JP2019517506A - Composition for the treatment of liver cancer comprising a blood vessel destroying agent - Google Patents

Abstract

The present invention relates to (S) -N- (4- (3- (1H-1,2,4-triazol-1-yl) -4- (3,4,5-trimethoxybenzoyl) phenyl) thiazole-2. And -yl) -2-amino-3-methylbutanamide or a pharmaceutically acceptable salt thereof. The composition of the present invention exerts an excellent liver cancer therapeutic effect. [Selected figure] Figure 1

Description

  The present invention relates to a composition for the treatment of liver cancer containing a blood vessel destroying agent (VDA) and a method for treating liver cancer using the same.

  Liver cancer is a malignancy with a poor prognosis, which is the second leading cause of death from cancer and the third leading in the world. Radical surgery is not possible in more than 70% of liver cancer patients, and even after radical resection, there is a 50% or more chance of recurrence at another site within five years. In addition, the probability of responding to systemic anti-cancer therapy in advanced liver cancer is currently very low at less than 10%.

Vascular Disrupting Agent (VDA) aims to rapidly and selectively block formed tumor blood vessels by selectively destroying microtubules in the cytoskeleton of vascular endothelial cells. It can induce ischemic necrosis of cells in the center. Therefore, the method of blood vessel destruction using a blood vessel destruction agent (VDA) has recently emerged as a new anticancer strategy. Therefore, the present applicant has developed a compound of the following chemical formula 1 as such a blood vessel destructive agent (International Publication Patent Publication WO2009-119980).

  The compound of formula I is a tubulin polymerization inhibitor having a dual action mechanism of rapid collapse of existing tumor blood vessels by microtubule destabilization and apoptosis by cell cycle arrest. However, there is a problem that the tumor can rapidly re-grow from a viable rim when treated alone with a blood vessel destroying agent (VDA) including the compound of the above-mentioned chemical formula. It seriously reduces the therapeutic utility of these drugs.

  Therefore, the present inventors are able to treat a liver cancer which is one of malignancies by solving the problem of tumor regrowth while taking advantage of the blood vessel destruction agent as an anticancer agent and Various studies have been attempted to provide a therapeutic method.

International Patent Publication WO 2009-119980

  An object of the present invention is to provide a composition for liver cancer treatment or liver cancer treatment assistance containing a blood vessel destructive agent (VDA).

  Another object of the present invention is to provide a combination for the treatment of liver cancer comprising a blood vessel destroying agent (VDA) and sorafenib.

  Another object of the present invention is to provide a composition for chemical embolization comprising a blood vessel rupturing agent (VDA).

  As a result of intensive research efforts by the present inventors to achieve the above object, the present inventors have completed the preparation of a pharmaceutical composition for the treatment of liver cancer or for assisting the treatment of liver cancer containing a blood vessel destroying agent (VDA).

In the present invention, the compound used as the blood vessel destruction agent is (S) -N- (4- (3- (1H-1,2,4-triazol-1-yl)-) represented by the following chemical formula 1 4- (3,4,5-trimethoxybenzoyl) phenyl) thiazol-2-yl) -2-amino-3-methylbutanamide or a pharmaceutically acceptable salt thereof.

  The composition of the present invention exhibits excellent liver cancer therapeutic activity and exhibits an effect with a low possibility of tumor recurrence. Therefore, the composition of the present invention can be applied for liver cancer treatment or as an adjunct to liver cancer treatment.

It is a figure which shows the result of the bioluminescence imaging (BLI) of the parenteral administration control group and each experimental group in a Hep3B xenograft mouse model. It is the graph which compared the relative BLI signal strength of the parenteral administration control group and each experimental group in the Hep3B xenograft mouse model. It is the figure which showed the H & E and TUNEL staining image of a parenteral administration control group and each experimental group. It is the graph which compared the apoptosis of the parenteral administration control group and each experimental group. It is a figure which showed the CD31 stained image of a parenteral administration control group and each experimental group. It is the graph which compared the microvessel density (MVD; microvessel density) of a parenteral administration control group and each experimental group. It is a figure which shows the result of the bioluminescence imaging (BLI) of the oral administration control group and each experimental group in a Hep3B xenograft mouse model. It is the graph which compared the relative BLI signal strength of the oral administration control group and each experimental group in the Hep3B xenograft mouse model. It is the graph which compared the apoptosis of the oral administration control group and each experimental group. It is a figure showing a CD31 stained image of an oral administration control group and each experimental group. It is the graph which compared the microvascular area (MVA; microvessel area) of the oral administration control group and CD of the CD31 staining. It is the figure which showed the staining image by meca-32 of the oral administration control group and each experimental group. It is the graph which compared the microvascular area (MVA) of the oral administration control group and the experimental group which were stained with meca-32. The experimental group was injected into the rabbit VX2 liver cancer model by hepatic artery chemoembolization (TACE), and it is a graph comparing the viable part (viable portion) of the tumor (A: Lipiodol administration group, B: Lipiodol + VDA administration) Group C: Lipiodol + doxorubicin administration group D: Lipiodol + VDA + doxorubicin administration group)

  In the present invention, pharmaceutically acceptable salts mean salts commonly used in the pharmaceutical industry, for example, inorganic ion salts prepared with calcium, potassium, sodium or magnesium, etc .; hydrochloric acid, nitric acid, phosphoric acid, Inorganic acid salts prepared with bromic acid, iodic acid, perchloric acid or sulfuric acid etc .; acetic acid, trifluoroacetic acid, citric acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, mandelic acid, propione Organic acid salts prepared with acid, lactic acid, glycolic acid, gluconic acid, galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carboxylic acid or vanillic acid; methanesulfonic acid, ethanesulfonic acid, benzene Sulfonic acid produced with sulfonic acid, p-toluenesulfonic acid or naphthalenesulfonic acid Amino acids salts prepared with glycine, arginine, lysine etc .; or amine salts produced with trimethylamine, triethylamine, ammonia, pyridine, picoline etc, etc., but the salts mentioned in the present invention according to these salts mentioned The type is not limited.

  In the present invention, (S) -N- (4- (3- (1H-1,2,4-triazol-1-yl) -4- (3,4,5-trimethoxybenzoyl) phenyl) thiazole-2 The salt of (-yl) -2-amino-3-methylbutanamide may be the hydrochloride salt.

  In the present invention, the composition of the present invention may be applied for treatment of liver cancer or for treatment of liver cancer. Although the liver cancer includes both primary liver cancer or metastatic liver cancer, the composition of the present invention may be applied to primary liver cancer. The liver cancer also includes both hepatocellular carcinoma and cholagiocarcinoma, but the composition of the present invention may be applied to hepatocellular carcinoma. Also, the liver cancer may be early liver cancer, advanced liver cancer or end-stage liver cancer, and the composition of the present invention may be applied to advanced liver cancer.

  The pharmaceutical composition of the present invention can be formulated in unit volume using a pharmaceutically acceptable carrier according to a method that can be easily implemented by those skilled in the art to which the present invention belongs. It may be manufactured in the form or in a multi-volume container.

  The pharmaceutically acceptable carrier is generally used for formulation, and includes lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginic acid, gelatin, calcium silicate, microcrystalline cellulose Polyvinyl pyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil (mineral oil) and the like, but it is not limited thereto. The pharmaceutical composition of the present invention may further comprise, in addition to the above-mentioned components, lubricants, wetting agents, sweetening agents, flavoring agents, emulsifying agents, suspending agents, preservatives and the like. Pharmaceutically acceptable compatible carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

  The present invention provides a method of treating liver cancer comprising administering the pharmaceutical composition of the present invention to an individual in need thereof. In the present invention, the term "individual" includes mammals, in particular humans.

  According to one embodiment of the present invention, the present invention provides a combination of a vascular disrupting agent and sorafenib.

Specifically, the present invention provides (1) (S) -N- (4- (3- (1H-1,2,4-triazol-1-yl) -4- (4) represented by the following chemical formula 1) 3,4,5-Trimethoxybenzoyl) phenyl) thiazol-2-yl) -2-amino-3-methylbutanamide or a pharmaceutically acceptable salt thereof, and (2) sorafenib represented by the following chemical formula 2 Or a combination for the treatment of liver cancer comprising a pharmaceutically acceptable salt thereof.

  Antiangiogenic agents (Antiangiogenic agents) have the effect of suppressing tumor angiogenesis, and have traditionally been widely used in anti-cancer strategies. Sorafenib (sorafenib), developed by Bayer, is known to have two properties: direct anti-cancer effect and anti-angiogenic. The anti-cancer effect of sorafenib is partially mediated through the inhibition of B-RAF kinase and appears to have anti-angiogenic properties by the inhibition of VEGFR-2 and PDGFR-β. However, it has been revealed that when an anti-angiogenic agent such as sorafenib is treated alone, only one aspect of tumor angiogenesis is blocked, so that the anti-cancer effect is insufficient.

  However, in the combination of the present invention, when the compound of the compound formula (vasculolytic agent) which is an anticancer agent of another therapeutic mechanism and sorafenib (angiogenesis inhibitor) are co-administered, the synergistic complementation effect is very important. It was confirmed to have excellent liver cancer therapeutic activity. Furthermore, since there are few viable tumors, it is also possible to solve the tumor recurrence problem that occurs when a single compound of Formula 1 or sorafenib is administered.

  In the present invention, as pharmaceutically acceptable salts of sorafenib, all salts commonly used in the pharmaceutical industry can be used, and p-toluenesulfonate can be used.

  The combination of the present invention may comprise two separate formulations and may consist of a single formulation.

  The combination of the present invention can be administered orally or parenterally (for example, intravenously, subcutaneously, intraperitoneally or topically) depending on the intended method. In the present invention, the (S) -N- (4- (3- (1H-1,2,4-triazol-1-yl) -4- (3,4,5-trimethoxybenzoyl) phenyl) thiazole-described above. 2-yl) -2-amino-3-methylbutanamide or a pharmaceutically acceptable salt thereof may be administered parenterally or orally, preferably orally. Also, the sorafenib or pharmaceutically acceptable salt thereof may be orally administered.

  In the combination of the present invention, the suitable dose of the active ingredient may vary depending on the patient's weight, age, sex, health condition, diet, administration time, administration method, excretion rate and disease severity etc. is there. (S) -N- (4- (3- (1H-1,2,4-triazol-1-yl) -4- (3,4,5-trimethoxybenzoyl) phenyl) thiazole-2-thiazole of the present invention The daily dose of yl) -2-amino-3-methylbutanamide or a pharmaceutically acceptable salt thereof is about 1.0 to 40.0 mg, preferably 2.5 to 25.0 mg. Also, the daily dose of sorafenib or a pharmaceutically acceptable salt thereof of the present invention is about 50 to 1500.0 mg, preferably 100 to 1000 mg.

  Also, in the combination of the present invention, the adapted administration cycle of the active ingredient may be determined according to the dose. (S) -N- (4- (3- (1H-1,2,4-triazol-1-yl) -4- (3,4,5-trimethoxybenzoyl) phenyl) thiazole-2-thiazole of the present invention I) -2-amino-3-methylbutanamide or a pharmaceutically acceptable salt thereof may be administered once a day to once a week, once a week depending on the route of administration Five doses may be administered. In addition, sorafenib or a pharmaceutically acceptable salt thereof of the present invention may be administered twice a day to once a week, and may be administered once to seven times a week depending on the administration route. It may well be administered once or twice daily.

  According to one embodiment of the present invention, the combination according to the invention is (S) -N- (4- (3- (1H-1,2,4-triazol-1-yl) -4- (3, When 4,5-trimethoxybenzoyl) phenyl) thiazol-2-yl) -2-amino-3-methylbutanamide or a pharmaceutically acceptable salt thereof is administered parenterally, the administration cycle is one week. , (S) -N- (4- (3- (1H-1,2,4-triazol-1-yl) -4- (3,4,5-trimethoxybenzoyl) phenyl) thiazol-2-yl) -2-Amino-3-methylbutanamide or its pharmaceutically acceptable salt is administered once daily on the first day, sorafenib or pharmaceutically acceptable salt thereof is given on the first day to the sixth day every week It may be administered once daily to the eyes.

  According to another embodiment of the present invention, the combination of the present invention is (S) -N- (4- (3- (1H-1,2,4-triazol-1-yl) -4- (3) (4,5-trimethoxybenzoyl) phenyl) thiazol-2-yl) -2-amino-3-methylbutanamide or a pharmaceutically acceptable salt thereof is orally administered (S) -N- ( 4- (3- (1H-1,2,4-triazol-1-yl) -4- (3,4,5-trimethoxybenzoyl) phenyl) thiazol-2-yl) -2-amino-3-methyl Butanamide or its pharmaceutically acceptable salt is administered once a week (three times a week) on Days 1, 3 and 5 weekly, and Sorafenib or a pharmaceutically acceptable salt thereof once a day (weekly) 7) may be administered. Also according to one embodiment of the present invention, (S) -N- (4- (3- (1H-1,2,4-triazol-1-yl) -4- (3,4,5-tri) 20 minutes to 1 hour after oral administration of methoxybenzoyl) phenyl) thiazol-2-yl) -2-amino-3-methylbutanamide or a pharmaceutically acceptable salt thereof, sorafenib or pharmaceutically Acceptable salts can be administered.

  Also, the combination of the present invention has a very good liver cancer therapeutic activity and there are very few viable cancer cells, so the possibility of cancer recurrence is extremely low. Therefore, the combination of the present invention can be usefully used for liver cancer treatment.

  According to another embodiment of the present invention, the present invention provides a composition for chemical embolization comprising a blood vessel destroying agent.

Specifically, the present invention provides (1) (S) -N- (4- (3- (1H-1,2,4-triazol-1-yl) -4- (4) represented by the following chemical formula 1) 3,4,5-Trimethoxybenzoyl) phenyl) thiazol-2-yl) -2-amino-3-methylbutanamide or a pharmaceutically acceptable salt thereof, and (2) doxorubicin represented by the following chemical formula 3 Provided is a composition for chemical embolization comprising (doxorubicin) or a pharmaceutically acceptable salt thereof, and (3) lipiodol.

  Two of the video-guided treatments applied to liver cancer are representative, but one is a treatment method in which an anticancer agent is locally injected into the liver cancer via the hepatic artery and parallel embolization to the hepatic artery is performed. Hepatic artery chemoembolization (TACE), and the other is radiofrequency ablation (RFA).

  The above-mentioned hepatic artery chemoembolization (TACE) uses a strategy that maximizes the local anticancer effect on liver cancer by injecting an anticancer drug into the hepatic artery and blocking the hepatic artery using an embolic substance, and many In the case of palliative therapy (palliative therapy). Because normal liver tissue supplies over 70% of the blood through the portal vein, liver cancer tissue supplies over 90% of the blood through the hepatic artery, so hepatic artery chemoembolization (TACE) Therefore, when anticancer agents are injected through the hepatic artery, relatively high concentrations of anticancer agents can be administered to liver cancer tissues as compared to normal liver tissues, and in particular, direct injection through such blood vessels is The amount of anticancer agent accumulated in liver cancer tissue can be increased by about 100 times compared to systemic injection. Nevertheless, one of the main causes of recurrence is the formation of new blood vessels around liver cancer tissue in a reaction to overcome hypoxia induced by chemoembolization. It has become clear that

  However, in the composition for chemical embolization of the present invention, a synergistic effect is obtained when a combination of a compound of the formula 1 (vasculolytic agent), which is an anticancer agent with another therapeutic mechanism, and doxorubicin is used in combination for topical administration to liver cancer cells. It shows very good liver cancer therapeutic activity. Furthermore, since there are few viable tumors, it is also possible to solve the tumor recurrence problem that occurs when a single compound of Formula 1 or doxorubicin is administered.

  In the chemical embolic composition of the present invention, lipiodol plays various roles. Specifically, lipiodol plays a role in delivering an anticancer agent in a composition for chemical embolization to liver cancer cells. Lipiodol also plays a role as a contrast agent. In addition, since lipiodol has an embolism function to the hepatic artery, it can enhance the embolic effect of an embolic substance such as gelatin. Therefore, in the composition for chemical embolization of the present invention, lipiodol transmits the anticancer agent of the present invention to liver cancer cells, functions as a contrast agent to enable the treatment of chemical embolization, and exerts an embolic effect as well. Help treat liver cancer.

  The composition for chemical embolization of the present invention may further comprise an aqueous contrast agent. Doxorubicin is a hydrophilic substance and is difficult to dissolve in lipiodol. Thus, it is preferred to further include an aqueous contrast agent to dissolve doxorubicin.

  According to an embodiment of the present invention, in the composition of the present invention (S) -N- (4- (3- (1H-1,2,4-triazol-1-yl) -4- (3,4) 5,6-Trimethoxybenzoyl) phenyl) thiazol-2-yl) -2-amino-3-methylbutanamide or a pharmaceutically acceptable salt thereof and a weight ratio of doxorubicin or a pharmaceutically acceptable salt thereof May be 5: 1 to 1: 5. Preferably, it may be 2: 1 to 1: 2.

  According to another embodiment of the present invention, the (S) -N- (4- (3- (1H-1,2,4-triazol-1-yl) -4- (3,4,5-triol) is Preferably, 5.0 to 30.0 mg of methoxybenzoyl) phenyl) thiazol-2-yl) -2-amino-3-methylbutanamide or a pharmaceutically acceptable salt thereof is contained per 1 mL of lipiodol.

  According to yet another embodiment of the present invention, the doxorubicin or pharmaceutically acceptable salt thereof is preferably contained in an amount of 5.0 to 50.0 mg per mL of the aqueous contrast agent.

  The composition for chemical embolization of the present invention can be used for various chemical embolization. Therefore, the composition for chemical embolization of the present invention can locally administer the embolism to a desired site, and is most preferably locally administered to the liver by hepatic artery chemoembolization (TACE).

  In addition, the composition for chemical embolization of the present invention has extremely excellent liver cancer therapeutic activity, in particular, and since there are few viable cancer cells, the possibility of cancer recurrence is extremely low. Therefore, the composition for chemoembolization of the present invention can be usefully used for the treatment of liver cancer.

  After injecting the chemical embolic composition of the present invention, the embolic material can be injected to embolize at the desired site. The embolic material is preferably in the form of gelatin.

  In the composition for chemical embolization of the present invention, the aforementioned (S) -N- (4- (3- (1H-1,2,4-triazol-1-yl) -4- (3,4,5-triol) is mentioned. A compatible dose of methoxybenzoyl) phenyl) thiazol-2-yl) -2-amino-3-methylbutanamide or a pharmaceutically acceptable salt thereof and doxorubicin or a pharmaceutically acceptable salt thereof is The range varies depending on the weight, age, sex, health condition, diet, administration time, administration method, excretion rate, severity of disease, etc. of the patient. (S) -N- (4- (3- (1H-1,2,4-triazol-1-yl) -4- (3,4,5-trimethoxybenzoyl) phenyl) thiazole-2-thiazole of the present invention The daily dosage of (I) -2-amino-3-methylbutanamide or a pharmaceutically acceptable salt thereof is about 1.0 to 40.0 mg / kg, preferably 2.5 to 25.0 mg It is / kg. In addition, the daily dose of doxorubicin or a pharmaceutically acceptable salt thereof of the present invention is about 10.0 to 1000.0 mg / kg, preferably 20.0 to 800.0 mg / kg.

  Hereinafter, the configuration and effects of the present invention will be described in more detail through examples. These examples are merely for illustrating the present invention, and the scope of the present invention is not limited by these examples.

Example 1 Confirmation of Synergistic Effect of Parenteralally Administered Compound of Formula 1 (VDA) and Sorafenib experimental method
Preparation of cell line and infection of Hep3B cells Hep3B, which is a human hepatocellular carcinoma cell, was purchased from Korea Cell Line Bank (Seoul, Korea). Hep3B was made infected with a lentivirus containing the firefly luciferase reporter gene and clones expressing the reporter strongly were isolated to establish the Hep3B + luc cell line.

  Stable expression of luciferase (Luciferase) was confirmed by Bioluminescence Imaging (BLI) (PerkinElmer, Waltham, Mass., USA). The Hep3B + luc cell line is Dulbecco's Modified Eagle's Medium (Dulbecco's) containing 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA) and 1% antibiotics (Gibco, USA) Maintained in modified Eagle medium (DMEM, Welgene Inc, Seoul, Korea).

Preparation of animal tumor model Male Balb / c nude mice (6 weeks old) were purchased from Orient Bio (Seoul, Korea).

1 × 10 ⁶ Hep3B + luc cells were inoculated into the right flank of each animal via a 27-gauge needle syringe. When the tumor diameter exceeded 5 mm at about 14 days after inoculation, it was used for the experiment.

Preparation of Active Ingredient The compound of Formula 1 (VDA) was prepared by the method described in International Patent Publication WO 2009-119980, and was prepared by dissolving in 0.9% saline.

  Dissolve sorafenib p-toluenesulfonate (LC laboratories, Woburn, Mass., USA) in Cremophor EL / ethanol (50:50; Sigma Cremophor EL, 95% ethyl alcohol) at 4 times the concentration used and then dilute with water. Prepared on the day of use.

Observation of in vivo bioluminescence imaging
Bioluminescence imaging (BLI) was performed before drug administration (Day 0), 2 days after drug administration (Day 2), 6 days after (Day 6), 10 days after (Day 10) and 14 days after (Day 14). After anesthetizing the mice, images were obtained with an IVIS spectrum system (PerkinElmer, Waltham, Mass., USA). Mice were injected intraperitoneally with D-luciferin (0.375 mg / ml, Promega, Madison, Wis., USA) dissolved in phosphate buffered saline (PBS). Bioluminescence was reported as the sum of measured photons per second at a given region of interest (ROI). For measurement of in vivo bioluminescence, ROIs having the same size and shape, including the tumor of each animal, were used. Quantification of BLI data was performed by Living Image software (version 2.50.1, Perkin Elmer, Waltham, Mass., USA).

Confirmation of Liver Cancer Therapeutic Activity The anti-cancer effect of the compound of Formula 1 and sorafenib was evaluated in the Hep3B xenograft model. The experimental groups were randomly divided into the following groups.

  Control group: vehicle (saline, intraperitoneal injection).

  -Compound of the formula 1 (VDA) alone administration group: compound of the formula 1 (5 mg / kg, once weekly, intraperitoneally administered on the first day)

  -Sorafenib alone administration group: Sorafenib (30 mg / kg, 6 times a week, orally administered once daily from day 1 to day 6)

  -Combined administration group: Compound of chemical formula 1 (5 mg / kg, once a week, intraperitoneally administered on day 1), sorafenib (30 mg / kg, 6 times a week, orally administered once daily from day 1 to 6) )

  All experimental groups were dosed for 2 weeks. Toxicity was observed by measuring the body weight of the mice, and tumor growth was measured with calipers during the experimental period. Tumor volume was calculated by the following formula.

Tumor volume = (length × width ² ) / 2

Histopathological study (Histopathological studies)
At the end of the experiment (Day 14) all mice were sacrificed for tissue processing, immunohistochemical staining and histological analysis.

In tissue processing, experimental animals were deeply anesthetized and transcardially perfused with saline through a ventricular catheter and then treated with 10% paraformaldehyde (PFA) for 10 minutes by gravity flow. Tumor tissues were collected, fixed in formalin and embedded in paraffin. The largest part of the tumor was cut to a thickness of 4 μm.
Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) using a kit (Millipore, Billerica, Mass., USA) to assess the extent of tumor apoptosis. Staining was performed. Hematoxylin (Hematoxylin) and eosin (H & E) staining was performed on representative areas of harvested tumor tissue. The ratio of apoptotic sites was calculated using the software (NIH-Image J, Bethesda, MD, USA).

  To assess changes in tumor blood vessels, immunohistochemical staining of CD31 was performed using a primary rabbit antibody (Abcam, Cambridge, MA) to mouse CD31. Visualization was performed for 30 minutes using DakoEnVision + System HRP labeled polymer anti-rabbit (Dako). Images were observed with a light microscope (model DM2500, leica, Germany).

  Measurement of the immunostained area was performed based on the Weidner method. Three areas were observed at low magnification (40 × and 100 ×) using a Leica microscope (model DM2500, leica, Germany). The number of microvessels in each area was measured in the area with a 200 × field (20 × objective, 10 × eyepiece). Image analysis on the number of blood vessels was performed using software (Image analysis, Leica, Germany). In this software, one cell in a stained lumen was calculated as one point. The mean number of microvessels in all measured areas was determined as microvessel density (MVD).

Statistical analysis
Quantitative data were expressed as mean ± standard error. Statistical significance of differences in tumor volume, bioluminescence imaging, apoptosis rate, and microvessel density between each group was analyzed using Mann-Whitney's U test. A p-value less than 0.05 was considered significant. All data were calculated using commercial software (SPSS, version 21.0 for Windows, SPSS, Chicago, IL, USA).

  2. Experimental result

Liver cancer therapeutic activity of compound of formula 1 (VDA) in Hep3B xenograft model
To evaluate the anti-tumor effect of Compound 1 (VDA) alone, xenograft mice were administered Compound 1 (VDA), and body weight and tumor growth were observed during the experimental period. No weakened mice were observed during the dosing period. The change in body weight was not significant (p> 0.05) before and 14 days after administration in the compound of the Formula 1 (VDA) administration group, the sorafenib administration group, and the combination administration group. In terms of tumor growth inhibitory effect, the combination administration group was similar to sorafenib alone administration group. This suggests that the tumor growth inhibitory effect of the compound of the formula 1 (VDA) alone administration group is not significant.

During the BLI signal observation experiment, the relative BLI signal in the control group continued to increase. This shows strong growth of Hep3B-luc tumor. The BLI signal in the sorafenib single dose group gradually decreased for 6 days and recovered significantly after 6 days. The relative BLI signal in the compound of the Formula 1 (VDA) administration group decreased in the initial stage after the administration of the compound of the Formula 1 (Day 1 and Day 8), which persisted for 2 days and recovered significantly after 2 days. This suggests that the compound of formula 1 exhibits rapid vascular shut down and cytotoxic effects at an early stage after administration. However, in the combination administration group, the relative BLI signal was significantly reduced in the early stage of treatment, and showed a tumor suppression effect for 10 days after administration (see FIG. 1).

  Also, the relative BLI signal strength matches the result of the BLI signal (see FIG. 2). These results suggest that the combination of the compound of formula 1 and sorafenib has a synergistic effect. Specifically, the compound of formula 1 induces a rapid therapeutic effect, and sorafenib maintains a tumor suppressive effect.

Correlation Between BLI Parameters and Histological Results A series of histopathological studies were performed to confirm the results of the BLI signal. Tumor apoptosis area was quantified using TUNEL assay 14 days after administration. As a result, it is the highest in the combined administration group (43.0 ± 14.0), and then the compound alone administration group of the chemical formula 1 (38.0 ± 13.7) and the sorafenib alone administration group (18.3 ± 6.0) , In the order of the control group (11.1 ± 3.7). However, the result of apoptosis in the compound alone administration group of the formula 1 and the result of apoptosis in the combination administration group are not so different. This suggests that the effect of sorafenib on tumor cell growth is cytostatic rather than cytotoxic (see FIG. 4). In addition, as seen from the microscopic H & E staining and TUNEL staining, in the sorafenib single administration group and the control group, the tumor cells are scattered as a whole in the form of dots, whereas in the compound single administration group of the chemical formula 1, the tumor The cells are mainly in the form of being collected in apoptotic tumor cells. In addition, apoptosis in the combined administration group showed a level similar to that in the compound alone administration group of the chemical formula 1 (see FIG. 3). From these results, it was confirmed that the tumor-disrupting effect of the compound of Formula 1 shows a broad spectrum of apoptosis, and that the tumor growth-suppressing property of sorafenib exhibits a tumor growth-suppressing effect.

  Also, quantification of anti-CD31 stained histological specimens and tumor microvessel density (MVD) was obtained 14 days after a single dose of drug. Tumor blood vessels in the control group were abundant and distributed throughout the tumor. However, the experimental group showed a blood vessel image different from that of the control group. Specifically, the blood vessels in the experimental group had a short length and were distributed at the rim of the tumor. And the comparison between the experimental groups was based on the density of blood vessels (see FIG. 5). For quantification of tumor MVD, MVD is highest in the saline group (3180 ± 465), followed by the compound alone administration group of chemical formula 1 (2334 ± 60), sorafenib alone administration group (1871 ± 225), combined administration group The order was (1122 ± 212) (see FIG. 6). Statistical significance was observed in all experimental groups. Also from these results, it can be confirmed that the combination of the vascular destruction property of the compound of formula 1 and the anti-angiogenic property of sorafenib exhibits a synergistic effect.

Example 2 Confirmation of Synergistic Effect of Orally Administered Compound of Formula 1 (VDA) and Sorafenib Experimental Methods The preparation of cell lines, tumor models of animals and active ingredients was carried out as in Example 1.

Observation of in vivo bioluminescence imaging
Bioluminescence imaging (BLI) is before drug administration (Day 0), 2 days after drug administration (Day 2), 4 days after (Day 4), 8 days after (Day 8), 11 days after (Day 11), 15 days after (Day 15), 18 days after Day 18), 20 days later (Day 20). After anesthetizing the mice, images were obtained with an IVIS spectrum system (PerkinElmer, Waltham, Mass., USA). Mice were injected intraperitoneally with D-luciferin (1.5 mg / ml, Promega, Madison, Wis., USA) dissolved in phosphate buffered saline (PBS). Bioluminescence was reported as the sum of measured photons per second in a given region of interest (ROI). For measurement of in vivo bioluminescence, an ROI having the same size and shape, including a tumor of each animal, was used. Quantification of BLI data was performed by Living Image software (version 2.50.1, Perkin Elmer, Waltham, Mass., USA).

Confirmation of Liver Cancer Therapeutic Activity The anti-cancer effect of the compound of Formula 1 and sorafenib was evaluated in the Hep3B xenograft model. The experimental groups were randomly divided into the following different administration groups.

  Control group: vehicle (saline, orally administered).

Compound (VDA) alone administration group of chemical formula 1: compound of chemical formula 1 (4 mg / kg, orally administered three times a week on day 1, day 3, day 5)

-Sorafenib alone administration group: Sorafenib (15 mg / kg, orally administered 7 times a week)

  -Combined administration group: Compound of Chemical Formula 1 (4 mg / kg, orally administered three times a week on the first, third, and fifth days), sorafenib (15 mg / kg, orally administered once daily seven times weekly)

  Oral administration was performed at 2 pm every day, and in the combination administration group, sorafenib was dosed 30 minutes after administration of the compound of formula 1.

  All experimental groups were dosed for 3 weeks. Toxicity was observed by measuring the body weight of the mice, and tumor growth was measured with calipers during the experimental period. Tumor volume was calculated by the following formula.

Tumor volume = (length × width ² ) / 2

Histopathological study (Histopathological studies)
At the end of the experiment (Day 21) all mice were sacrificed for tissue processing, immunohistochemical staining and histological analysis.

  Treatment of the excised tumor tissue was performed according to the method described in Example 1.

  Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) using a kit (Millipore, Billerica, Mass., USA) to assess the extent of tumor apoptosis. Staining was performed. Hematoxylin (Hematoxylin) and eosin (H & E) staining was performed on representative areas of harvested tumor tissue. The ratio of apoptotic sites was calculated using the software (NIH-Image J, Bethesda, MD, USA).

  Immunohistochemical staining using primary rabbit antibody (Abcam, Cambridge, Mass.) Against mouse CD31 and primary rat antibody against meca-32 (Novus Biologicals, Littleton, Colorado, USA) to assess changes in tumor blood vessels Did. Color development of CD31 was performed for 30 minutes by DakoEnVision + System HRP labeled polymer anti-rabbit (Dako). Images were observed with a light microscope (model DM2500, leica, Germany). For meca-32, observation was performed using Alexa Fluor 488 anti-rat antibody.

  Microvessel area (MVA) was assessed by immunochemical staining of CD31 and meca-32. Three areas were observed at low magnification (40 × and 100 ×) using a Leica microscope (model DM2500, leica, Germany). The number of microvessels in each area was measured in the area with a 200 × field (20 × objective, 10 × eyepiece). Image analysis on the number of blood vessels was performed using software (Image analysis, Leica, Germany).

Statistical analysis

  Quantitative data were expressed as mean ± standard error. The tumor volume, bioluminescence imaging (bioluminescence imaging) between each group were evaluated by repeated measures analysis of variance (RMANOVA) test, and the statistical significance of differences in apoptosis rate and microvessel area was analyzed by one-way analysis of variance (one- The analysis was performed using a way analysis of variance (abbreviation: one-way ANOVA) test. A p-value less than 0.05 was considered significant. All data were calculated using commercial software (SPSS, version 21.0 for Windows, SPSS, Chicago, IL, USA).

2. Experimental result
BLI signal observation

  During the experimental period, the relative BLI signal in the control group increased to day 11, then traffic congestion at that level was observed, and in the second half of the dosing period it was observed that the signal intensity in the central part of the cancer decreased. This is believed to be due to natural necrosis that occurs as the cancer grows (see FIG. 7).

  In the compound administration group of the chemical formula 1, it was observed that the signal intensity decreased rapidly until day 2 and then increased. In the sorafenib administration group, it was observed that the signal intensity gradually dropped over the entire administration period. In the combination group, the signal intensity decreased rapidly, and the decreased condition persisted throughout the entire dosing period (see FIG. 8).

  From these results, it is understood that the compound of Formula 1 induces a rapid therapeutic effect and sorafenib maintains the tumor suppressive effect, as in the experimental results of Example 1. In addition, when the compound of Formula 1 is orally administered, it is found that the same excellent effects as in the case of administration by injection are exhibited.

Correlation Between BLI Parameters and Histological Results A series of histopathological studies were performed to confirm the results of the BLI signal. Tumor apoptosis area was quantified using TUNEL assay 21 days after administration. As a result, the level of apoptosis is highest in the combination group (34.18 ± 5.99), followed by the compound single administration group of the formula 1 (16.24 ± 6.16), and the sorafenib single administration group (10.36 ±). 3.62), the control group (3.74 ± 2.08) in that order (see FIG. 9).

  In addition, anti-CD31 stained histological specimens (Figure 10) and their quantification of MVA in tumors (Figure 11), and meca-32 stained histological specimens (Figure 12) and their quantification of tumors (Figure 12) Figure 13) was obtained 21 days after administration of each drug. According to MVA analysis using CD31, the tumor MVA is (16339 ± 3614), (14452 ± 2709) and (14934 ± 3154) in the control group, the compound alone administration group of the chemical formula 1 and the sorafenib alone administration group, respectively. While the combination administration group shows (7161 ± 4522), and it was confirmed that the MVA was significantly lower than all the other groups (see FIG. 11).

  According to MVA analysis using meca-32, the MVD of the tumor is highest in the control group (17183 ± 5508), followed by sorafenib alone (14897 ± 3556), and compound 1 alone (12871 ± 3433). The order was the lowest in the combination administration group (7631 ± 2872) (see FIG. 13).

From these results, it can be seen that the combination of the vascular destruction property of the compound of Formula 1 and the anti-angiogenic property of sorafenib exhibits a synergistic effect.
In addition, differences in blood vessel morphology in each group were observed. Specifically, in the compound administration group of the chemical formula 1, the number of blood vessels decreased and the lumen of blood vessels narrowed (see FIGS. 10 and 12). This is a good example of the angiolytic activity of compounds of Formula 1. Furthermore, the combination of the compound of the formula 1 and sorafenib having antiangiogenic effect is considered to exhibit a synergistic effect by changing the number and form of blood vessels in liver cancer.

<Example 3> Confirmation of liver cancer therapeutic effect and hepatotoxicity of the composition for chemical embolization of the present invention Experimental Method The rabbit VX2 carcinoma liver tumor model (Rabbit VX2 carcinoma liver tumor model) established in a representative animal model of liver cancer was used. After incision of the abdomen of a rabbit weighing 3 kg (New Zealand White, male) to expose the left lobe of the liver, injection of a tumor chip (tumor chip) of VX2 carcinoma prepared for 1 mm ³ in size is performed. It was implanted on the liver surface using a needle. Non-contrast CT and perfusion MR were performed 2-3 weeks after tumor implantation to confirm the presence or absence of tumor formation.

Setting of Experiment Group The experiment group of 10 animals was randomly divided into the following experiment groups A to D.

  -Experimental group A: Lipiodol administration group (Lipiodol 0.2 mL + contrast agent [GE healthcare Visipaque 270] 0.05 mL)

  -Experimental group B: Lipiodol / compound of formula 1 (VDA) administration group (lipiodol 0.2 mL / compound of formula 1 (VDA) 1.26 mg + contrast agent [GE healthcare Visipaque 270] 0.05 mL)

  -Experimental group C: Lipiodol / doxorubicin administration group (Lipiodol 0.2 mL + contrast agent [GE healthcare Visipaque 270] 0.05 mL / 1 mg doxorubicin)

  Experimental group D: Lipiodol / compound of formula 1 (VDA) / doxorubicin administration group (lipiodol 0.2 mL / compound of formula 1 (VDA) 1.26 mg + imaging agent [GE healthcare care Visipaque 270] 0.05 mL / doxorubicin 1 mg)

Drug administration After the microcatheter (microcatheter) is advanced to the left hepatic artery through the celiac trunk accessed through the auricular artery of the rabbit ear, the experimental group A The drug of ~ D was carefully injected. Before and after TACE, non-enhanced CT was performed to evaluate presence or absence of a tumor and lipiodol deposited on the tumor after TACE.

2. Experimental result
Confirmation of Liver Cancer Therapeutic Effect One week after administration, liver tissue was removed to prepare a pathological slide. H & E staining and TUNEL staining were performed to confirm the site of tumor necrosis. The necrotic tumor area was calculated using the Image J program to express the viable site of the tumor as a percentage. The results are shown in Table 1 below and FIG.

  As shown in Table 1 above, in the case of experimental group A administered only lipiodol without anticancer activity, the viable tumor site was 47.06%, and lipiodol and the compound of formula 1 (VDA) were coadministered In the case of the experimental group B, the viable tumor site was 27.48%, and in the case of the experimental group C coadministered with lipiodol and doxorubicin, it was confirmed that the viable tumor site was only 14.36%. . From these results, it is understood that experimental groups A to C have a high possibility of cancer recurrence. On the other hand, in the case of experimental group D coadministered with lipiodol, the compound of chemical formula 1 (VDA) and doxorubicin, since the viable tumor site was 0.66% and most of the tumor was removed, the recurrence of cancer was observed It was confirmed to be significantly lower than Groups A-C.

  In addition, although experimental groups A to C have poor reproducibility of the tumor treatment effect so that the standard deviation of the results exceeds 10%, experimental group D has only about 1% of the standard deviations of the results and has good reproducibility. He was treated for liver cancer.

Toxicity confirmation

  Blood was collected before drug injection, 1 day after drug injection, 3 days after drug injection, and 7 days after drug injection to measure AST and ALT, which can evaluate the degree of acute liver injury.

  As a result, it was shown that the degree of acute liver injury in the experimental group D was the highest, but recovered to a level similar to that in the other experimental groups after 7 days. Furthermore, no death of the rabbit was observed during the experiment, which confirms that the liver damage in experimental group D is tolerable.

Claims (22)

(S) -N- (4- (3- (1H-1,2,4-triazol-1-yl) -4- (3,4,5-trimethoxybenzoyl) phenyl) represented by the following chemical formula 1 A pharmaceutical composition for the treatment of liver cancer or for assisting the treatment of liver cancer, which comprises thiazol-2-yl) -2-amino-3-methylbutanamide or a pharmaceutically acceptable salt thereof.
  (S) -N- (4- (3- (1H-1,2,4-triazol-1-yl) -4- (3,4,5-trimethoxybenzoyl) phenyl) thiazol-2-yl)- The pharmaceutical composition according to claim 1, wherein the pharmaceutically acceptable salt of 2-amino-3-methylbutanamide is a hydrochloride salt.   The pharmaceutical composition according to claim 1, wherein the liver cancer is primary liver cancer.   The pharmaceutical composition according to claim 1, wherein the liver cancer is advanced liver cancer.   The pharmaceutical composition according to claim 1, wherein the liver cancer is hepatocellular carcinoma (HCC). (1) (S) -N- (4- (3- (1H-1,2,4-triazol-1-yl) -4- (3,4,5-trimethoxybenzoyl) represented by the following chemical formula 1 ) Phenyl) thiazol-2-yl) -2-amino-3-methylbutanamide or a pharmaceutically acceptable salt thereof;
(2) Sorafenib represented by the following chemical formula 2 or a pharmaceutically acceptable salt thereof:
Combinations for the treatment of liver cancer, including   7. A combination according to claim 6, wherein the pharmaceutically acceptable salt of sorafenib is p-toluenesulfonate.   (S) -N- (4- (3- (1H-1,2,4-triazol-1-yl) -4- (3,4,5-trimethoxybenzoyl) phenyl) thiazol-2-yl) The combination according to claim 6, wherein -2-amino-3-methylbutanamide or a pharmaceutically acceptable salt thereof is administered parenterally or orally.   7. A combination according to claim 6, wherein the sorafenib or pharmaceutically acceptable salt thereof is administered orally.   (S) -N- (4- (3- (1H-1,2,4-triazol-1-yl) -4- (3,4,5-trimethoxybenzoyl) phenyl) thiazol-2-yl) 7. The combination according to claim 6, wherein -2-amino-3-methylbutanamide or a pharmaceutically acceptable salt thereof is administered once to five times weekly.   The combination according to claim 6, wherein the sorafenib or pharmaceutically acceptable salt thereof is administered once to seven times weekly.   The combination according to claim 11, wherein the sorafenib or a pharmaceutically acceptable salt thereof is administered once or twice daily.   (S) -N- (4- (3- (1H-1,2,4-triazol-1-yl) -4- (3,4,5-trimethoxybenzoyl) phenyl) thiazol-2-yl) -2-amino-3-methylbutanamide or its pharmaceutically acceptable salt is administered once to five times weekly, and the sorafenib or pharmaceutically acceptable salt thereof is administered once to seven times weekly 7. A combination according to claim 6, which is   (S) -N- (4- (3- (1H-1,2,4-triazol-1-yl) -4- (3,4,5-trimethoxybenzoyl) phenyl) thiazol-2-yl)- When 2-amino-3-methylbutanamide or a pharmaceutically acceptable salt thereof is administered parenterally, the administration cycle is one week, and (S) -N- (4- (3- (1- (1H-1) , 2,4-triazol-1-yl) -4- (3,4,5-trimethoxybenzoyl) phenyl) thiazol-2-yl) -2-amino-3-methylbutanamide or pharmaceutically acceptable thereof 7. The combination according to claim 6, wherein once a week one salt is administered on the first day of the week, sorafenib or a pharmaceutically acceptable salt thereof is administered once a day from the first day to the sixth day of the week. .   (S) -N- (4- (3- (1H-1,2,4-triazol-1-yl) -4- (3,4,5-trimethoxybenzoyl) phenyl) thiazol-2-yl)- When 2-amino-3-methylbutanamide or a pharmaceutically acceptable salt thereof is orally administered, (S) -N- (4- (3- (1H-1,2 4-triazole-1-) Weekly) 1, 3 and pharmaceutically acceptable salts thereof, and (iii) 4- (3,4,5-trimethoxybenzoyl) phenyl) thiazol-2-yl) -2-amino-3-methylbutanamide or The combination according to claim 6, which is administered once a day (3 times a week) and 5 times a day (7 times a week) sorafenib or a pharmaceutically acceptable salt thereof.   (S) -N- (4- (3- (1H-1,2,4-triazol-1-yl) -4- (3,4,5-trimethoxybenzoyl) phenyl) thiazol-2-yl)- 10. The method according to claim 9, wherein after oral administration of 2-amino-3-methylbutanamide or a pharmaceutically acceptable salt thereof, sorafenib or a pharmaceutically acceptable salt thereof is administered after 20 minutes to 1 hour. Combination of (1) (S) -N- (4- (3- (1H-1,2,4-triazol-1-yl) -4- (3,4,5-trimethoxybenzoyl) represented by the following chemical formula 1 ) Phenyl) thiazol-2-yl) -2-amino-3-methylbutanamide or a pharmaceutically acceptable salt thereof;
(2) doxorubicin represented by the following chemical formula 3 or a pharmaceutically acceptable salt thereof;
(3) With lipiodol (lipiodol),
And a composition for chemical embolization.   18. The composition of claim 17, further comprising an aqueous contrast agent.   (S) -N- (4- (3- (1H-1,2,4-triazol-1-yl) -4- (3,4,5-trimethoxybenzoyl) phenyl) thiazol-2-yl)- 18. The weight ratio of 2-amino-3-methylbutanamide or pharmaceutically acceptable salt thereof to doxorubicin or pharmaceutically acceptable salt thereof is 2: 1 to 1: 2. Composition as described.   (S) -N- (4- (3- (1H-1,2,4-triazol-1-yl) -4- (3,4,5-trimethoxybenzoyl) phenyl) thiazol-2-yl) The composition according to claim 17, wherein -2-amino-3-methylbutanamide or a pharmaceutically acceptable salt thereof comprises 5.0 to 30.0 mg per mL of lipiodol.   18. The composition of claim 17, wherein the doxorubicin or pharmaceutically acceptable salt thereof comprises 5.0 to 50.0 mg per mL of aqueous imaging agent.   The composition according to claim 17, used for hepatic artery chemoembolization (TACE).