KR101952805B1 - Compositions for treating liver cancer comprising a vascular disrupting agent - Google Patents

Abstract

The present invention relates to a process for the preparation of (S) -N- (4- (3- (1H-1,2,4-triazol-1-yl) -4- (3,4,5-trimethoxybenzoyl) -2-yl) -2-amino-3-methylbutanamide or a pharmaceutically acceptable salt thereof. The composition of the present invention shows excellent liver cancer treatment effect.

Description

[0001] The present invention relates to a composition for treating liver cancer,

The present invention relates to a composition for treating liver cancer including a blood vessel blocking agent (VDA) and a method for treating liver cancer using the same.

Liver cancer is the second most malignant tumor in Korea and the third most common malignant tumor in the world. The incidence of recurrence at other sites is more than 50% within 5 years even if 70% or more of patients with liver cancer can not undergo curative surgery and curative resection is performed. In addition, the probability of responding to systemic chemotherapy in advanced liver cancer is very low, within 10%.

The Vascular Disrupting Agent (VDA) is intended to rapidly and selectively block tumor vessels formed by selectively destroying the microtubules of the cytoskeleton of vascular endothelial cells, and induces ischemic necrosis of cells in the center of the tumor can do. Therefore, the vascular occlusion method using the vascular blocker (VDA) is emerging as a new anti-cancer strategy. Thus, the present applicant has developed a compound of the following formula (I) as an anti-vascular agent (International Patent Publication No. WO 2009-119980).

(I)

The compound of formula (I) is a tubulin polymerization-inhibitor having a dual action mechanism of apoptosis by rapid collapse of an already existing tumor vessel by microtubule destabilization and cell cycle arrest. However, when a vascular blocker (VDA) alone, including the compound of the above formula, is treated alone, there is a problem in that tumors can regrow quickly from a viable rim, and as a result, the therapeutic utility of such drugs is deteriorated .

Accordingly, the present inventors have made various studies to provide a composition and a treatment method for treating liver cancer, which is one of malignant tumors, by solving the problem of tumor regrowth while taking advantage of the advantage of an anti-cancer agent.

International Patent Publication No. WO 2009-119980

It is an object of the present invention to provide a composition for the treatment of liver cancer or a composition for the treatment of liver cancer containing a vascular blocker (VDA).

Another object of the present invention is to provide a combination for the treatment of liver cancer comprising vaso-occlusive agents (VDA) and sorapenib.

It is still another object of the present invention to provide a chemical color exclusive composition comprising a vascular blocker (VDA).

As a result of efforts of the present inventors to accomplish the above object, the present inventors have completed a pharmaceutical composition for the treatment of liver cancer or an adjuvant for the treatment of liver cancer containing a vascular blocker (VDA).

In the present invention, the compound used as the vasoconstrictor 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.

[Chemical Formula 1]

In the present invention, the pharmaceutically acceptable salt means a salt commonly used in the pharmaceutical industry. Examples of the salt include inorganic ion salts such as calcium, potassium, sodium or magnesium, hydrochloric acid, nitric acid, phosphoric acid, , Iodic acid, perchloric acid, sulfuric acid and the like; There may be mentioned acetic acid, trifluoroacetic acid, citric acid, maleic acid, succinic acid, benzoic acid, tartaric acid, fumaric acid, mandelic acid, propionic acid, lactic acid, glycolic acid, gluconic acid, galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, Organic acid salts prepared from acids, ascorbic acid, carbonic acid, or vanillyric acid; Methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, or naphthalenesulfonic acid; Amino acid salts prepared with glycine, arginine, lysine and the like; Or an amine salt prepared by using trimethylamine, triethylamine, ammonia, pyridine, picoline, or the like. However, the types of salts defined in the present invention are not limited by the listed salts.

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

In the present invention, the composition of the present invention can be applied for liver cancer treatment or liver cancer treatment. The liver cancer includes both primary liver cancer or metastatic liver cancer, but the composition of the present invention can be applied to primary liver cancer. In addition, the liver cancer includes all hepatocellular carcinoma or cholangiocarcinoma, but the composition of the present invention can be applied to hepatocellular carcinoma. In addition, 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 may be prepared in unit dosage form by formulating it using a pharmaceutically acceptable carrier according to a method which can be easily carried out by a person skilled in the art to which the present invention belongs, Into the container.

Such pharmaceutically acceptable carriers are those conventionally used in the field of manufacture and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, But are not limited to, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc., in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington ' s Pharmaceutical Sciences (19th ed., 1995).

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

In accordance with one embodiment of the present invention, the present invention provides a combination of a vascular blocker and sorapenib.

Specifically, the present invention provides: (1) A process for producing (S) -N- (4- (3- (1H-1,2,4-triazol- 2-amino-3-methylbutanamide or a pharmaceutically acceptable salt thereof, and (2) a salt of sorapenib or a salt thereof represented by the following formula (2) Or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of liver cancer.

[Chemical Formula 1]

(2)

Antiangiogenic agents have been shown to inhibit angiogenesis in tumors and have been traditionally used for anticancer strategies. Sorafenib, developed by Bayer, is known to have two properties, direct anti-cancer effect and neovascularization inhibition. The anticancer effect of sorafenib is partially mediated through the inhibition of B-RAF kinase and appears to have neovascular inhibition properties due to inhibition of VEGFR-2 and PDGFR-β. However, when the neovascularization inhibitor such as sorafenib is treated alone, it has been found that the anticancer effect is insufficient because it blocks only one side of tumor angiogenesis.

However, in the combination of the present invention, when the compound of the formula (anti-vascular agent) and the sorapenib (neovascularization inhibitor), which are anticancer agents of other therapeutic mechanisms, were administered together, . Furthermore, since there are few viable tumors, the problem of recurrence of tumors occurring when the compound of formula (I) alone or sorapenib is administered alone can be solved.

In the present invention, the pharmaceutically acceptable salt of sorapenib may be any salt commonly used in the pharmaceutical industry, and p-toluenesulfonate may be used.

The combination of the present invention may comprise two separate preparations and may be composed of one formulation.

The combination of the present invention can be administered orally or parenterally (for example, intravenously, subcutaneously, intraperitoneally or topically) according to the desired method. In the present invention, the (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 may be administered parenterally or orally, preferably orally. In addition, the sorafenib or a pharmaceutically acceptable salt thereof may be orally administered.

In the combination of the present invention, the appropriate dosage of the active ingredients varies depending on the patient's body weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and severity of disease. (S) -N- (4- (3- (1H-1,2,4-triazol-1-yl) -4- (3,4,5-trimethoxybenzoyl) phenyl) thiazole -2-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. In addition, the daily dose of sorapenib or a pharmaceutically acceptable salt thereof of the present invention is about 50 to 1500.0 mg, preferably 100 to 1000 mg.

Further, in the combination of the present invention, a suitable administration period of the above-mentioned effective ingredients may be determined depending on the dose. (S) -N- (4- (3- (1H-1,2,4-triazol-1-yl) -4- (3,4,5-trimethoxybenzoyl) phenyl) thiazole -2-yl) -2-amino-3-methylbutanamide or a pharmaceutically acceptable salt thereof may be administered once a day to once a week and may be administered once a week, It may be administered 5 times. In addition, the sorafenib or pharmaceutically acceptable salt thereof of the present invention may be administered once to twice a day to once a week and may be administered once to seven times a week depending on the route of administration, It may be administered once or twice daily.

According to one embodiment of the present invention, the combination of the present invention is (S) -N- (4- (3- (1H-1,2,4-triazol- , 5-trimethoxybenzoyl) phenyl) thiazol-2-yl) -2-amino-3-methylbutanamide or a pharmaceutically acceptable salt thereof is parenterally administered, (S) -N- (4- (3- (1H-1,2,4-triazol-1-yl) -4- (3,4,5-trimethoxybenzoyl) phenyl) thiazole- 2-yl) -2-amino-3-methylbutanamide or a pharmaceutically acceptable salt thereof is administered once, and from 1 day to 6 days, sorapenib or a pharmaceutically acceptable salt thereof is administered once a day .

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- Yl) -2-amino-3-methylbutanamide or a pharmaceutically acceptable salt thereof is orally administered, (3- (1H-1,2,4-triazol-1-yl) -4- (3,4,5-trimethoxybenzoyl) phenyl) thiazol- Methylbutanamide or a pharmaceutically acceptable salt thereof is administered three times a week once every day on days 1, 3, and 5, and sorafenib or a pharmaceutically acceptable salt thereof is administered 7 times a day once a day Lt; / RTI > (S) -N- (4- (3- (1H-1,2,4-triazol-1-yl) -4- (3,4,5-tri Methoxybenzoyl) phenyl) thiazol-2-yl) -2-amino-3-methylbutanamide or a pharmaceutically acceptable salt thereof is administered 20 minutes to 1 hour after oral administration of sorapenib or its pharmaceutical It may be to administer an acceptable salt.

In addition, the combination of the present invention is extremely excellent in hepatocarcinoma therapeutic activity, and the cancer recurrence possibility is remarkably low because there are few surviving cancer cells. Therefore, the combination of the present invention can be usefully used for the treatment of liver cancer.

According to another embodiment of the present invention, the present invention provides a composition dedicated to color chemistry comprising a vasoconstrictor.

Specifically, the present invention provides: (1) A process for producing (S) -N- (4- (3- (1H-1,2,4-triazol- 4,5-trimethoxybenzoyl) phenyl) thiazol-2-yl) -2-amino-3-methylbutanamide or a pharmaceutically acceptable salt thereof; (2) doxorubicin represented by the following formula (3) or a pharmaceutically acceptable salt thereof; And (3) a lipidol.

[Chemical Formula 1]

(3)

Two of the image-guided therapeutic methods applied to liver cancer are representative. One is transarterial chemoembolization (TACE), which is a treatment for locally injecting an anticancer agent into hepatocarcinoma through hepatic artery and performing hepatic embolization, Is radiofrequency ablation (RFA).

Carotid artery chemoembolization (TACE) uses a strategy to maximize the local anti-cancer effect on hepatocarcinoma by injecting anticancer drugs into the hepatic artery and blocking the hepatic artery by using embolization materials. In many cases, it plays a role as palliative therapy . Although normal liver tissue receives more than 70% of blood through the portal vein, 90% or more of the blood is supplied through the hepatic artery. Therefore, when carotid artery chemoembolization (TACE) In contrast, hepatocellular carcinoma can be administered at a relatively high concentration. In particular, direct injection through the blood vessels can increase the amount of anticancer agent accumulated in liver cancer by 100 times as compared with that during systemic injection. Nonetheless, the emergence of new neovasculature around liver cancer as a response to overcome hypoxia induced by chemoembolization has been identified as one of the major causes of recurrence.

However, in the composition for exclusive use of chemotherapy of the present invention, when the compound of the formula (1) (anti-vascular agent) and doxorubicin are combined and administered locally to liver cancer cells, the hepatocarcinoma treatment activity is excellent due to the synergistic effect. Furthermore, since there are few viable tumors, the problem of recurrence of tumors that occur upon administration of the compound of formula (I) or doxorubicin alone can be solved.

In the chemical color only composition of the present invention, lipiodol plays various roles. Specifically, lipiodol plays a role of transporting anticancer drugs in the chemo-specific composition to liver cancer cells. Lipiodol also acts as an contrast agent. In addition, Lipiodol has the ability to embolize hepatic artery, so it can amplify the embolization effect of embolization materials such as gelatin. Therefore, in the chemical color exclusive composition of the present invention, lipiodol transmits chemotherapeutic agents of the present invention to hepatocellular carcinoma cells, enables chemoembolization through the function as a contrast agent, and exhibits an embolization effect to assist liver cancer treatment.

The chemical color exclusive composition of the present invention may further comprise a water-based contrast agent. Doxorubicin is a hydrophilic substance, and it is not easy to dissolve in lipiodol. Therefore, it is preferable to further include a water-based contrast agent in order to dissolve doxorubicin.

According to an embodiment of the present invention, there is provided a process for preparing (S) -N- (4- (3- (1H-1,2,4-triazol- 5-trimethoxybenzoyl) phenyl) thiazol-2-yl) -2-amino-3-methylbutanamide or a pharmaceutically acceptable salt thereof and doxorubicin or a pharmaceutically acceptable salt thereof in a weight ratio of 5: 1: 5. Preferably, it may be from 2: 1 to 1: 2.

According to another embodiment of the present invention, there is provided a process for preparing (S) -N- (4- (3- (1H-1,2,4-triazol-1-yl) -4- Phenyl) thiazol-2-yl) -2-amino-3-methylbutanamide or a pharmaceutically acceptable salt thereof is preferably included in an amount of 5.0 to 30.0 mg per mL of lipiodol.

According to another embodiment of the present invention, it is preferred that the doxorubicin or a pharmaceutically acceptable salt thereof is contained in an amount of 5.0 to 50.0 mg per 1 mL of the aqueous contrast medium.

The chemical color only composition of the present invention can be used for various chemical embolization. Therefore, the chemical color composition of the present invention can be administered topically to a desired site of embolization, and is most preferably administered locally to liver via carotid artery chemoembolization (TACE).

In addition, the composition for exclusive use of color of the present invention is particularly excellent in liver cancer therapeutic activity, and the possibility of cancer recurrence is remarkably low because there are few surviving cancer cells. Therefore, the chemical color exclusive composition of the present invention can be usefully used for the treatment of liver cancer.

After injection of the chemical color-only composition of the present invention, an embolization substance may be injected to cause embolization at a desired site. Preferably, the embolus substance is in a gelatin form.

(S) -N- (4- (3- (1H-1,2,4-triazol-1-yl) -4- Methoxybenzoyl) phenyl) thiazol-2-yl) -2-amino-3-methylbutanamide or a pharmaceutically acceptable salt thereof and doxorubicin or a pharmaceutically acceptable salt thereof, , Sex, health condition, diet, time of administration, administration method, excretion rate, and severity of disease. (S) -N- (4- (3- (1H-1,2,4-triazol-1-yl) -4- (3,4,5-trimethoxybenzoyl) phenyl) thiazole -2-yl) -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 / 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.

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

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a graph showing the results of a bioluminescence image (BLI) of a parenterally administered control group and each experimental group in a Hep3B xenograft mouse model.
2 is a graph comparing the relative BLI signal intensities of the parenteral administration control group and each experimental group in the Hep3B xenograft mouse model.
FIG. 3 shows H & E and TUNEL staining images of the parenteral administration control group and each experimental group.
4 is a graph comparing apoptosis of the parenteral administration control group and each experimental group.
5 shows CD31 staining images of the parenteral administration control group and each experimental group.
6 is a graph comparing the microvessel density (MVD) of the parenteral administration control group and each experimental group.
FIG. 7 is a graph showing the results of a bioluminescence image (BLI) of an oral administration control group and each experimental group in a Hep3B xenograft mouse model.
8 is a graph comparing the relative BLI signal intensities of the oral administration control group and each experimental group in the Hep3B xenograft mouse model.
9 is a graph comparing the cell death of the oral administration control group and each experimental group.
10 shows CD31 staining images of the oral administration control group and each experimental group.
FIG. 11 is a graph comparing the microvessel area (MVA) of the oral administration control group stained with CD31 and each experimental group.
12 is a diagram showing a staining image of meca-32 of the oral administration control group and each experimental group.
FIG. 13 is a graph comparing the microvessel regions (MVA) of meca-32-stained oral control group and each experimental group.
FIG. 14 is a graph comparing viable portions of tumors obtained by injecting an experimental group into a rabbit VX2 liver cancer model with carotid artery chemoembolization (TACE) (A: Lipiodol administration group, B: Lipiodol + VDA, C: Lipiodol + Doxorubicin group, D: lipiodol + VDA + doxorubicin group)

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

< Example  1> Parenterally administered  The compound of formula (1) VDA ) And Sora Fenib's Synergistic effect  Confirm

1. Experimental Method

Cell line preparation and Hep3B  Infection of cells

Hep3B, a human hepatocellular carcinoma cell, was purchased from Korean Cell Line Bank (Seoul, Korea). Hep3B was constructed by infection with a lentivirus containing the firefly luciferase reporter gene, and the reporter strongly expressing clone was isolated to establish the Hep3B + luc cell line.

The stable expression of luciferase was confirmed by bioluminescence imaging (BLI) (PerkinElmer, Waltham, Mass., USA). Hep3B + luc cell lines were cultured in Dulbecco's modified Eagle medium containing 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA) and 1% antibiotics (Gibco, USA) medium) (DMEM, WelgeneInc, Seoul, Korea).

Animal tumor model preparation

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

The mice were inoculated with 1 x 10 ⁶ Hep3B + luc cells to the right side of each animal through a 27-gauge needle syringe. When the tumors exceeded 5 mm in diameter at about 14 days after inoculation, they were used in the experiment.

Preparation of active ingredient

The compound (VDA) of formula (1) was prepared by the method disclosed in WO 2009-119980 and dissolved in 0.9% saline.

The sorafenib p-toluenesulfonate (LC laboratories, Woburn, Mass., USA) was dissolved in Cremophor EL / ethanol (50:50; Sigma Cremophor EL, 95% ethyl alcohol) at 4 times the concentration used, .

In vivo  Bioluminescence imaging ( 약물 imaging ) observe

The bioluminescence images (BLI) were taken before (Day 0), 2 days (Day 2), 6 days (Day 6), 10 days (Day 10) and 14 days (Day 14). Mice were anesthetized and imaged with an IVIS spectrum system (PerkinElmer, Waltham, MA, USA). Mice were intraperitoneally injected with D-luciferin (0.375 mg / ml; Promega, Madison, WI, USA) dissolved in phosphate buffered saline (PBS). Bioluminescence has been reported as the sum of photons measured per second at a given region of interest (ROI). For in vivo bioluminescence measurements, ROIs of the same size and shape, including the tumor of each animal, were used. Quantification of BLI data was performed using Living Image software (version 2.50.1, Perkin Elmer, Waltham, Mass., USA).

Identification of liver cancer treatment activity

The anticancer effect of the compound of formula 1 and sorapenib was evaluated in the Hep3B xenograft model. The experimental group was randomly divided into the following different treatment groups.

- Control group: vehicle (saline, intraperitoneal)

- Compound (1) (VDA) alone: Compound (1) (5 mg / kg, once a week, intraperitoneally administered on day 1)

- sorafenib alone: sorafenib (30 mg / kg, 6 times per week, once daily on days 1 to 6)

- Concomitantly administered group: Compound 1 (5 mg / kg, once per week, per day), sorafenib (30 mg / kg, 6 times a week,

All experimental groups were dosed for 2 weeks. Toxicity was monitored by mouse weight measurement, and tumor growth was measured using a caliper during the experiment. Tumor volume was calculated according to the following equation.

Tumor volume = (length x width ² ) / 2

Histopathological study ( Histopathological studies )

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

In the tissue treatment, 10% paraformaldehyde (PFA) was treated with gravity flow for 10 min after deep anesthesia with ventilator catheter with saline. Tumor tissues were pooled, fixed with 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) staining was performed in a kit (Millipore, Billerica, MA) to assess the extent of tumor apoptosis , USA). Hematoxylin and eosin (H & E) staining were performed on representative areas of collected tumor tissue. The percentage of apoptotic sites was calculated using software (NIH-Image J, Bethesda, MD, USA).

To assess changes in tumor vascularity, immunohistochemical staining of CD31 was performed using primary rabbit antibody against mouse CD31 (Abcam, Cambridge, Mass.). Color development was performed with DakoEnVision + System HRP labeled polymer anti-rabbit (Dako) for 30 min. Images were observed using an optical microscope (model DM2500, leica, Germany).

Immunostaining was measured based on the Weidner method. Three sites were observed at low magnification (40X and 100X) using a Leica microscope (model DM2500, leica, Germany). Each microvessel count was measured in this region in a 200 X field (20 X objective, 10 X alternative). Image analysis for vascular numbers was performed using software (Image analysis, Leica, Germany). In this software, one cell of a stained lumen was calculated at one point. The mean microvessel counts in all measured areas were determined by microvessel density (MVD).

Quantitative analysis ( Statistical 분석 )

Quantitative data were expressed as mean ± standard error. Statistical significance of differences in tumor volume, bioluminescence imaging, apoptosis rate, and vascular density in each group was analyzed using Mann-Whitney's U-test. A p value of 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 results

Hep3B  In a xenograft model, the compound of formula (1) VDA ) Liver cancer treatment activity

To evaluate the antitumor effect of the compound (VDA) alone, xenograft mice were given the compound of formula (VDA) and body weight and tumor growth were observed during the experiment. There was no weakened mouse during the administration period. The body weight change was not significant (p> 0.05) before and 1 day after administration of compound (1) (VDA) alone, sorapenib alone or in combination. In terms of inhibiting tumor growth, the combination administration group was similar to sorapenib alone administration group. This suggests that the inhibitory effect of the compound (VDA) alone on the tumor growth is not significant.

BLI  Signal observation

During the experimental period, the relative BLI signal of the control group continued to increase. This reflects the vigorous growth of Hep3B-luc tumors. In the case of sorafenib alone, these values gradually decreased for 6 days and significantly recovered after 6 days. In the group of the compound of formula 1 (VDA) alone, the relative BLI signal dropped early after the administration of the compound of formula 1 (Day 1 and Day 8), which lasted for 2 days and significantly recovered after 2 days. This suggests that there is a rapid shutdown effect and a cytotoxic effect at the early stage of administration of the compound of formula (1). However, in the co-administered group, the relative signal was significantly lowered at the early stage of treatment and showed an inhibitory effect for 10 days after administration (see FIG. 1).

Also, the relative BLI signal strength is consistent with the BLI signal results (see FIG. 2). These results reflect the synergistic effect of the compound of formula (I) and sorapenib in combination. Specifically, the compound of formula (I) induces a rapid therapeutic effect, and sorapenib appears to maintain the inhibitory effect.

BLI  Correlation between Parameters and Histological Results

To confirm the BLI signal results, a series of histopathological studies were performed. The cytotoxic area of the tumor was quantified using the TUNEL assay at 14 days after administration. (43.0 ± 14.0), followed by compound (1) alone (38.0 ± 13.7), sorapenib alone (18.3 ± 6.0) and control (11.1 ± 3.7) in the combined treatment group. However, the results of the cell death of the compound of formula (I) alone and the combination treatment group were not significantly different, suggesting that the effect of sorapenib on tumor cell growth was cytostatic rather than cytotoxic (See Fig. 4). In microscopic H & E staining and TUNEL staining, the tumors were scattered dot-like in the case of sorafenib alone and in the control group, whereas in the case of the compound of formula 1 alone, In addition, in the combination administration group, apoptosis was similar to that of the compound of the formula (I) alone (see FIG. 3). These results show that the compound of Chemical Formula 1 showed extensive cell death due to the tumor destruction effect and showed the tumor growth inhibition effect due to the tumor growth inhibition property of sorapenib.

In addition, quantification of MVD of anti-CD31 stained histological specimens and tumors was obtained at 14 days after a single dose of the drug. The number of tumor vessels in the control group was large and distributed throughout the tumor. However, blood vessels appeared differently in the experimental group. In the experimental group, the blood vessels were short and distributed at the margin of the tumor. And the comparison of the experimental groups was made with the density of the blood vessels (see FIG. 5). The MVD of the tumor was highest in the control group (3180 ± 465), followed by the compound of the formula 1 alone (2334 ± 60), sorafenib alone (1871 ± 225) (See Fig. 6). Statistical significance was observed in all experimental groups. These results also indicate that the combination of the blood vessel destruction properties of the compound of formula (1) and the novel blood vessel inhibiting properties of sorapenib shows a synergistic effect.

< Example  2 > The orally administered compound of formula (1) VDA ) Sora Fenib's Synergistic effect Confirm

1. Experimental Method

The cell line, animal tumor model, and active ingredient were prepared in the same manner as in Example 1.

In vivo  Bioluminescence imaging ( 약물 imaging ) observe

The bioluminescence image (BLI) was measured before the administration of the drug (Day 0), 2 days after the drug administration (Day 2), 4 days (Day 4), 8 days (Day 8), 11 days Day (Day 15), 18 days later (Day 18), and 20 days later (Day 20). Mice were anesthetized and imaged with an IVIS spectrum system (PerkinElmer, Waltham, MA, USA). Mice were intraperitoneally injected with luciferin (1.5 mg / ml; Promega, Madison, WI, USA) dissolved in phosphate buffered saline (PBS). Bioluminescence has been reported as the sum of photons measured per second at a given region of interest (ROI). In vivo luminescence ( in For vivo bioluminescence measurements, ROIs of the same size and shape, including the tumor of each animal, were used. Quantification of BLI data was performed using Living Image software (version 2.50.1, Perkin Elmer, Waltham, Mass., USA).

Identification of liver cancer treatment activity

The anticancer effect of the compound of formula 1 and sorapenib was evaluated in the Hep3B xenograft model. The experimental group was randomly divided into the following different treatment groups.

- Control: vehicle (saline oral administration)

- Compound (1) (VDA) alone: Compound (1) (4 mg / kg, 1 day, 3 days, 5 times daily Oral administration three times a week)

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

- Combined administration group: Compound 1 (4 mg / kg, 1 day, 3 days, 5 times a week Oral administration), Sorapenib (15 mg / kg,

Oral administration was carried out at 2:00 pm daily and sorafenib was administered 30 minutes after the administration of the compound of formula 1 in the combination administration group.

All experimental groups were dosed for 3 weeks. Toxicity was monitored by mouse weight measurement, and tumor growth was measured using a caliper during the experiment. Tumor volume was calculated according to the following equation.

Tumor volume = (length x width ² ) / 2

Histopathological study ( Histopathological studies )

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

The extracted tumor tissue treatment was carried out according to the method described in Example 1.

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

Immunohistochemical staining was performed using a primary rabbit antibody against mouse CD31 (Abcam, Cambridge, MA) and a primary rat antibody against meca-32 (Novus Biologicals, Littleton, Colorado, USA) Respectively. CD31 was stained with DakoEnVision + System HRP labeled polymer anti-rabbit (Dako) for 30 min. Images were observed using an optical microscope (model DM2500, leica, Germany). meca-32 was observed using an Alexa Fluor 488 anti-rat antibody.

The microvascular area (MVA) was assessed by CD31 and meca-32 immunochemical staining. Three sites were observed at low magnification (40X and 100X) using a Leica microscope (model DM2500, leica, Germany). Each microvessel count was measured in this region in a 200 X field (20 X objective, 10 X alternative). Image analysis of blood vessel numbers was performed using software (Image analysis, Leica, Germany).

Quantitative analysis ( Statistical 분석 )

Quantitative data were expressed as mean ± standard error. Tumor volume, bioluminescence imaging of each group was evaluated by RMANOVA test, and statistical significance of differences in apoptosis rate, vascular area was analyzed using one-way ANOVA test. A p value of 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 results

BLI  Signal observation

During the experimental period, the relative BLI signal of the control group increased up to Day 11, stagnated, and the signal intensity at the center of the cancer was lowered in the latter half of the administration period. This is thought to be due to the natural necrosis caused by the enlargement of cancer (see FIG. 7).

In the compound group of the formula (1), the signal intensity rapidly decreased until Day 2, but then increased. In patients treated with sorafenib, the signal intensity gradually decreased over the entire administration period. In the combination group, the signal intensity rapidly decreased, and the state remained reduced throughout the entire administration period (see FIG. 8).

These results, like the results of the experiment of Example 1, show that the compound of Chemical Formula 1 induces a rapid therapeutic effect and sorapenib maintains its inhibitory effect. In addition, when the compound of the formula (1) is orally administered, it can be seen that the same effect as that in the case of administration as an injection is exhibited.

BLI  Correlation between Parameters and Histological Results

To confirm the BLI signal results, a series of histopathological studies were performed. The cytotoxic area of the tumor was quantified using the TUNEL assay at day 21 after administration. As a result, the degree of apoptosis was the highest (34.18 ± 5.99) in the combination treatment group, followed by the treatment with the compound of Formula 1 alone (16.24 ± 6.16), sorapenib alone (10.36 ± 3.62) (See Fig. 9).

In addition, quantitative analysis of anti-CD31-stained histological specimens (FIG. 10) and subsequent MVA of tumors (FIG. 11) and meca-32 stained histological specimens (FIG. 12) Were obtained on the 21st day after each administration of the drug. In the MVA analysis using CD31, MVA of the tumor was (16339 ± 3614), (14452 ± 2709) and (14934 ± 3154) in the control group, the compound 1 alone group and the sorapenib alone group, respectively (7161 ± 4522), respectively, and the MVA is remarkably lower than all other groups (see FIG. 11).

In MVA analysis using meca-32, the MVD of tumor was the highest (17183 ± 5508), followed by sorapenib alone (14897 ± 3556) and the compound of formula 1 alone (12871 ± 3433) (7631 ± 2872) (see FIG. 13).

These results also show that the combination of the blood vessel destruction properties of the compound of formula 1 and the novel blood vessel inhibiting properties of sorapenib shows a synergistic effect.

There was also a difference in the shape of the blood vessels. In the group to which the compound of the formula (1) was administered, not only the number of blood vessels was decreased but also the vascular lumen was narrowed (see FIGS. 10 and 12). This is a good example showing the blood vessel destroying activity by the compound of the formula (1). In addition, the combination of the compound of formula (1) and sorafenib with neovascular inhibitory effect is thought to increase the combined effect by changing the number and shape of blood vessels in liver cancer.

< Example  3> Chemical color only  The effect of the composition on liver cancer treatment and Hepatotoxicity Confirm

1. Experimental Method

Rabbit VX2 carcinoma liver tumor model established as a representative animal model of liver cancer was used. The tumor chip of VX2 carcinoma prepared in 1 mm ³ size was placed on the liver surface using a needle. After 2 to 3 weeks of tumor placement, non-contrast CT and perfusion MR were performed to confirm tumor formation.

Experimental group  Set

The experimental group was randomly divided into 10 experimental groups A to D below.

- Experimental group A: Lipiodol-treated group (Lipiodol 0.2 mL + Contrast media [GE healthcare Visipaque 270] 0.05 mL)

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

- Experimental group C: Lipiodol / doxorubicin treated group (Lipiodol 0.2 mL + Contrast media [GE healthcare Visipaque 270] 0.05 mL / doxorubicin 1 mg)

- Experimental group D: Lipiodol / Compound of formula 1 (VDA) / Doxorubicin (0.2 mL of lipiodol / 1.26 mg of compound of formula 1 (VDA) + Contrast media (GE healthcare Visipaque 270)

Administration of medicines

The microcatheter was introduced into the left hepatic artery through the celiac trunk through the auricular artery of the rabbit ear and then carefully injected into the experimental groups A to D. Before and after TACE, non-enhanced CT was performed to evaluate the presence or absence of tumor and Lipiodol deposited in tumor after TACE.

2. Experimental results

Checking the effect of liver cancer treatment

One week after administration, hepatic tissue was removed and a pathological slide was prepared. H & E staining, and TUNEL staining were performed to identify areas of necrosis of the tumor. The area was calculated by the Image J program and the viable portion of the tumor was expressed as a percentage. The results are shown in Table 1 and Fig. 14 below.

[Table 1]

As shown in Table 1, in the case of the experimental group A in which only the lipiodol without anticancer activity was administered, the viable site was 47.06%. In the case of the experimental group B in which the lipiodol and the compound of formula 1 (VDA) were administered in combination, the viable site was 27.48 , And that the survival potential of the experimental group C administered with the combination of lipiodol and doxorubicin was only 14.36%. From these results, it can be seen that the experimental groups A to C have a high possibility of cancer recurrence.

On the other hand, in the case of the experimental group D in which the combination of the lipiodol, the compound of formula (1) (VDA) and doxorubicin was administered, the viability was 0.66%, and most of the tumors were removed, have.

In addition, the reproducibility of the treatment effect was not good in the experimental groups A to C so that the standard deviation of the results exceeded 10%. However, in the experimental group D, the standard deviation was only about 1%, so the liver cancer was reproducibly treated.

Toxicity check

Blood samples were taken before, 1, 3, and 7 days after the drug injection to measure AST and ALT, which can assess the extent of acute liver injury.

As a result, the acute liver injury was the highest in the experimental group D, but the recovery was similar to that of the other experimental group after 7 days. In addition, since no deaths of rabbits were observed during the experiment, liver damage in the experimental group D was confirmed to be acceptable.

Claims (23)

delete delete delete delete delete (1) Synthesis of (S) -N- (4- (3- (1H-1,2,4-triazol-1-yl) -4- (3,4,5-trimethoxy Benzoyl) phenyl) thiazol-2-yl) -2-amino-3-methylbutanamide or a pharmaceutically acceptable salt thereof; And
[Chemical Formula 1]

(2) sorapenib or a pharmaceutically acceptable salt thereof represented by the following formula (2);
(2)

As an active ingredient. 7. The pharmaceutical composition according to claim 6, wherein the pharmaceutically acceptable salt of sorapenib is p-toluenesulfonate. 7. The method according to claim 6, wherein the (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 parenterally or orally administered. 7. The pharmaceutical composition according to claim 6, wherein the sorapenib or a pharmaceutically acceptable salt thereof is orally administered. 7. The method according to claim 6, wherein the (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 administered once to five times per week. 7. The pharmaceutical composition according to claim 6, wherein the sorafenib or a pharmaceutically acceptable salt thereof is administered once to seven times a week. 12. The pharmaceutical composition according to claim 11, wherein the sorafenib or a pharmaceutically acceptable salt thereof is administered once or twice daily. 7. The method according to claim 6, wherein the (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 administered once to 5 times a week, and the sorapenib or a pharmaceutically acceptable salt thereof is administered weekly 0.0 &gt; 1 &lt; / RTI &gt; to 7 times. 7. The compound according to claim 6, which is (S) -N- (4- (3- (1H-1,2,4-triazol-1-yl) -4- (3,4,5-trimethoxybenzoyl) phenyl When administered parenterally, the administration period is one week, and when the (S) -N- (thiazol-2-yl) -2-amino-3-methylbutanamide or its pharmaceutically acceptable salt is administered parenterally, 4-triazol-1-yl) -4- (3,4,5-trimethoxybenzoyl) phenyl) thiazol- 3-methylbutanamide or a pharmaceutically acceptable salt thereof is administered once, and somapaperib or a pharmaceutically acceptable salt thereof is administered once a day from day 1 to day 6. 7. The compound according to claim 6, which is (S) -N- (4- (3- (1H-1,2,4-triazol-1-yl) -4- (3,4,5-trimethoxybenzoyl) phenyl Yl) -2-amino-3-methylbutanamide or a pharmaceutically acceptable salt thereof is orally administered to a patient in need of such treatment an effective amount of (S) -N- (4- (3- 4-triazol-1-yl) -4- (3,4,5-trimethoxybenzoyl) phenyl) thiazol-2-yl) -2-amino-3-methylbutanamide or a pharmaceutically acceptable salt thereof Wherein the salt is administered three times per week on days 1, 3, and 5, and sorafenib or a pharmaceutically acceptable salt thereof is administered 7 times per week once a day. 10. The compound according to claim 9, which 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 administered orally 20 minutes to 1 hour after administration of sorafenib or a pharmaceutically acceptable salt thereof &Lt; / RTI &gt; delete delete delete delete delete delete (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 as an active ingredient,
Wherein the pharmaceutical composition is administered in combination with sorapenib or a pharmaceutically acceptable salt thereof represented by the following formula (2):
[Chemical Formula 1]

(2)

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