anti-fibrotic effect of the compound of the present disclosure was examined by Sirius red staining of the liver tissue. Sirius red positive areas indicate collagen deposition. For BDL mice and DDC mice, hematoxylin and eosin staining was also performed at the same time. A significant decrease in the Sirius red positive area, ie, a decrease in the fibrotic area, was observed in all mouse model mice (FIGS. 1 to 3), confirming the anti-fibrotic effect of the compound of the present disclosure.

Example 2
Preclinical test (evaluation in PBC model mice) 2 (cholestasis improving effect)
As in Example 1, the cholestasis-improving effect of the compound of the present disclosure was investigated using BDL mice, Mdr2-KO mice, and DDC mice as PBC model mice. Since bile acid (BA) metabolism is involved in liver fibrosis due to cholestasis, the amount of BA (total bile acids (TBA), cholic acid (CA), and chenodeoxycholic acid (CDCA)) was measured. The results are shown in FIG. 4 (Mdr2-KO mouse), FIG. 5 (BDL mouse), and FIG. 6 (DDC mouse). In Mdr2-KO mice, hepatic TBA and CA levels were unaffected by compounds of the present disclosure, whereas CDCA levels were decreased. Serum TBA, CA and CDCA levels were reduced by the compounds of the present disclosure (Figure 4). Similarly, compounds of the present disclosure also reduced liver TBA, CA, and CDCA levels in BDL mice (Figure 5), and reduced liver and serum TBA, CA, and CDCA levels in DDC mice (Figure 6). ).

Example 3
Preclinical test (evaluation in PBC model mice) 3 (hepatic function improvement effect)
As in Example 1, the liver function improving effect of the compound of the present disclosure was investigated using BDL mice, Mdr2-KO mice, and DDC mice as PBC model mice. Serum alanine aminotransferase (ALT) is an enzyme produced by liver cells, and when the liver cells are destroyed, it is released into the blood, resulting in increased levels. Alkaline phosphatase (ALP) is an enzyme that breaks down phosphoric acid compounds. It is produced in the liver, kidneys, intestinal mucosa, bones, etc., and is processed in the liver and released into bile. When the bile duct is blocked by gallstones, cholangitis, or biliary tract cancer, resulting in poor bile flow (cholestasis) or when liver function declines, ALP in bile flows backwards into the blood. Therefore, ALP is greatly elevated in cholestasis. Bilirubin includes indirect bilirubin before being processed by the liver, and direct bilirubin that is processed into bile (total bilirubin), and direct bilirubin is excreted from the biliary tract. When liver function is impaired, indirect bilirubin cannot be processed, so a large amount of indirect bilirubin remains in the blood.On the other hand, when biliary system dysfunction causes insufficient bile excretion, direct bilirubin increases in the blood. do.
In this example, the amounts of ALT, ALP, and total bilirubin in the serum of each PBC model mouse were measured. The results are shown in FIG. 7 (Mdr2-KO mouse), FIG. 8 (BDL mouse), and FIG. 9 (DDC mouse). In all of the PBC model mice, reduction in liver damage was confirmed as shown by reductions in ALT, ALP, and T.Bil values.

Example 4
Preclinical test (evaluation in PBC model mice) 4 (improving effect on collagen gene expression level)
Using Mdr2-KO mice and DDC mice as PBC model mice, the effect of the compound of the present disclosure on improving collagen gene expression was investigated. Collagen, particularly type IV collagen, is known as a marker of liver fibrosis. Type IV collagen constitutes the basement membrane. Although there is no basement membrane in normal liver sinusoids, proliferation of the basement membrane occurs as liver fibrosis progresses, and the expression level of type IV collagen increases.
In this example, the expression level of collagen gene in hepatocytes was measured for each PBC model mouse. The results are shown in FIG. 10 (Mdr2-KO mouse) and FIG. 11 (DDC mouse). In all of the PBC model mice, reduction in liver fibrosis was confirmed, as indicated by a decrease in collagen gene expression.

Example 5
Clinical trial (evaluation in PBC patients) (cholestasis improving effect)
This example is based on the results of a Phase I study designed to examine the safety and tolerability of the disclosed compounds and to determine recommended doses in PBC patients.
The subject was a patient who was diagnosed with primary biliary cholangitis and who was diagnosed with advanced fibrosis (Scheuer stage III or higher) as a result of liver histological examination. As a dosing schedule, compounds of the present disclosure were administered intravenously twice a week (4 hours) for 12 weeks. However, the dose scheduled for the first cycle was administered once 7 days before the first cycle administration by continuous intravenous administration for 4 hours, and safety and pharmacokinetics were evaluated from the day of administration to the day after administration. . Dose levels were 2 doses (280 mg/m ² /4 hours, 380 mg/m ² /4 hours).
Serum total bile acid concentrations (TBA concentrations) were measured after the completion of a 12-week dosing schedule of compounds of the present disclosure. The results are shown in FIG.
A clear decrease in TBA concentration was observed as a result of the drug treatment, confirming the cholestasis-improving effect of the compound of the present disclosure.

Example 6 (evaluation in hepatitis patients) (improving effect on liver tissue hardness)
This example describes a phase I study aimed at evaluating the safety and pharmacokinetics of administering a compound of the present disclosure to patients with HCV or HBV cirrhosis and determining recommended doses of the compound of the present disclosure; Based on the results of a Phase II study aimed at evaluating the efficacy and safety of recommended doses of compounds of the present disclosure administered to patients with HCV or HBV cirrhosis.
As for the dosing schedule, in the phase I study, the drug was administered at three doses: (Level 1) 140 mg/m ² /4 hours, (Level 2) 280 mg/m ² /4 hours, and (Level 3) 380 mg/m ² /4 hours. The drug was administered intravenously for 4 consecutive hours twice a week (acceptable range of administration time: ±15 minutes). This was done with a single dose and 12 cycles (total 12 weeks). For single administration, the dose scheduled for the first cycle is administered intravenously for 4 consecutive hours (tolerable range of administration time: ±15 minutes) once 7 days before the first cycle administration, and from the day of administration to the day after administration. The safety and pharmacokinetics were evaluated.
In the Phase II (IIa) study, continuous intravenous administration was performed for 4 hours twice a week at the recommended dose determined in Phase I (Level 2). This was done for 12 cycles (total 12 weeks) (Protocol A).
In a Phase I study, liver tissue stiffness was measured using Fibroscan after the completion of a 12-week dosing schedule of compounds of the present disclosure. The results are shown in FIG.
A clear decrease in liver tissue stiffness was observed with the drug treatment, confirming the liver tissue stiffness improving effect of the compound of the present disclosure. Particularly good results were obtained with the level 2 dosing schedule.
In a Phase II study, serum albumin concentrations were measured using the Protocol A dosing schedule. The results are shown in Figure 14A. An improving effect on serum albumin concentration was confirmed by administering the compound of the present disclosure. Serum albumin concentrations were determined following the dosing schedule of compounds of the present disclosure in Protocol A. The results are shown in Figure 14B. It was confirmed that the improving effect on serum albumin concentration continued even 9 months after the start of administration of the compound of the present disclosure.
In a Phase II study, the FIB-4 index was examined after completion of the Protocol A dosing schedule. The results are shown in FIG. A significant decreasing trend was observed.
Liver tissue stiffness was measured using Fibroscan after completing the dosing schedule of the compounds of the present disclosure in Protocol A. The results are shown in FIG. It was confirmed that the effect of improving liver tissue hardness continued even 9 months after the start of administration of the compound of the present disclosure.
In a Phase II study, ALBI scores were examined 6 months after the end of the Protocol A dosing schedule. The results are shown in Figure 17-1. Figure 17-2 shows a case with Child-Pugh (CP) classification B. Six months after the end of administration of the compound of the present disclosure, improvement in liver function was suggested by improvement in ALBI score. In the Phase II study, serum albumin levels were determined 6 months after the end of the Protocol A dosing schedule. The results are shown in Figure 18-1. Figure 18-2 shows a case with Child-Pugh (CP) classification B. A case in which an albumin preparation was used during administration of the compound of the present disclosure (KOM-009), a case in which ascites puncture was performed 6 months after the completion of administration of the compound of the present disclosure (KOM-021), and a case with moderate or higher amount of ascites. This includes a case (KOM-033) in which an albumin preparation was used up to 28 days after the end of administration of the compound of the present disclosure. CP class B, excluding 3 cases where the evaluation of liver function was affected and 2 cases where results were not obtained 6 months after the end of administration of the compound of the present disclosure (KOM-012, KOM-018). Two of the four patients showed an improvement in their CP score (serum albumin level rating) from 2 points (2.8 to 3.5 g/dL) to 1 point (over 3.5 g/dL) 6 months after the end of treatment. Ta. Six months after the end of administration of the compound of the present disclosure, improvement in liver function was suggested due to improvement in serum albumin levels.
In a Phase II study, prothrombin time activity values were determined 6 months after the end of the Protocol A dosing schedule. The results are shown in Figure 19-1. Figure 19-2 shows a case with Child-Pugh (CP) classification B. A case in which an albumin preparation was used during administration of the compound of the present disclosure (KOM-009), a case in which ascites puncture was performed 6 months after the completion of administration of the compound of the present disclosure (KOM-021), and a case with moderate or higher amount of ascites. This includes a case (KOM-033) in which an albumin preparation was used up to 28 days after the end of administration of the compound of the present disclosure. CP class B, excluding 3 cases where the evaluation of liver function was affected and 2 cases where results were not obtained 6 months after the end of administration of the compound of the present disclosure (KOM-012, KOM-018). Three of the four patients showed an improvement in their CP score (prothrombin time activity score) from 2 points (40-70%) to 1 point (over 70%) 6 months after the end of treatment. Six months after the end of administration of the compound of the present disclosure, improvement in the prothrombin time activity value suggested improvement in liver function.

Example 7 (Evaluation in patients with hemophilia and cirrhosis caused by HIV/HCV co-infection)
This example is a phase I study aimed at examining the safety and tolerability of administering the compound of the present disclosure to patients with hemophilia and liver cirrhosis caused by HIV/HCV co-infection. Based on results.
The dosing schedule was two doses: (Level 1) 140 mg/m2/4 hours and (Level 2) 280 mg/m2/4 hours, twice a week, for 4 consecutive hours (administration time tolerance range: ±15 minutes). This was done with a single dose and 12 cycles (total 12 weeks). For single administration, the dose scheduled for the first cycle is administered intravenously for 4 consecutive hours (tolerable range of administration time: ±15 minutes) once 14 days before the first cycle administration, and from the day of administration to the day after administration. The safety and pharmacokinetics were evaluated.
After completing the 12-week dosing schedule of the compounds of the present disclosure, liver tissue stiffness was measured using Fibroscan and FIB-4 index and APRI were calculated. The results are shown in FIG. 20, FIG. 21A, and FIG. 21B, respectively. The liver tissue stiffness measured by Fibroscan showed a decrease from the baseline in both Level 1 and Level 2 of the FIB-4 index and APRI. and APRI showed a greater decline at level 2. After administering the compound of the present disclosure, a decrease in liver stiffness as determined by Fibroscan, and a decreasing trend in FIB-4 index and APRI score was confirmed.

Example 8 (Evaluation in patients with decompensated liver cirrhosis)
This example demonstrates the efficacy of compounds of the present disclosure when administered to patients with decompensated cirrhosis due to HCV or HBV infection, and patients with decompensated cirrhosis due to (suspected) non-alcoholic steatohepatitis; It is based on the design of a Phase II study aimed at evaluating safety and pharmacokinetics. The severity of decompensated cirrhosis is Child-Pugh classification B, and patients with decompensated cirrhosis due to HCV or HBV infection are classified into Cohort A, and patients with decompensated cirrhosis due to non-alcoholic steatohepatitis (including suspected). Patients will be divided into Cohort B and will receive the compound or placebo in a double-blind manner.
The administration schedule is 280 mg/m2, administered intravenously for 3 to 4 consecutive hours each time (acceptable range of administration time: ±15 minutes). Cohort A has 3 groups (the compound is administered twice a week, the compound and placebo are administered once a week, and the placebo is administered twice a week), and Cohort B has 2 groups (the compound is administered twice a week). Patients were randomly assigned to a group receiving twice a week administration of a placebo and a group receiving a placebo twice a week, and were administered for 24 weeks, with observation being made until 52 weeks after the start of administration.
Efficacy evaluation items include liver reserve index (Child-Pugh score, ALBI score, MELD score, Child-Pugh classification, mALBI grade, serum albumin level, serum bilirubin level, prothrombin activity value), liver fibrosis index (fibroscan Evaluate liver tissue stiffness, MRE liver tissue stiffness, serum fibrosis marker, FIB-4 index). In addition, the dose and frequency of administration of albumin preparations used to treat ascites, events associated with decompensated cirrhosis (esophageal/gastric variceal bleeding, ascites, etc.), and the severity of ascites and hepatic encephalopathy will also be evaluated.

4-({(6S,9S,9aS)-1-(benzylcarbamoyl)-2,9-dimethyl-4,7-dioxo-8-[(quinolin-8-yl)methyl]octahydro-2H-pyrazino[2 ,1-c][1,2,4]triazin-6-yl}methyl)phenyl dihydrogen phosphate is administered to humans at a dose of 100 to 400 mg/m ² once or twice a week, each time 2 to 5 It is suggested that when administered intravenously over time and the administration is continued for 2 to 6 months, it exhibits an excellent therapeutic effect on liver cirrhosis (hepatic fibrosis). Therefore, it is suggested that a pharmaceutical composition containing the compound is effective for preventing and/or treating liver diseases.

Claims (25)

1. 100 to 400 mg/m ² of 4-({(6S,9S,9aS)-1-(benzylcarbamoyl)-2,9-dimethyl-4,7-dioxo-8-[(quinolin-8-yl)methyl] Octahydro-2H-pyrazino[2,1-c][1,2,4]triazin-6-yl}methyl)phenyl dihydrogen phosphate, or a pharmaceutically acceptable salt thereof, is administered to humans once or twice a week. 4-({(6S,9S,9aS)-1 -(benzylcarbamoyl)-2,9-dimethyl-4,7-dioxo-8-[(quinolin-8-yl)methyl]octahydro-2H-pyrazino[2,1-c][1,2,4]triazine -6-yl}methyl)phenyl dihydrogen phosphate, or a pharmaceutically acceptable salt thereof, for the prevention and/or treatment of liver diseases.
2. 280 mg/m ² of 4-({(6S,9S,9aS)-1-(benzylcarbamoyl)-2,9-dimethyl-4,7-dioxo-8-[(quinolin-8-yl)methyl]octahydro- 2H-pyrazino[2,1-c][1,2,4]triazin-6-yl}methyl)phenyl dihydrogen phosphate, or a pharmaceutically acceptable salt thereof, is administered to humans twice a week for 4 hours each time. 2. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is administered intravenously over a period of 12 weeks.
3. The pharmaceutical composition according to claim 1, wherein the liver disease is accompanied by liver fibrosis or cirrhosis.
4. The pharmaceutical composition according to claim 3, wherein the liver fibrosis or cirrhosis is a disease caused by hepatitis B or C or primary biliary cholangitis (PBC).
5. The pharmaceutical composition according to claim 3, wherein the liver fibrosis or cirrhosis is a disease caused by hemophilia, HIV/HCV co-infection, or non-alcoholic steatohepatitis.
6. The pharmaceutical composition according to claim 3, wherein the liver disease is classified as A, B, or C according to the Child-Pugh score.
7. The pharmaceutical composition according to any one of claims 1 to 6, which is used for maintaining or improving liver tissue hardness.
8. 8. The pharmaceutical composition according to claim 7, wherein maintenance or improvement is evaluated during drug administration or at the end of drug administration compared to before the drug administration started.
9. 8. The pharmaceutical composition according to claim 7, wherein maintenance or improvement is evaluated 6 to 12 months after the end of drug administration compared to the time point at which drug administration ended.
10. 8. The pharmaceutical composition according to claim 7, wherein liver tissue hardness is indicated by Fibroscan measurement value.
11. The pharmaceutical composition according to claim 7, wherein liver tissue hardness is indicated by MR elastography measurements.
12. The pharmaceutical composition according to any one of claims 1 to 6, which is used for maintaining or improving a liver reserve index.
13. 13. The pharmaceutical composition according to claim 12, wherein maintenance or improvement is evaluated during drug administration or at the end of drug administration compared to before the drug administration started.
14. 13. The pharmaceutical composition according to claim 12, wherein maintenance or improvement is evaluated 6 to 12 months after the end of drug administration compared to the time point at which drug administration ended.
15. 13. The pharmaceutical composition according to claim 12, wherein the hepatic reserve index is represented by serum albumin concentration.
16. The pharmaceutical composition according to claim 12, wherein the hepatic reserve index is represented by serum bilirubin concentration, prothrombin activity value, or ALBI score.
17. [17] The pharmaceutical composition according to claim 12, wherein the hepatic reserve index is indicated by a change in Child Pugh classification (A, B, C) or a change in score.
18. The pharmaceutical composition according to any one of claims 1 to 6, which is used for maintaining or improving a cholestasis index.
19. 19. The pharmaceutical composition according to claim 18, wherein maintenance or improvement is evaluated 6 to 12 months after the end of drug administration compared to the time point at which drug administration ended.
20. The pharmaceutical composition according to claim 18, wherein the cholestasis index is expressed by serum total bile acid concentration.
21. 4-({(6S,9S,9aS)-1-(benzylcarbamoyl)-2,9-dimethyl-4,7-dioxo-8-[(quinolin-8-yl)methyl]octahydro-2H-pyrazino[2 ,1-c][1,2,4]triazin-6-yl}methyl)phenyl dihydrogen phosphate, or a pharmaceutically acceptable salt thereof, for assisting in the evaluation of the therapeutic effect on liver diseases, the method comprising: A method characterized by measuring at least one selected from hardness, liver reserve index, and cholestasis index.
22. 22. The method according to claim 21, wherein the measurement is performed 6 to 12 months after the end of drug administration compared to the time point at which drug administration ended.
23. 23. The method according to claim 21 or 22, wherein the liver tissue stiffness is indicated by a fibroscan measurement value.
24. 23. The method according to claim 21 or 22, wherein the hepatic reserve index is indicated by serum albumin concentration.
25. 23. The method according to claim 21 or 22, wherein the cholestasis index is indicated by serum total bile acid concentration.