JP7042836B2 - How to treat cancer - Google Patents

Description

Cancer mainly involves an increase in the number of abnormal cells derived from certain normal tissues, infiltration of the abnormal cells into adjacent tissues, or lymphatic or hematogenous spread (metastasis) to the lymph nodes to which malignant cells belong and distant sites. It is characterized by. Clinical data and molecular biology studies show that cancer is a multi-step process that begins with minor changes before neoplasm development and can progress to neoplasia under certain conditions.

Primary liver cancer is one of the most common forms of cancer in the world. There are two main types of liver cancer: hepatocellular carcinoma (HCC) (also known as malignant hepatoma) and bile duct cell carcinoma (also known as bile duct cancer (CCA)). HCC is the most common form of primary liver cancer and develops within hepatocytes. HCC most often occurs in men and patients suffering from cirrhosis. HCC is one of the most common cancers in the world and the third leading cause of cancer-related death. The disease is often diagnosed late in the course of clinical symptoms. As a result, only 10-15% of patients are candidates for curative surgery. For the majority of HCC patients, systemic chemotherapy or supportive therapy is the main treatment option.

In contrast, it is cholangiocellular carcinoma or cholangiocarcinoma that develops primarily in the bile duct epithelial cells (ie, bile duct cells) in the liver. This type of cancer is more common in women. The incidence of bile duct cancer (CCA) is increasing worldwide, making it already the second most common primary liver cancer. Patients with CCA have a poor prognosis because of the late diagnosis and the refractory tumor.

To date, the number of drugs that can effectively treat cancers such as HCC and / or CCA is limited. For example, patients with metastatic hepatocellular carcinoma or hepatocellular carcinoma usually only maintain a life expectancy of about 3-4 months if local treatment fails. Metastatic hepatocellular carcinoma or hepatocellular carcinoma is primarily treated systemically if topical treatment fails. The use of doxorubicin, the use of high doses of tamoxifen in combination with doxorubicin, or the use of EA-PFL (etopoxide, adrimycin, cisplatin, fluorouracil and leucovorin) are effective examples. Remission rates with these drugs can reach levels of 15-30%. However, patients with hepatocellular carcinoma usually do not receive systemic chemotherapy because they develop complications of cirrhosis and other complications (such as leukopenia, thrombocytopenia or liver dysfunction). Moreover, the efficacy of most chemotherapeutic agents is limited and has not been able to significantly improve patient survival. Despite continued efforts, the deterioration of the clinical course of most cancer patients underscores the need for more effective chemotherapy.

Bile duct cancer is a relatively rare neoplasm classified as adenocarcinoma (a cancer that forms a gland or secretes a significant amount of mucin). Its annual incidence is 1-2 cases per 100,000 in Western countries, but the rate of bile duct cancer has risen worldwide over the last few decades. Bile duct cancer is considered to be an incurable, rapidly dying cancer unless both the primary tumor and any of the metastases can be completely removed by surgery. There is no cure other than surgery, but the majority of people are in the advanced stages of the disease when they notice the symptoms and are inoperable at the time of diagnosis. People with bile duct cancer are generally managed with chemotherapy, radiation therapy and other palliative care measures rather than cure.

又はその薬学的に許容される塩若しくはアミノ酸抱合体から選択されるＦＸＲアゴニストを投与するステップを含む方法。
（項目２）
前記癌は、肝細胞癌、胆管癌、膵癌、腎臓癌、前立腺癌、食道癌、乳癌、胃癌、腎癌、唾液腺癌、卵巣癌、子宮体癌、膀胱癌及び肺癌から選択される、項目１に記載の方法。
（項目３）
前記癌は、肝細胞癌である、項目１又は２に記載の方法。
（項目４）
前記肝細胞癌は、初期段階肝細胞癌、非転移性肝細胞癌、原発性肝細胞癌、進行肝細胞癌、局所進行肝細胞癌、転移性肝細胞癌、寛解期の肝細胞癌又は再発肝細胞癌からなる群から選択される、項目３に記載の方法。
（項目５）
前記ＦＸＲアゴニストは、化合物１又はその薬学的に許容される塩である、項目１～４の何れか一項に記載の方法。
（項目６）
前記ＦＸＲアゴニストは、化合物１のナトリウム塩である、項目５に記載の方法。
（項目７）
前記ＦＸＲアゴニストは、化合物１のＮ，Ｎ－ジエタンアミン塩である、項目５に記載の方法。
（項目８）
前記ＦＸＲアゴニストは、化合物２又はその薬学的に許容される塩若しくはアミノ酸抱合体である、項目１～４の何れか一項に記載の方法。
（項目９）
前記ＦＸＲアゴニストは、化合物２のグリシン抱合体である、項目８に記載の方法。
（項目１０）
前記ＦＸＲアゴニストは、化合物２のタウリン抱合体である、項目８に記載の方法。
（項目１１）
前記ＦＸＲアゴニストは、化合物２のサルコシン抱合体である、項目８に記載の方法。
（項目１２）
癌の処置又は予防を、それを必要とする対象において行うための、化合物１若しくは２：

又はその薬学的に許容される塩若しくはアミノ酸抱合体から選択されるＦＸＲアゴニストと、薬学的に許容される添加剤とを含む医薬組成物。
（項目１３）
癌の処置又は予防を、それを必要とする対象において行うためのキットであって、化合物１若しくは化合物２：

又はその薬学的に許容される塩若しくはアミノ酸抱合体を含むキット。
（項目１４）
癌の処置又は予防を、それを必要とする対象において行うための医薬品の製造における、化合物１若しくは２：

又はその薬学的に許容される塩若しくはアミノ酸抱合体から選択されるＦＸＲアゴニストの使用。
（項目１５）
前記癌は、胆管癌である、項目１又は２に記載の方法。
（項目１６）
前記ＦＸＲアゴニストは、化合物１又はその薬学的に許容される塩である、項目１５に記載の方法。 The present invention addresses these needs. Therefore, it is an object of the present invention to provide an improved method for treating or preventing cancers such as hepatocellular carcinoma and cholangiocarcinoma.
In certain embodiments, for example, the following are provided:
(Item 1)
A method of treating or preventing cancer in a subject in need thereof, wherein a therapeutically effective amount of Compound 1 or 2:

Or a method comprising the step of administering an FXR agonist selected from a pharmaceutically acceptable salt or amino acid conjugate thereof.
(Item 2)
The cancer is selected from hepatocellular carcinoma, bile duct cancer, pancreatic cancer, kidney cancer, prostate cancer, esophageal cancer, breast cancer, gastric cancer, renal cancer, salivary adenocarcinoma, ovarian cancer, uterine body cancer, bladder cancer and lung cancer, item 1. The method described in.
(Item 3)
The method according to item 1 or 2, wherein the cancer is hepatocellular carcinoma.
(Item 4)
The hepatocellular carcinoma is early stage hepatocellular carcinoma, non-metastatic hepatocellular carcinoma, primary hepatocellular carcinoma, advanced hepatocellular carcinoma, locally advanced hepatocellular carcinoma, metastatic hepatocellular carcinoma, remission stage hepatocellular carcinoma or recurrence. Item 3. The method according to item 3, which is selected from the group consisting of hepatocellular carcinoma.
(Item 5)
The method according to any one of items 1 to 4, wherein the FXR agonist is Compound 1 or a pharmaceutically acceptable salt thereof.
(Item 6)
5. The method of item 5, wherein the FXR agonist is a sodium salt of compound 1.
(Item 7)
The method according to item 5, wherein the FXR agonist is an N, N-dietaneamine salt of Compound 1.
(Item 8)
The method according to any one of items 1 to 4, wherein the FXR agonist is Compound 2 or a pharmaceutically acceptable salt or amino acid conjugate thereof.
(Item 9)
8. The method of item 8, wherein the FXR agonist is a glycine conjugate of compound 2.
(Item 10)
8. The method of item 8, wherein the FXR agonist is a taurine conjugate of compound 2.
(Item 11)
8. The method of item 8, wherein the FXR agonist is a sarcosine conjugate of compound 2.
(Item 12)
Compound 1 or 2: for treating or preventing cancer in a subject in need thereof:

Alternatively, a pharmaceutical composition comprising an FXR agonist selected from a pharmaceutically acceptable salt or amino acid conjugate thereof and a pharmaceutically acceptable additive.
(Item 13)
A kit for treating or preventing cancer in a subject in need thereof, wherein Compound 1 or Compound 2:

Or a kit containing a pharmaceutically acceptable salt or amino acid conjugate thereof.
(Item 14)
Compound 1 or 2: in the manufacture of a pharmaceutical product for the treatment or prevention of cancer in a subject in need thereof:

Or the use of FXR agonists selected from their pharmaceutically acceptable salts or amino acid conjugates.
(Item 15)
The method according to item 1 or 2, wherein the cancer is bile duct cancer.
(Item 16)
15. The method of item 15, wherein the FXR agonist is Compound 1 or a pharmaceutically acceptable salt thereof.

FIG. 6 is a bar graph showing the effect of compound 1-Na and a control diet on the number of liver tumors in multidrug resistant protein 2 (Mdr2 ^{− / −} ) knockout mice. * P <0.01 for the control group. 3 is a bar graph showing the effect of compound 1-Na and a control diet on a percentage reduction in tumor (> 5 mm diameter) in Mdr2 ^-/- mice. 3 is a bar graph showing the effect of OCA (Compound 2) and control diet on the number of liver tumors in Mdr2 ^-/- knockout mice. 3 is a bar graph showing the effect of OCA and control diets on the percentage reduction of tumors (> 5 mm in diameter) in Mdr2 ^-/- mice. FIG. 6 is a bar graph showing the effect of compound 1-Na and a control diet on the number of liver tumors in farnesoid X receptor (FXR ^{− / −} ) mice. FIG. 6 is a bar graph showing the effect of compound 1-Na and a control diet on a percentage reduction in tumor (> 5 mm diameter) in FXR ^{− / −} mice. FIG. 6 is a bar graph showing the effect of compound 1-Na and a control diet on a percentage reduction in the number of liver tumors in FXR ^{− / −} mice. 3 is a bar graph showing the effect of compound 1-Na and a control diet on a percentage reduction in liver / body weight ratio in Mdr2 ^-/- mice. * P <0.01 for the control group. FIG. 6 is a bar graph showing the effect of compound 1-Na and control feed on a percentage reduction in liver / body weight ratio in FXR ^{− / −} mice. FIG. 6 is a bar graph showing the effect of compound 1-Na and control feed on alanine transaminase (ALT) levels in Mdr2 ^-/- mice and FXR ^-/- mice. * P <0.01 for the control group. FIG. 6 is a bar graph showing the effect of compound 1-Na and control feed on aspartate transaminase (AST) levels in Mdr2 ^-/- mice and FXR ^-/- mice. * P <0.01 for the control group. FIG. 6 is a bar graph showing the effect of compound 1-Na and control feed on ileal gene expression of fibroblast growth factor 15 (Fgf15) in Mdr2 ^-/- mice and FXR ^-/- mice. * P <0.01 for the control group. FIG. 6 is a bar graph showing the effect of compound 1-Na and control feed on ileal gene expression of low molecular weight heterodimer partners (Shps) in Mdr2 ^-/- mice and FXR ^-/- mice. * P <0.01 for the control group. It is a bar graph which shows the influence of compound 1-Na and a control feed on the expression reduction of cholesterol 7α hydroxylase (cyp7a1) in Mdr2 ^{− / −} mouse and FXR ^{− / −} mouse. * P <0.01 for the control group. FIG. 6 is a bar graph showing the effect of compound 1-Na and control feed on liver gene expression of low molecular weight heterodimer partner (Shp) in Mdr2 ^{− / −} mice and FXR ^{− / −} mice. * P <0.01 for the control group. It is a bar graph which shows the influence of compound 1-Na and a control feed on the liver gene expression of a bile acid excretion pump (Bsep) in Mdr2 ^{− / −} mouse and FXR ^{− / −} mouse. * P <0.01 for the control group. 3 is a bar graph showing the effect of compound 1-Na and control feed on total serum bile acid content in Mdr2 ^-/- mice. * P <0.01 for the control group. FIG. 6 is a bar graph showing the effect of compound 1-Na3 and control feed on total serum bile acid content in FXR ^{− / −} mice. Shows FXR mRNA microarray expression in all tissues of CCA tumors when compared to human peripheral tissues. FXR mRNA microarray expression in all tissues of CCA tumors grouped by tumor differentiation is shown. FXR mRNA expression (qPCR) in CCA tumors as compared to normal human liver tissue and tissues around human liver is shown. Figure shows FXR mRNA expression (qPCR) in CCA tumors when compared to the corresponding human liver peri-tissue. Shows TGR mRNA microarray expression in all tissues of CCA tumors when compared to human peripheral tissues. It shows the expression of TGR5 mRNA microarrays in all tissues of CCA tumors due to clinical pathological parameters, ie, anatomical site and perineural infiltration. TGR5 mRNA expression (qPCR) in CCA tumors when compared to human liver perimeter tissue is shown. TGR5 mRNA expression (qPCR) in CCA tumors when compared to the corresponding human liver perimeter tissue is shown. FXR mRNA expression (qPCR) in normal human bile duct cells and CCA cell lines is shown. TGR5 mRNA expression (qPCR) in normal human bile duct cells and CCA cell lines is shown. Representative MRI and liver images of untreated control mice, OCA-treated and compound 4 treated mice are shown. It is a bar graph which shows the tumor volume magnification change quantified by MRI. The mRNA expression levels of growth markers (ie, Ki67 and PCNA), bile markers (ie, CK19) and epithelial markers (ie, ZO-1) in hepatic sympatric CCA tumors are shown. Representative immunohistochemical images of growth markers (ie, Ki67 and PCNA) in liver sympathetic CCA tumors of untreated or treated with OCA or Compound 4 are shown. MRNA expression levels of proliferation markers after treatment with OCA and treatment with 10 or 25 μM OCA for 48 hours as compared to untreated control cells performed using CFSE cell proliferation dye staining by flow cytometry. It shows the proliferation of CCA cells (ie, EGI1). Representative microscopic images of migration and wound healing assays in CCA cells (ie EGI1), corresponding quantification and representative microscopic images of the transwell migration chamber at 24 hours are shown. A bar graph of oxygen consumption rate (OCR) by Seahorse using a mitochondrial stress test kit in CCA cells (ie EGI1) and metabolic parameters calculated after OCR measurements is shown. Quantification of flow cytometry-based apoptosis assays and representative histograms and corresponding pool data by Annexin V and propidium iodide staining in untreated or treated with 10 or 25 μM OCA (ie EGI1). show. It shows the mRNA expression level of the proliferation marker after treatment with compound 4 and the proliferation of CCA cells (ie EGI1) treated with 10 or 25 μM compound 3 for 48 hours as compared to untreated control cells. Representative microscopic images of migration assay, wound healing assay in CCA cells (ie EGI1) and corresponding microscopic images of transwell migration chambers at 24 hours in untreated and compound 4 treated cells. And the corresponding quantification. A bar graph of oxygen consumption rate (OCR) by Seahorse and metabolic parameters calculated after OCR measurements using a mitochondrial stress test kit in CCA cells untreated or treated with compound 4 (25 μM) (ie EGI1) is shown. The mRNA expression levels of growth markers (ie, Cdc25a, cyclin D1 and cyclin D3) in hepatic sympathetic CCA tumors are shown.

又はその薬学的に許容される塩若しくはアミノ酸抱合体である。 The present application relates to a method of treating or preventing cancer in a subject in need thereof, comprising the step of administering a therapeutically effective amount of a farnesoid X receptor (FXR) agonist. In one embodiment, the FXR agonist is compound 1 or compound 2:

Or a pharmaceutically acceptable salt or amino acid conjugate thereof.

In one embodiment, the cancer is selected from the group consisting of hepatocellular carcinoma, pancreatic cancer, kidney cancer, prostate cancer, esophageal cancer, breast cancer, gastric cancer, renal cancer, salivary adenocarcinoma, ovarian cancer, uterine body cancer, bladder cancer and lung cancer. To. In one embodiment, the cancer is hepatocellular carcinoma. In one embodiment, the cancer is pancreatic cancer. In one embodiment, the cancer is kidney cancer. In one embodiment, the cancer is prostate cancer. In one embodiment, the cancer is esophageal cancer. In one embodiment, the cancer is breast cancer. In one embodiment, the cancer is gastric cancer. In one embodiment, the cancer is renal cancer. In one embodiment, the cancer is salivary gland cancer. In one embodiment, the cancer is ovarian cancer. In one embodiment, the cancer is endometrial cancer. In one embodiment, the cancer is lung cancer.

In one embodiment, the FXR agonist is Compound 1 or a pharmaceutically acceptable salt thereof. In another embodiment, the FXR agonist is a sodium salt of compound 1 (ie, compound 1-Na). In yet another embodiment, the FXR agonist is the N, N-dietanamine salt of Compound 1 (ie, Compound 1-DEA).

In another embodiment, the FXR agonist is Compound 2 or a pharmaceutically acceptable salt or amino acid conjugate thereof. In one embodiment, the FXR agonist is a glycine conjugate of compound 2. In one embodiment, the FXR agonist is a taurine conjugate of compound 2. In one embodiment, the FXR agonist is a sarcosine conjugate of compound 2.

The present invention relates to compound 1 or a pharmaceutically acceptable salt thereof or compound 2 or a pharmaceutically acceptable salt thereof in the manufacture of a pharmaceutical product for treating or preventing cancer in a subject in need thereof. Further on the use of amino acid conjugates.

The present invention relates to Compound 1 or a pharmaceutically acceptable salt thereof and Compound 2 or a pharmaceutically acceptable salt thereof for use in performing treatment or prevention of cancer in a subject in need thereof. Further related to amino acid conjugates.

The present invention relates to Compound 1 or a pharmaceutically acceptable salt thereof and Compound 2 or a pharmaceutically acceptable salt or amino acid conjugate thereof for treating or preventing cancer in a subject in need thereof. Also related to pharmaceutical compositions containing, pharmaceutically acceptable additives.

The present invention is a kit for treating or preventing cancer in a subject in need thereof, wherein the compound 1 or a pharmaceutically acceptable salt thereof and the compound 2 or a pharmaceutically acceptable salt thereof are used. Further on kits containing amino acid conjugates.

The present invention is at least partially based on the finding that Compounds 1 and 2 were effective in treating cancer in animal models that predict cancer. As described in the Examples below, we found that the compounds of the invention suppressed tumor growth in a mouse model of spontaneous liver cancer formation.

Definitions For convenience, the specific terms used in this specification, examples and the appended claims are collected herein.

The term "cancer" as used herein refers to any of the diseases characterized by the presence of cancerous tissue in a subject.

As used herein, "cancerous tissue" refers to tissue that contains malignant neoplastic cells and expresses cell overgrowth and / or hyperproliferative cells. The cancerous tissue can be a primary malignancy originating from the developing tissue or organ, or a metastatic malignant tumor that grows in the body tissue that was not the source of the primary tumor.

As used herein, the term "tumor" may include solid tumors or cancers of hematopoietic origin. In some embodiments, the tumor may be characterized by the ability to invade surrounding tissues, metastasize to other parts of the body and / or angiogenic activity. Exemplary tumors result from hepatocellular carcinoma, gastric cancer, renal cancer, prostate cancer, adrenal cancer, pancreatic cancer, breast cancer, bladder cancer, salivary adenocarcinoma, ovarian cancer, endometrial cancer and lung cancer.

As used herein, the term "invasive" refers to the process of spreading cells, cell populations or malignant tumors from one site to an adjacent site.

As used herein, the term "metastatic" refers to the process of spreading a cell, a population of cells, or a malignant tumor from one site to one that is not adjacent to the original site.

As used herein, "hepatocellular carcinoma", "HCC" and "malignant hepatoma" are used interchangeably and refer to primary tumors and secondary (metastatic) tumors originating from liver tissue. The term "treatment-resistant hepatocellular carcinoma" as used herein refers to hepatocellular carcinoma that does not respond favorably to antineoplastic treatment. Thus, "treatment-resistant hepatocellular carcinoma", as used herein, is hepatocellular carcinoma that is either incapable of responding favorably to treatment or is resistant, or instead favorably to treatment. Refers to hepatocellular carcinoma that recurs or reoccurs after responding.

As used herein, "bile duct cancer," or "CCA," or "intrahepatic cell carcinoma," or cholangiocarcinoma, is a mutant epithelial cell (or epithelial differentiation) that occurs in the bile duct that drains bile from the liver into the small intestine. It is a form of cancer composed of characteristic cells). The rate of CCA is elevated when the liver is in a cholestasis state. Accumulation of bile acids in the liver does not directly induce carcinogenesis, but promotes carcinogenic support by promoting bile duct cell proliferation and inflammation and by reducing FXR-dependent chemical protection. Sometimes.

を指し、これは、６α－エチル－３α，７α，２３－トリヒドロキシ－２４－ノル－５β－コラン－２３－水素スルフェートとしても知られている。６α－エチル－３α，７α，２３－トリヒドロキシ－２４－ノル－５β－コラン－２３－スルフェートナトリウム」としても知られている「化合物１－Ｎａ」又は「１－Ｎａ」は、互換的に使用され、化合物１のナトリウム塩を指す。本明細書で使用する場合、６α－エチル－３α，７α，２３－トリヒドロキシ－２４－ノル－５β－コラン－２３－スルフェートＮ，Ｎ－ジエチルエタンアミン」としても知られている「化合物１－ＤＥＡ」又は「１－ＤＥＡ」は、互換的に使用され、化合物１のＮ，Ｎ－ジエチルエタンアミン塩を指す。化合物１－Ｎａ及び化合物１－ＤＥＡの構造を以下に示す。

As used herein, the term "Compound 1" is used.

Also known as 6α-ethyl-3α, 7α, 23-trihydroxy-24-nor-5β-cholane-23-hydrogen sulfate. "Compound 1-Na" or "1-Na", also known as "6α-ethyl-3α, 7α, 23-trihydroxy-24-nor-5β-cholane-23-sulfate sodium", are compatible. Used and refers to the sodium salt of compound 1. As used herein, "Compound 1-" also known as "6α-ethyl-3α, 7α, 23-trihydroxy-24-nor-5β-cholane-23-sulfate N, N-diethylethaneamine". "DEA" or "1-DEA" is used interchangeably and refers to the N, N-diethylethaneamine salt of Compound 1. The structures of compound 1-Na and compound 1-DEA are shown below.

を指し、これは、オベチコール酸（ＯＣＡ）、６－ＥＣＤＣＡ、６－α－エチルケノデオキシコール酸又は６α－エチル－３α，７α－ジヒドロキシ－５β－コラン－２４－酸としても知られている。 "Compound 2", as used herein, is used herein.

Also known as obeticholic acid (OCA), 6-ECDCA, 6-α-ethylkenodeoxycholic acid or 6α-ethyl-3α, 7α-dihydroxy-5β-cholane-24 acid.

を指し、これは、３α，７α，１１β－トリヒドロキシ－６α－エチル－５β－コラン－２４－酸としても知られている。 As used herein, "Compound 3" is used.

Also known as 3α, 7α, 11β-trihydroxy-6α-ethyl-5β-cholane-24-acid.

を指し、これは、６α－エチル－２３（Ｓ）－メチル－３α，７α，１２α－トリヒドロキシ－５β－コラン－２４－酸としても知られている。米国特許第８，１１４，８６２号明細書に記載されている化合物４は、ＴＧＲ５モジュレーターである。実施形態の１つでは、ＴＧＲ５モジュレーターは、アゴニストである。 As used herein, "Compound 4" is used.

Also known as 6α-ethyl-23 (S) -methyl-3α, 7α, 12α-trihydroxy-5β-cholane-24-acid. Compound 4 described in US Pat. No. 8,114,862 is a TGR5 modulator. In one of the embodiments, the TGR5 modulator is an agonist.

The term "TGR5 modulator" means any compound that interacts with the TGR5 receptor. Interactions are not limited to compounds that act as antagonists, agonists, partial agonists or inverse agonists of the TGR5 receptor.

In general, the term "agonist" means a compound that enhances the activity of another molecule or receptor site. Agonists, by classical definition, are orthosteric agonists, allosteric agonists, inverse agonists, co-agonists, which have the property of binding to a receptor, altering the state of the receptor and exerting a biological effect. Bring. Therefore, agonism is defined as the nature of an agonist or ligand that produces a biological effect. More specifically, a TGR5 agonist is a receptor ligand or compound that binds to TGR5 and increases the concentration of cyclic adenosine monophosphate (cAMP) in cells expressing the receptor by at least 20%.

The expression "compound of the invention" as used herein includes compounds 1, 1-Na, 1-DEA, 2 and 3 or pharmaceutically acceptable salts or amino acid conjugates thereof.

The term "treating", as used herein, refers to any sign of response or cancer remission in treatment. Treatment can include, for example, reducing or alleviating the severity of one or more symptoms of cancer, or the patient experiences symptoms such as a disease, defect, disorder or adverse condition. It can include reducing the frequency. "Treatment" can also refer to reducing or eliminating the pathology of parts of the body such as cells, tissues or body fluids such as blood.

As used herein, the term "preventing" refers to the partial or complete prevention of cancer in an individual or population or part of the body such as cells, tissues or body fluids (eg, blood). The term "prevention" does not establish a requirement for complete prevention of a disease or condition in an individual or individual cells, tissues or body fluids throughout the treated population. The term "treat or prevent" is used herein to refer to a method that results in a certain level of treatment or remission of cancer, including, but not entirely limited to, cancer prevention, for that purpose. Intent on the various outcomes that have been achieved.

The term "therapeutically effective amount" as used herein shrinks and / or slows the growth of tumors (such as suppressing tumor growth) or other unwanted cells in cancer. Refers to an effective amount containing an amount sufficient to prevent or delay proliferation. In some embodiments, the effective amount is sufficient to delay the onset of cancer. In some embodiments, the effective amount is sufficient to prevent or delay recurrence. The effective amount can be administered in a single dose or in multiple doses. In the case of HCC or CRC, an effective amount of drug or composition may (i) reduce the number of tumor cells; (ii) reduce the size of the tumor; (iii) inhibit the infiltration of cancer cells into peripheral organs to some extent. Can be delayed, slowed, preferably arrested; (iv) can inhibit tumor metastasis (ie, can be slowed to some extent, preferably arrested); (v) can inhibit tumor growth; (Vi) The onset and / or recurrence of the tumor can be prevented or delayed; and / or (vii) one or more of the symptoms associated with the cancer can be alleviated to some extent.

As used herein, the term "regimen" refers to a protocol for the administration and / or time of administration of a compound of the invention for treating cancer. Regimen can include periods of active dosing and periods of rest, as is known in the art. The aggressive dosing period comprises administration of the compounds of the invention over a defined time course, including, for example, the number and duration of dosing of the composition. Some regimens can include one or more rest periods in which the compound is not actively administered, and optionally includes periods during which the effectiveness of such compounds may be minimal.

As used herein, "combination therapy" refers to the fact that a compound of the invention can be administered in combination with another therapeutic agent. "In combination with" means that one treatment mode is added to another treatment mode, for example, the compound of the present invention described in the present specification is administered to another therapeutic agent for the same subject. In addition, it refers to administration. Thus, "in combination with" refers to administering to a subject one treatment mode before, during or after delivery of the second treatment mode.

As used herein, "pharmaceutically acceptable" refers to a material that is not biologically or otherwise inconvenient, for example, the material being used in a pharmaceutical composition administered to a patient. It can be incorporated, does not cause significant and undesired biological effects, and does not interact in a harmful manner with any of the other constituents of the composition in which it is contained. The pharmaceutically acceptable carrier or additive meets the required criteria for toxicity and manufacturing testing and / or is on the Inactive Ingredient Guide prepared by the US Food and Drug Administration. included.

The term "about" is used herein when referring to measurable values such as quantity and length of time to carry out the methods disclosed or to make the disclosed compounds. And ± 20% or ± 10% from the specified values, ± 5% in some embodiments, some, so that such variations are appropriate for use and in the claimed method. Variations of ± 1% in embodiments and ± 0.1% in some embodiments shall be included.

又はその薬学的に許容される塩若しくはアミノ酸抱合体からなる群から選択されるＦＸＲアゴニストを投与するステップを含む方法に関する。一実施形態では、ＦＸＲアゴニストは、化合物１である。一実施形態では、ＦＸＲアゴニストは、化合物１－Ｎａである。別の実施形態では、ＦＸＲアゴニストは、化合物１－ＤＥＡである。一実施形態では、ＦＸＲアゴニストは、化合物２である。一実施形態では、ＦＸＲアゴニストは、化合物３である。 Methods of Treating Cancer The present invention is at least partially based on the finding that the compounds of the invention are effective in treating cancer in a mouse model that predicts human cancer. Accordingly, the present application is a method of treating or preventing cancer in a subject in need thereof and in therapeutically effective amounts of Compounds 1, 1-Na, 1-DEA, 2 and 3: 3.

Or a method comprising the step of administering an FXR agonist selected from the group consisting of a pharmaceutically acceptable salt or amino acid conjugate thereof. In one embodiment, the FXR agonist is compound 1. In one embodiment, the FXR agonist is compound 1-Na. In another embodiment, the FXR agonist is compound 1-DEA. In one embodiment, the FXR agonist is compound 2. In one embodiment, the FXR agonist is compound 3.

In the methods described herein, exemplary cancers are hepatocellular carcinoma, bile duct cancer, pancreatic cancer, prostate cancer, esophageal cancer, breast cancer, gastric cancer, renal cancer, salivary adenocarcinoma, ovarian cancer, uterine body cancer, bladder cancer and Selected from the group consisting of lung cancer. Appropriate treatment regimens for cancer depend on the type of cell from which the tumor is derived, the stage and severity of the malignant tumor, and the genetic abnormalities that contribute to the tumor.

The cancer staging system indicates the degree of cancer progression. In general, staging systems represent the extent of cancer spread and include patients with similar prognosis and treatment in the same staging group. In general, tumors that have become invasive or metastasized have a poor prognosis. In a type of staging system, cases are classified into four stages, represented by the Roman numerals I-IV. At stage I, the cancer is often localized and usually curable. Stage II and IIIA cancers are usually more advanced and may invade surrounding tissues and spread to the lymph nodes. Stage IV cancers include metastatic cancer that has spread to sites outside the lymph nodes.

Another staging system is the category, TNM staging, which stands for tumor, lymph node and metastasis. In this system, malignant tumors are represented according to the severity of each category. For example, T classifies the degree of the primary tumor by 0 to 4, 0 represents a malignant tumor having no invasive activity, and 4 represents a malignant tumor that has invaded other organs by spreading from the primary site. N represents the degree of lymph node metastasis, 0 represents a malignant tumor without lymph node metastasis, and 4 represents a malignant tumor with extensive lymph node metastasis. M classifies the degree of metastasis into 0 and 1, where 0 represents a malignant tumor without metastasis and 1 represents a malignant tumor that has metastasized.

These staging systems or variants of these staging systems or other suitable staging systems can be used to represent tumors. Depending on the stage and characteristics of the cancer, there are few options available for treating the cancer. Treatment includes surgery, treatment with sorafenib and targeted treatment. In general, surgery is the first choice for the treatment of localized cancer in the early stages. Additional systemic treatments can be used to treat invasive and metastatic tumors.

According to one aspect of the present invention, there is provided a method for treating hepatocellular carcinoma (or malignant hepatoma). Specifically, the method comprises treating a subject having hepatocellular carcinoma and in need of treatment with a therapeutically effective amount of a compound of the invention. That is, the present invention is directed to the use of the compound of the present invention for producing a pharmaceutical product for treating a hepatocellular carcinoma patient identified or diagnosed as having hepatocellular carcinoma. In another embodiment, the treatment method optionally comprises the step of diagnosing or identifying the patient as having hepatocellular carcinoma. The identified patient is then treated or administered with a therapeutically effective amount of the compound of the invention. Hepatocellular carcinoma can be diagnosed by any conventional diagnostic method known in the art, including ultrasound, CT scan, MRI, α-fetoprotein testing, des-γ carboxyprothrombin screening and biopsy.

The present invention also provides a method of treating treatment-resistant hepatocellular carcinoma, comprising treating a patient identified as having treatment-resistant hepatocellular carcinoma with a therapeutically effective amount of a therapeutically effective amount of a compound of the invention. In certain embodiments, the patient consists of sorafenib, regorafenib, anthracyclines (eg, doxorubicin, daunorubicin, epirubicin, idarubicin), platinum preparations (eg, cisplatin, carboplatin, oxaliplatin, picoplatin), 5-FU and capecitabine. Has hepatocellular carcinoma that is refractory to treatment involving one or more drugs selected from the group. The present invention comprises refractory hepatocellular carcinomas such as sorafenib, regorafenib, anthracyclines (eg doxorubicin, daunorubicin, epirubicin, idarubicin), platinum formulations (cisplatin, carboplatin, oxaliplatin, picoplatin), 5-FU and capecitabine. The use of the compounds of the invention for producing pharmaceuticals for treating hepatocellular carcinoma refractory to one or more selected drugs is also covered.

Patients undergoing initial treatment to detect treatment-resistant hepatocellular carcinoma can be carefully monitored for signs of resistance, non-responsiveness, or recurrence of hepatocellular carcinoma. This may include, for example, one or more drugs selected from the group consisting of sorafenib, regorafenib, doxorubicin, daunorubicin, epirubicin, idarubicin, cisplatin, carboplatin, oxaliplatin, picoplatin, 5-FU, tegafur and capecitabine. This can be achieved by monitoring the patient's cancer response to the procedure. Response to initial treatment, lack of response, or recurrence of cancer can be determined by any suitable method performed in the art. For example, this can be achieved by assessing tumor size and number of tumors. An increase in tumor size or instead an increase in tumor number indicates that the tumor has not responded to chemotherapy or has recurred. Judgment was made by Theasse et al, J. et al. Natl. Cancer Inst. It can be done according to the "RECIST" criteria detailed in 92: 205-216 (2000).

According to still another aspect of the invention, there is provided a method of preventing or delaying the onset of hepatocellular carcinoma (or hepatocellular carcinoma) or preventing or delaying the recurrence of hepatocellular carcinoma, wherein the method is: Includes the step of treating a patient in need of prevention or delay with a prophylactically effective amount of a compound of the invention.

Subjects infected with hepatitis B or hepatitis C or have cirrhosis are known to be at increased risk of developing hepatocellular carcinoma. In addition, people with acute and chronic hepatic porphyria (acute intermittent porphyria, late cutaneous porphyria, hereditary coproporphyria, diverse porphyria) and hypertyrosinemia type I also have hepatocellular carcinoma. There is a high risk of developing porphyria. All of these people may be candidates for the method of the invention to prevent or delay the development of hepatocellular carcinoma using prophylactically effective amounts of the compounds of the invention. In addition, patients with a family history of hepatocellular carcinoma can be identified for the application of the methods of the invention to prevent or delay the development of hepatocellular carcinoma. To prevent or delay the recurrence of hepatocellular carcinoma, patients with hepatocellular carcinoma who have been treated and are in remission or in a stable or non-progressive state are treated with prophylactically effective amounts of the compounds of the invention. Therefore, the recurrence or recurrence of hepatocellular carcinoma can be effectively prevented or delayed.

According to one aspect of the present disclosure, there is provided a method of treating bile duct cancer (CCA). Specifically, the method comprises treating a subject having bile duct cancer and in need of treatment with a therapeutically effective amount of a compound of the invention. That is, the present invention relates to the use of the compound of the present invention for producing a pharmaceutical product for treating a patient with bile duct cancer identified or diagnosed as having bile duct cancer. In another embodiment, the treatment method optionally comprises the step of diagnosing or identifying the patient as having bile duct cancer. The identified patient is then treated or administered with a therapeutically effective amount of the compound of the invention. Cholangiocarcinoma is any conventional diagnostic method known in the art, including ultrasound, CT scan, MRI, carcinoembryonic antigen (CEA) and sugar chain antigen 19-9 (CA19-9) screening and biopsy. Can be diagnosed with.

Yet another aspect of the invention provides a method of preventing or delaying the onset of bile duct cancer or preventing or delaying the recurrence of bile duct cancer, the method requiring prevention or delay. It comprises treating a patient with a prophylactically effective amount of a compound of the invention.

To prevent or delay the recurrence of bile duct cancer, patients with bile duct cancer who have been treated and are in remission or in a stable or non-progressive state are treated with prophylactically effective amounts of the compounds of the invention. The recurrence or recurrence of bile duct cancer can be effectively prevented or delayed.

According to another aspect of the present invention, there is provided a method of inhibiting the proliferation and migration of CCA cells with an FXR agonist.

According to another aspect of the invention, there is provided a method of inhibiting the proliferation and migration of CCA cells with a therapeutically effective amount of a compound of the invention. According to another aspect of the invention, a method of inhibiting the progression of CCA is provided, the method comprising treating a patient in need of the inhibition with a therapeutically effective amount of a compound of the invention.

One embodiment is a method of treating pancreatic cancer by administering a therapeutically effective amount of a compound of the invention. Another embodiment is a method of treating prostate cancer by administering a therapeutically effective amount of a compound of the invention. Another embodiment is a method of treating kidney cancer by administering a therapeutically effective amount of a compound of the invention. Another embodiment is a method of treating prostate cancer by administering a therapeutically effective amount of a compound of the invention. Yet another embodiment is a method of treating esophageal cancer by administering a therapeutically effective amount of a compound of the invention. Yet another embodiment is a method of treating breast cancer by administering a therapeutically effective amount of a compound of the invention. One embodiment is a method of treating gastric cancer by administering a therapeutically effective amount of a compound of the invention. Another embodiment is a method of treating renal cancer by administering a therapeutically effective amount of a compound of the invention. Yet another embodiment is a method of treating salivary gland cancer by administering a therapeutically effective amount of a compound of the invention. Yet another embodiment is a method of treating ovarian cancer by administering a therapeutically effective amount of a compound of the invention. One embodiment is a method of treating endometrial cancer by administering a therapeutically effective amount of a compound of the invention. Another embodiment is a method of treating bladder cancer by administering a therapeutically effective amount of a compound of the invention. Yet another embodiment is a method of treating lung cancer by administering a therapeutically effective amount of a compound of the invention.

If the compounds of the invention are useful in the treatment of the cancers described herein, such compounds can be administered in combination with a second agent. Second agents useful in the methods of treating cancer provided herein include any known class of anti-cancer agents such as radiation therapy, surgery, alkylating agents, antimetabolites, anthracycs, campotecins. , Vinca alkaloids, taxanes or platinum and other antineoplastic agents known in the art. The classification of such anti-cancer and anti-neoplastic agents is known in the art and is used in accordance with its explicit usual meaning.

Exemplary anti-cancer agents include, but are not limited to, abraxane; avilateron; ace-11; acralubicin; acibicin; acodazole hydrochloride; acronin; actinomycin; acylfluben; adecipenor; adzelesin; adriamycin; aldesroykin; all. -Trans-retinoic acid (ATRA); altretamine; ambamustine; ambomycin; amethanetron acetate; amidox; amistin; aminoglutetimid; aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole; Aphidicholine glycine acid; aprinic acid; ara-CDP-DL-PTBA; arginine deaminase; ARRY-162; ARRY-300; ARRY-142266; AS703026; asparaginase; asperlin; aslaculin; atamestane; attrimstine; avastin; axinastatin 1; Axinastatin 2; Axinastatin 3; Azacetron; Azatoxin; Azatyrosine; Azacitidine; AZD8330; Azetepa; Azotomycin; Baranol; Batimastat; BAY 11-7082; BAY 43-9006; BAY 869766; Bendamustine; Bendamustine; Benzodepa; Staulosporin; β-alethin; β-clamycin B; bethuric acid; b-FGF inhibitor; bicartamide; bisantren; bisaziridinyl spermin; bisnaphthide; bisnaphthide dimesylate; Busulfan; Biselecin; Breflate; Voltezomib; Brequinalsodium; Bropyrimin; Budotitanium; Butionine sulfoxymin; Briostatin; Cactinomycin; Carsterone; Carsipotiol; Carmustine C; Camptothecin derivative; Capecitabin; Carboxamide-amino-triazole; Carboxy Amidotriazole; CaRest M3; CARN 700; Carasemido; Calvetimer; Carboplatin; Carmustine; Carbisin Hydrochloride; Calzeresin; Castanospermin; Seclopin B; Sedefingol; Celecoxib; Cetrorelix; Cisplatin; CI-1040; cis-porphyrin; fludarabine; clomiphene analog; clotrimazole; chorismycin A; chorismycin B; combretastatin A4; combretastatin analog; conagenin; clambecidin 816; chrysnator; mesylic acid Chrisnatol; cryptophycin 8; cryptophycin A derivative; clacin A; cyclopentaanthraquinones; cycloplatam; cisplatin; cyclophosphamide; cytarabine; cytarabine ocphosphate; cytolytic factor; cytostatin; dacarbadine; dactinomycin Daunorubicin; daunorubicin hydrochloride; decarbazine; dacriximab; dasatinib; decitarabine; dehydrodidemnin B; deslorerin; dexamethasone; dexifosfamide; dexrazoxane; dexverapamil; dexolmaplatin; Didemnin B; didox; diethylnorspermin; dihydro-5 azacitidine; dihydrotaxol; 9-dioxamycin; diphenylspiromustin; docosanol; drasetron; docetaxel; doxorubicin; doxorubicin hydrochloride; doxifludarabine; droroxyphen; Dromostanolone propionate; dronabinol; duazomycin; duocarumycin SA; ebuserene; ecomstin; edelhosin; edrecolomab; edatorexate; eflornitine hydrochloride; eflornitin; elemene; emiteflu; elsamitorcin; enroplatin; empromart; Epristeride; Elbrosol; Elibrin; Esorbicin Hydrochloride; Estramstin; Estramstin Sodium Phosphate; Ethanidazole; Etoposide; Etoposide Phosphate; Etoprin; Exemestane; Flavopyridol; Frezerastin; Fluasterone; Floxuridine; Fludarabine Phosphate; Fludarabine; Fluorodaunorubicin Hydrochloride; Horphenimex; Formestane; Fluorouracil; Floxouridine; Flulocytarabine; Triesin Sodium; Hostriecin; Hotemstin; Gadrinium texaphyllin; Gallium Nitrate; Galositabin; Ganirelix; Geratinase Inhibitors; Gemcitabine; Gerdanamycin; Gosipole; GDC-0973; GSK11120212 / Tramethinib; Herceptin; Hydroxyurea; Bisacetamide; Hypericin; Ibandronic acid; Ibrutinib; Idarubicin; Idarubicin hydrochloride; Ifosfamide; Canphosfamide; Ilmohosin; Iproplatin; Idoxyphen; Idramanton; Ilmohosin; Iromastat; 201); yobenguan; iododoxorubicin; ipomeanol; irinotecan; irinotecan hydrochloride; irsogladine; isovengazole; isohomohalicondorin B; itasetron; imofosine; rlL. sub. 2); Interferon Alpha-2a; Interferon Alpha-2b; Interferon Alpha-n1; Interferon Alpha-n3; Interferon Beta-1a; Interferon Gamma-1b; Jaspraquinolide; Kahalalide F; Lameralin N Triacetate; Lanleothide; Reina Mycin; renoglastim; sulfated lentinan; leptolstatin; retrozole; leuprorelin; levamisol; renalidemid; lembatinib; ariarosol; rissoclinamide 7; lovaplatin; lobbricin; rometrexol; lonidamine; losoxantrone; lobastatin; Lysophylline; lanleotide acetate; laparib; retrozol; leucovorin; leuprolide acetate; lyarozole hydrochloride; rometrexol sodium; romustin; rosoxantrone hydrochloride; pomalidemid; LY294002; mytancin; mannostatin A; marimastert; Matrilysin inhibitor; menogalyl; melvalon; metererin; methioninase; methoclopramide; MIF inhibitor; mifepriston; myrtehosine; mirimostim; mitogazone; mitoxantrone; mitonafide; mitoxantrone; mophalotene; Mechloretamine hydrochloride; Megustrol acetate; Melengestrol acetate; Melfaran; Mercaptopurine; Metotrexate; Mettrexate sodium; Metpurin; Metsuredepa; Mitindomid; Mitocalcin; Mitochromin; Mitogirin; Mitomarcin; Mitomycin; Mycophenolic acid; Nafaleline; Nagrestop; Napabin; Nafterpine; Nartgrastim; Nedaplatin; Nemorphicin; Neridronic acid; Niltamide; Nisamicin; Nitrogen monoxide modulator; Nitrullynin; Obrimersen (genasens); octreotide; oxenone; olaparib (limperza); oligonucleotides; onapriston; ondancetron; olacin; oral cytokine inducer; ormaplatin; oxysla Oxaloplatin; Osateron; Oxaliplatin; Oxanomycin; Parauamine; Palmitoyl lysoxin; Pamidronic acid; Panaxitriol; Panomiphen; Parabactin; PARP (poly ADP-ribose polymerase) inhibitor; Polysodium polysulfate; pentostatin; pentrozole; perflubron; perphosphamide; perylyl alcohol; phenadinomycin; phenylacetate; phosphatase inhibitor; picibanil; pyrocarpine hydrochloride; pirarubicin; pyritrexim; placetin A; placetin B; porphyromycin Prednison; Prostaglandin J2; Pyrazoloacridine; Paclitaxel; PD035901; PD184352; PD318206; PD98059; Periomycin; Pentamustine; Pepromycin sulfate; PKC412; Pipobroman; Piposulfan; Pyroxanthron hydrochloride; Plicamycin; Promethan; Phyllotoxin; Polyphenol E; Porphimer Sodium; Porphyromycin; Predonimustin; Procarbazine; Procarbazine Hydrochloride; Puromycin; Puromycin Hydrochloride; Pyrazofluin; Rohitzkin; Lomustine; Lokinimex; Rubiginone B1; Luboxyl; Ribopurine; Lomidepsin; Lucaparib; Saffingol; Saffingol Hydrochloride; Cytopine; Sarcophytol A; Sargramostim; (Solverol); Sonermin; Soraphenib; Snitinib; Sparphoic acid; Plicamycin D; Spiromustine; Sprenopentin; Spongitatin 1; Spongitatin 2; Spongitatin 3; Spongitatin 4; Spongitatin 5; Spongitatin 6; spongistatin 7; spongistatin 8; and spongistatin 9; squalamine; stipamide; stromerycin inhibitor; sulfinosin; sladista; slamin; swinesonin; SB239603; selmethinib / AZD6244; simtrazen; SP600125; spalho Setona Triu Mu; sparsomycin; spirogermanium hydrochloride; spiroplatin; streptnigrin; streptozosine; slophenul; talimstin; tamoxyphenmethiodide; tarazoparib (BMN 673); tauromustin; tazarotene; tecogalan sodium; tegafur; Temozoromide; teniposide; tetrachlorodecaoxide; tetrazomine; tariblastin; thiocholalin; thrombopoetin; simulfacin; thymopoetin receptor agonist; thymotrinan; tyrapazamine; titanosen dichloride; topcentin; tremiphen; trethinoin; triacetyluridine; Tryptreline; tropisetron; turosteride; tyrphostins; talysomycin; TAK-733; taxotere; tegafur; teloxanthrone hydrochloride; teloxylone; test lactone; thiamipulin; thioguanine; thiotepa; thiazofurin; tyrapazamine; tremiphencitrate; trussutzumab; Tronacetate; Tricilibine Phosphate; Trimetrexate; Glucronate Trimetrexate; Tryptrelin; Tubrozole Hydrochloride; Tumor Necrosis Factor-Associated Antiproliferative ligand (TRAIL); UBC Inhibitor; Ubenimex; U0126; B; veralasole; veriparib (ABT-888); veramine; verteporfin; binorelbin; vinxartine; vinblastine; vinblastine; vinblastine sulfate; vindesine sulfate; vindesine; vindesine sulfate; vinesine sulfate; vindesine sulfate; vindesine sulfate; Examples include tartrate; vinlosine sulfate; vinzolysine sulfate; borozole; Waltmannin; XL518; zanoteron; xeniplatin; dilascolb; dinostatin stimalamer; dinostatin; and sorbicin hydrochloride.

Other exemplary anticancer agents include elbrozole (eg, R-55104); drastatin 10 (eg, DLS-10 and NSC-376128); mibobline isethionate (eg, CI-980); NSC-639829; discodelmolide (eg, eg). , NVP-XX-A-296); ABT-751 (Abbott; eg E-7010); Altorhyrtin A; Altorhyrtin C; Semadotin hydrochloride (eg LU-103973 and NSC). -D-669356); CEP9722; Epothilone A; Epothilone B; Epothilone C; Epothilone D; Epothilone E; Epothilone F; Epothilone BN-Oxide; 21-Hydroxyepothilone D; 26-fluoroepothilone; Auristatin PE (eg, NSC-654663); Sobridotin (eg, TZT-1027); LS-4559-P (Pharmacia; eg, LS-4757); LS-4578 (Pharmacia; for example, LS-477-P); LS-4477 (Pharmacia); LS-4559 (Pharmacia); RPR-11278 (Aventis); DZ-3358 (Daiichi Sankyo Co., Ltd.); FR-18287 (Fujisawa) Yakuhin Kogyo Co., Ltd .; for example, WS-9265B; GS-164 (Takeda Yakuhin Kogyo Co., Ltd.); GS-198 (Takeda Yakuhin Kogyo Co., Ltd.); KAR-2 (Hungarian Academy of Sciences); BSF-223651 (BASF; For example, ILX-651 and LU-223651); SAH-49960 (Lily / Novartis); SDZ-268970 (Lily / Novartis); AM-97 (Armad / Kyowa Hakko Kirin Co., Ltd.); AM-132 (Armad); AM -138 (Armad / Kyowa Hakko Kirin Co., Ltd.); IDN-5005 (Indena); cryptophysin 52 (eg, LY-355703); AC-7739 (Ajinomoto Pharmaceutical Co., Ltd .; eg, AVE-8063A and CS-39.HCl). ); AC-7700 (Ajinomoto Pharmaceutical Co., Ltd .; for example, AVE-8062; AVE-8062A; CS-39-L-Ser.HCl; and RPR-258062A); (Vitylevuamide); Tubricin A; Canadensol; CA-170 (Curis, Inc. ); Centauresin (eg, NSC-106969); T-138067 (Tularik; eg, T-67; TL-138067 and TI-138067); COBRA-1 (Parker Medicine Institute; eg, DDE-261 and WHI-). 261); H10 (Kansas State University); H16 (Kansas State University); Oncosidin A1 (eg, BTO-956 and DIME); DDE-313 (Parker Hugges Institute; ); SPA-1 (Parker Huges Institute; eg SPIKET-P); 3-IAABU (Cytoskeleton / Mt. Sinai School of Medicine; eg MF-569); Narcosine (eg NSC-5366); D-24851 (Asta Medica); A-105972 (Abbott); Hemiasterlin; 3-BAABU (Cytoskeleton / Mt. Sinai School of Medicine; for example, MF-191); TMPN (Arizona Station) Acetonate; T-138026 (Tularik); Monsatorol; inanocine (eg, NSC-698666); 3-IAABE (Cytoskeleton / Mt.Sinai School of Medicine); A-2047 607 (Tularik; eg, T-960607); RPR-115781 (Aventis); Eloiterobins (eg, desmethyleroyterobin; Desateylleutherobin; Isoeroyterobin A; and Z-eroiterobin) Cariveoside; Caribeolin; Haricondorin B; D-64131 (Asta Medicine); D-68144 (Asta Medicine); Diazonamide A; A-293620 (Abbott); NPI-2350 (Neeus); Nolide A; TUB-245 (Aventis); A-259754 (Abbott); Diozostatin; (-)-Phenylahistin (eg NSCL-96F037); D-6268 (Asta Medica); D-62366 (Asta) Medica); Myoseberin B; D-43411 (Zentaris; eg, D-81862); A-289099 (Abbott); A-318315 (Abbott); HTI-286 (eg, SPA-110; Trifluoroacetate) (Wyeth). ); D-82317 (Zentaris); D-82318 (Zentaris); SC-12983 (NCI); Sodium resverastatin phosphate; BPR-OY-007 (National Health Research Institutes); and SSR-250411 (Sanofi)) Goselelin; Leuprolide; Tryptride; Homoharingtonin; Topotecan; Itraconazole; Deoxyadenosine; Sertralin; Pitabatatin; Clofamidin; 5-Nonyloxytryptamine; Bemurafenib; Dabrafenib; Gefitinib (Iressa); Erlotinib (Tarceva); Erlotinib (Tarceva); (Tykerb); Panitummab (Vectuximab); Vandetanib (Caprelsa); Afatinib / BIBW2992; CI-1033 / Canertinib; Neratinib / HKI-272; CP-714714; TAK-285; AST-1306; ARRY334543; AG-1478; dacomitinib / PF299804; OSI-420 / desmethylerlotinib; AZD8931; AEE726; peritinib / EKB-569; CUDC-101; WZ8040; WZ4002; WZ3146; AG-490; XL647; Aza-2'-deoxycitidine; 17-N-allylamino-17-demethoxygeldanamycin (17-AAG); 20-ep-1,25 dihydroxyvitamin D3; 5 ethynyluracil; and BMS-599626.

One embodiment is a method of treating a patient with hepatocellular carcinoma (arbitrarily refractory to treatment) by administering the compound of the present invention in combination with capecitabine and / or PLX4032 (Plexixikon).

Another embodiment is a method for treating hepatocellular carcinoma (arbitrarily refractory to treatment) by administering the compound of the present invention in combination with capecitabine, Xeloda and / or CPT-11.

Another embodiment is a method of treating hepatocellular carcinoma (arbitrarily refractory to treatment) by administering the compound of the present invention in combination with capecitabine, Xeloda and / or CPT-11.

In another embodiment, a patient with hepatocellular carcinoma (optionally refractory to treatment) or a patient with unresectable or metastatic hepatocellular carcinoma is administered by administering the compound of the present invention in combination with capecitabine and irinotecan. It is a method of treatment.

Another embodiment is a method of treating a patient with unresectable or metastatic hepatocellular carcinoma by administering the compound of the present invention in combination with interferon alpha or capecitabine.

Another embodiment is a method of treating a patient with pancreatic cancer by administering the compound of the present invention in combination with gemcitabine.

One embodiment is a method of treating a patient with CCA by administering the compound of the present invention in combination with another one agent or a plurality of agents or a combination thereof.

One embodiment is a method of treating a patient with CCA by using the compound of the present invention in combination with gemcitabine, cisplatin or a combination thereof.

Pharmaceutical composition A "pharmaceutical composition" is a pharmaceutical product containing the compound of the present invention in a form suitable for administration to a subject. In one embodiment, the pharmaceutical composition is in bulk or unit dosage form. It may be advantageous to formulate the composition in dosage unit form for ease of administration and uniform dose. The specifications of the dosage unit form are determined by the unique characteristics of the active reagent and the specific therapeutic effect to be achieved and are directly dependent on them.

Candidate formulations include those suitable for oral, sublingual, buccal, parenteral (eg, subcutaneous, intramuscular or intravenous), transrectal, topical (including percutaneous), intranasal and inhalation. Can be mentioned. The optimal mode of administration for a particular patient will depend on the nature and severity of the disease being treated or the nature of the treatment used and the nature of the active compound, but if possible, oral administration may be used to prevent and prevent cancer. Can be used for treatment. Suitable formulations for oral administration are tablets, capsules, cachets, lozenges, etc., each as a separate unit containing a predetermined amount of active compound, as a powder or granule, a solution in an aqueous or non-aqueous solution. Alternatively, it may be provided as a suspending agent or as an oil in water or an emulsion in water in oil.

Suitable formulations for sublingual or buccal administration include troche agents containing the compounds of the present invention and flavor bases such as sugar and gum arabic or tragant, and active compounds such as gelatin and glycerin or sucrose gum arabic. Examples thereof include pastel agents contained in the active base.

A suitable formulation for parenteral administration usually contains a sterile aqueous solution containing a predetermined concentration of the active compound, which solution can be isotonic with the blood of the recipient of interest. Other formulations suitable for parenteral administration include formulations containing physiologically suitable co-solvents and / or surfactants and complexing agents such as cyclodextrins. The oil-in-water emulsion is also a suitable formulation for parenteral formulations. Such solutions can be administered intravenously, but can also be administered by subcutaneous or intramuscular injection.

Suitable formulations for transrectal administration may be provided as a unit dose suppository containing the compound of the invention in one or more solid carriers forming a suppository base, such as cocoa butter.

Suitable formulations for topical or intranasal application include ointments, creams, lotions, pastes, gels, sprays, aerosols and oils. Suitable carriers for such formulations include petrolatum, lanolin, polyethylene glycol, alcohols and combinations thereof.

The formulations of the invention were obtained by any suitable method (usually the compounds of the invention were uniformly and tightly mixed with the liquid and / or micronized solid carrier in the required proportions and then as needed. It can be prepared (by molding the mixture into the desired shape).

For example, a tablet may compress a homogeneous mixture containing the active ingredient of a powder or granules and one or more optional ingredients such as a binder, a lubricant, an inert diluent or a surfactant dispersant. Or by molding a homogeneous mixture of powdered active ingredient and inert liquid diluent. Suitable formulations for administration by inhalation include microparticulate powders or mists that can be produced using various types of quantitative pressurized aerosols, nebulizers or inhalers.

For oral intrapulmonary administration, the particle size of the powder or droplets is typically in the range of about 0.5-10 μm or 1-5 μm to ensure delivery into the bronchial tree. For nasal administration, particle sizes in the range of about 10-500 μm can be used to ensure retention in the nasal cavity.

The metered dose inhaler is typically a pressurized aerosol dispenser containing a suspension or solution formulation containing the compound of the invention in a liquefied propellant. In use, these devices dispense the pharmaceutical product through a valve designed to deliver a measured volume, typically about 10-150 μm, to produce a particulate spray containing the active ingredient. Suitable propellants include certain chlorofluorocarbon compounds such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane and mixtures thereof. The pharmaceutical product may further contain one or more co-solvents, such as ethanol surfactants such as oleic acid or sorbitan trioleate, antioxidants and suitable flavoring agents.

A nebulizer is a commercially available device that converts a solution or suspension of an active ingredient into a therapeutic aerosol mist by accelerating a compressed gas (usually air or oxygen) through a narrow Venturi orifice or by ultrasonic agitation. .. A suitable formulation for use in an atomizer consists of the active ingredient in a liquid carrier and may contain up to about 40% w / w of the formulation and less than about 20% w / w. The carrier is usually water or a diluted aqueous alcohol solution, and is preferably isotonic with body fluid by adding, for example, sodium chloride. Optional additives include preservatives (eg, hydroxy-methyl benzoate), antioxidants, flavoring agents, volatile oils, buffers and surfactants if the formulation is not prepared sterilized. ..

Suitable formulations for administration by aeration include finely divided powders that can be taken up and delivered into the nasal cavity using an inhaler or by inhaling through the nose. In an inhaler, the powder is usually contained in capsules or cartridges made of gelatin or plastic, which are perforated or opened in situ, inhaled or manually operated. With a pump, the powder is delivered by air that is drawn in and passes through the device. The powder used in the inhaler consists solely of the active ingredient or a powder blend containing the active ingredient, a suitable powder diluent such as lactose, and an optional surfactant. The active ingredient usually constitutes about 0.1-100% w / w of the pharmaceutical product.

In a further embodiment, the invention provides a pharmaceutical composition comprising the compound of the invention as an active ingredient in combination with and / or in admixture with at least one pharmaceutical carrier or diluent.

The carrier must be pharmaceutically acceptable and compatible with the other components in the composition, i.e., must not adversely affect the other components in the composition. The carrier can be solid or liquid and is preferably formulated as a unit dose formulation, eg, a tablet which can contain from about 0.05 to 95% by weight of the active ingredient. If desired, other physiologically active ingredients may also be incorporated into the pharmaceutical compositions of the present invention.

In addition to the components specifically described above, the pharmaceutical product of the present invention may contain other agents known to those skilled in the art of pharmaceuticals, taking into consideration the type of the pharmaceutical product of interest. For example, a formulation suitable for oral administration may contain a flavoring agent, and a formulation suitable for intranasal administration may contain a flavoring agent.

In one embodiment, the pharmaceutical composition comprises from about 0.1-1500 mg, 0.2-1200 mg, 0.3-1000 mg, 0.4-800 mg, 0.5-600 mg, 0.6- In a dosage form containing 500 mg, 0.7 to 400 mg, 0.8 to 300 mg, 1 to 200 mg, 1 to 100 mg, 1 to 50 mg, 1 to 30 mg, 4 to 26 mg, 5 to 25 mg, or 5 to 10 mg in a daily total amount. Be administered.

The compounds of the present invention may be used in combination with other drugs for treating hepatocellular carcinoma (such as anticancer chemotherapeutic agents, hormones, biological response regulators and other angiogenesis inhibitors), or for immunotherapy or immunotherapy. It can be used in combination with gene therapy.

Example 1. Synthesis of Compound 1 Compound 1 can be prepared by a method known in the art (eg, the method described in US Pat. No. 7,923,244). For example, compound 1 can be prepared by the process shown in Scheme 1 and disclosed in WO 2014/066819.

Step 1 is a step of esterifying compound 2 to obtain compound 4. Step 2 is a reaction for forming compound 5 from compound 4. Step 3 is a step of protecting the hydroxy group at the C3 position of compound 5 to obtain compound 6. Step 4 is a step of obtaining compound 7 by oxidative cleavage of compound 6. Step 5 is a step of reducing compound 7 to obtain compound 8. Step 6 is a step of sulfonation of compound 8 to obtain a sodium salt (1-Na) of compound 1. The sodium salt of compound 1 is converted to its free acid form (ie, compound 1) or other salt form (eg, compound 1-DEA or N, N-dietaneamine salt of compound 1) according to procedures known in the art. can do.

Example 2. Synthesis of Compound 2 Compound 2 is a method described in conventional methods (eg, US Patent Application Publication No. 2009/0062526, US Pat. No. 7,138,390 and International Publication No. 2006/122977). ), For example, can be prepared by 6-step synthesis following product compound 1 (obeticholic acid or OCA) shown in Scheme 2 below.

The above process is a 6-step synthesis. Step 1 is a step of esterifying the C-24 carboxylic acid of 7-ketritocholic acid (KLCA) with methanol in the presence of an acidic catalyst and heat to produce the methyl ester compound 7. Step 2 is the formation of a silyl enol ether which produces compound 8 from compound a by using a strong base and then treating with chlorosilane. Step 3 is a step of forming compound 10 by subjecting silyl enol ether of compound 8 and acetaldehyde to an aldol condensation reaction. Step 4 is a step of saponifying the C-24 methyl ester of compound 10 to produce compound 11. Step 5 is a step of hydrogenating the 6-ethylidene moiety of compound 11 to produce compound 12. Step 6 is a step of selectively reducing the 7-keto group of compound 12 to a 7α-hydroxy group to produce compound 1.

Example 3. Liver cancer-forming multidrug-resistant proteins 2 (Abcb4) in Mdr2 ^{− / −} mice and FXR ^{− / −} mice are members of the superfamily of ATP-binding cassette (ABC) transporters. Multidrug-resistant protein two-gene knockout mice (Mdr2 ^-/- ) provide an in vivo model of spontaneous liver cancer formation (Katzenellengoben, et al. Mol. Cancer Res. 2007, 5, 11, 1159-1170). Mice deficient in the Abc4 protein encoded by the multidrug-resistant protein 2 gene develop chronic periductal inflammation and cholestasis liver disease, resulting in hepatocellular carcinoma.

Farnesoid X receptor protein (FXR) is a nuclear receptor that functions as a bile acid sensor that regulates bile acid homeostasis. This receptor is highly expressed in the liver and other organs. FXR knockout (FXR ^{− / −} ) mice develop hepatocellular adenoma and hepatocellular carcinoma after 15 months of age (Yang, et al. Cancer Res., 2007, 67, 863).

The effects of OCA, compound 1-Na and control diet on the development of hepatocellular carcinoma in Mdr2 ^-/- mice and FXR ^-/- mice were evaluated. OCA is an FXR agonist and compound 1-Na is an FXR / TGR5 dual agonist. The efficacy of compound 1-Na on FXR is about 10 times stronger than OCA.

Test Design Mdr2 ^-/- mice and FXR ^-/- mice were randomly divided into three experimental groups. Mice were fed a specific rodent diet, ie, a diet supplemented with OCA, compound 1-Na or a control diet for 15 months. All mice were bred in a temperature controlled room (23 ° C.) with a light / dark cycle of 12 hours in the absence of pathogens and allowed to drink freely. The University of Bari's Ethical Committee of the University of Bari has approved this experimental composition, which is based on the internationally recognized guidelines for animal breeding by the Italian Ministry of Health. Was also approved. After 16 months, the mice were disposed of and serum, liver and intestines were collected. The total number of liver tumors was counted and the diameter of each tumor was measured.

Treatment group 1: Control feed mice (n = 4) were fed a control feed for 15 months.
Group 2: OCA
Mice (n = 8) were fed a control diet supplemented with OCA at a dose of 10 mg / kg for 15 months.
Group 3: Compound 1-Na
Mice (n = 15) were fed a control diet supplemented with compound 1-Na at a dose of 10 mg / kg for 15 months.

Results Hepatocyte dysplasia develops due to liver inflammation and toxicity caused by bile salts in Mdr2 ^-/- mice. By 16 months of age, nearly 100% of Mdr2 ^-/- control mice developed liver tumors. 16-month-old FXR ^{− / −} mice developed spontaneous hepatocellular carcinoma.

Tumor Reduction and Size Figures 1A and 2A show the effect of Compound 1-Na, OCA and controls on tumor number reduction in Mdr2 ^-/- mice. Compound 1-Na clearly prevented the development of hepatocellular carcinoma in this study compared to controls. Statistic significance was largely observed for the compound 1-Na group (p = 0.055) but was not achieved due to the small number of control animals used in this study (n = 2). Clearly identifiable liver tumors were found in Mdr2 ^- control mice and mice treated with OCA, but microtumors were found in the compound 1-Na group. FIGS. 1B and 2B show the effect of Compound 1-Na, OCA and controls on the percentage reduction of tumors larger than 5 mm in diameter. Approximately 80% of the tumors found in the OCA and control groups exceeded 5 mm in diameter in Mdr2 ^-/- mice. Conversely, FXR ^{− / −} mice treated with compound 1-Na, OCA and controls showed several large liver tumors, which were largely FXR-dependent in preventing the development of hepatocellular carcinoma. (FIGS. 3A, 3B and 4).

Liver weight / body weight (LW / BW)
5A and 5B illustrate the effects of Compound 1-Na and controls on liver weight and percentage of body weight. Consistent with previously generated data, Mdr2 ^-/- mice treated with compound 1-Na had a significantly reduced LW / BW ratio compared to control and OCA-treated Mdr2 ^-/- mice. No difference in LW / BW ratio was observed in the FXR ^{− / −} group.

Biochemical parameters Compound 1-Na and compound 1-Na for levels of the liver enzymes alanine aminotransferase (ALT) and aspartate aminotransferase (AST) to assess liver injury in Mdr2 ^{− / −} mice and FXR ^{− / −} mice. The effect of the control was analyzed. As shown in FIGS. 6A and 6B, treatment with compound 1-Na significantly reduced ALT and AST levels in Mdr2 ^-/- mice. However, no difference in ALT and AST levels was observed in FXR ^{− / −} treated mice.

Expression of FXR target gene in the ileum To demonstrate the involvement of FXR in the prevention of hepatocellular carcinoma, the effects of compound 1-Na and controls on the expression of the FXR target gene in the ileum were evaluated. As expected, both OCA and compound 1-Na stimulated gene expression of fibroblast growth factor 15 (Fgf15) and small molecule heterodimer partner (Shp) only in the Mdr2 ^{− / −} group (FIG. 7A). And 7B).

Liver FXR Target Gene Expression Cholesterol 7α-hydroxylase (cyp7a1) is a rate-determining enzyme in the classical biosynthetic pathway that converts cholesterol to bile acids. Both compounds 1-Na and OCA inhibited Cyp7a1 gene expression only in Mdr2 ^-/- mice (FIG. 8). A bile acid efflux pump (Bsep) is a membrane protein that actively transports bile acids using the energy of ATP hydrolysis. As shown in FIGS. 9A and 9B, administration of compound 1-Na induced Bsep activation of the liver in Mdr2 ^-/- mice. OCA did not promote hepatic Bsep induction, which in contrast to compound 1-Na, which efficiently activates FXR in the intestine and liver, where OCA increased hepatic activity in Mdr2 ^-/- mice. It suggests that it is difficult to hold.

Total serum bile acid compound 1-Na significantly reduced serum bile acid levels in Mdr2 ^-/- mice (FIG. 10A). The FXR dependence of this finding was confirmed by observing no reduction in FXR ^{− / −} mice (FIG. 10B).

Example 4. Different Effects of FXR or TGR5 Activation in Bile Duct Cancer Progression FXR and TGR5 Expression in CCA Human Biopsy Samples and Controls by Two Techniques (mRNA Microarray and qPCR) and Two Different Patient Cohorts (Copenhagen and San Sebastian) ) And in different human CCA cell lines as compared to normal human bile duct cells (NHC) (by qPCR). Proliferation of sympathetic model human CCA in immunocompromised mice with continuous administration of specific FXR or TGR5 agonists (OCA or Compound 4, respectively; 0.03% in solid feed for 2 months; Intercept Pharmaceuticals). It was evaluated by magnetic resonance imaging (MRI). Different effects of FXR or TGR5 activation were also evaluated in vitro for CCA and NHC cell proliferation, migration and mitochondrial energy metabolism.

Results In both the Copenhagen cohort and the San Sebastian cohort (by mRNA microarray and qPCR, respectively), in human CCA tissue samples, FXR was downregulated and TGR5 was elevated compared to normal perihepatic tissue and normal intrahepatic bile duct. Be controlled. By controlling proliferation, migration and mitochondrial energy metabolism, FXR activation inhibits CCA progression, whereas TGR5 activation can promote CCA progression. Controlling the activity of FXR and TGR5 has potential as a therapeutic strategy for CCA. In vitro, it was also found that in different human CCA cell lines, FXR was downregulated and TGR5 was upregulated compared to NHC.

As shown in FIGS. 11A-11D, FXR expression is reduced in CCA tumors and correlates with tumor differentiation: FIG. 11A shows the entire tissue (n) of CCA tumors as compared to human peripheral tissue (n = 60). = 104) represents the FXR mRNA microarray expression (Copenhagen cohort) (Man-Whitney test); FIG. 11B shows the degree of tumor differentiation: well-differentiated (n = 10), moderately-differentiated (n = 34) or poorly-differentiated. FXR mRNA microarray expression (Copenhagen cohort) (Man-Whitney test) in all tissues of CCA tumors grouped by (n = 9); FIG. 11C shows normal human liver tissue (n = 20) and human liver peripheral tissue. FXR mRNA expression (qPCR) (San Sebastian cohort) (Man-Whitney test) in CCA tumor (n = 5) as compared to (n = 7); and FIG. 11D shows the corresponding human liver peri-tissue (n = 7). Represents FXR mRNA expression (qPCR) (San Sebastian cohort) (paired t-test) in CCA tumors when compared to n = 4).

As shown in FIGS. 12A-12D, TGR5 expression is increased in CCA tumors, peripulmonary CCA is higher than intrahepatic CCA, and correlates with perineuronal infiltration (FIG. 12A). FIG. 12B represents TGR5 mRNA microarray expression (Copenhagen cohort) (Mann-Whitney test) in all tissues (n = 104) of CCA tumors when compared to human peripheral tissues (n = 60). FIG. 12C shows the clinicopathological parameters: anatomical site [peri-hilar (n = 36) or intrahepatic (n = 68)] and perineuronal infiltration [negative (n = 50) or positive (n = 42). )] Shows TGR5 mRNA microarray expression (Copenhagen cohort) (Man-Whitney test) in all tissues of CCA tumors. FIG. 12C represents TGR5 mRNA expression (qPCR) (San Sebastian cohort) (Mann-Whitney test) in CCA tumors (n = 15) when compared to human liver peri-tissue (n = 27). TGR5 mRNA expression (qPCR) (San Sebastian cohort) (Wilcoxon matched pair code order test) in CCA tumors when compared to the corresponding human liver peri-tissue (n = 11) is shown in FIG. 12D.

As shown in FIGS. 13A and 13B, in CCA cell lines, FXR expression is reduced and TGR5 expression is increased when compared to normal human bile duct cells: FIG. 13A shows normal human bile duct cells (n = 6). ) And CCA cell lines (n = 5 and 6 respectively) show FXR mRNA expression (qPCR) (Man-Whitney test or unpaired t-test), FIG. 13B shows normal human bile duct cells (n = 6) and CCA. TGR5 mRNA expression (qPCR) (unpaired t-test or Man-Whitney test) in cell lines (n = 5 and 4 respectively) is shown.

Continuous administration of the FXR agonist OCA to an orthotopic CCA mouse model inhibited tumor growth compared to untreated animals. This effect was associated with reduced expression of PCNA and Ki67 in the tumors of treated animals. As shown in FIGS. 14A-14D, the FXR agonist obeticholic acid (OCA) inhibited tumor growth in vivo. This was associated with reduced expression of proliferation, bile and epithelial markers. EGI1 cells were subcutaneously injected into male CD1 nude mice. As the tumor grew, the tumor was orthotopically transplanted into the liver of a male CD1 nude mouse. Two weeks later, MRI analysis was performed and treatment was started in the feed. Tumor growth was monitored by MRI at 1 and 2 months. FIG. 14A shows representative MRI and liver images of untreated control mice, mice treated with OCA and mice treated with Compound 4. The bar graph in FIG. 14B shows MRI quantified tumor volume magnification changes (control n = 8, OCA n = 6 and compound 4 n = 9 (Mann-Whitney test, unilateral). FIG. 14C is liver sympatrogenic. Represents mRNA expression levels of growth markers (ie Ki67 and PCNA), bile markers (ie CK19) and epithelial markers (ie ZO-1) in CCA tumors. N = 5-8 (Mann-Whitney or unpaired t). Test, unilateral). Representative immunohistochemical images of growth markers (ie Ki67 and PCNA) in liver sympathetic CCA tumors of untreated or treated with OCA or Compound 4 are shown in FIG. 14D. In contrast, no effect on CCA tumor growth was observed in animals continuously treated with compound 4, which is a TGR5 agonist.

FIG. 17 shows the effect of OCA and Compound 4 on the expression of growth markers for liver xenograf tumors. MRNA expression levels of growth markers (ie, Cdc25a, cyclin D1 and cyclin D3) in hepatic sympathetic CCA tumors. n = 5-8 (Mann-Whitney or unpaired t-test, one side).

Activation of FXR with OCA in vitro inhibited CCA cell proliferation and migration. These phenomena were associated with decreased mitochondrial energy metabolism (ie, decreased basal respiration, maximal respiration and ATP production-related respiration) when compared to controls. On the other hand, FXR activation did not affect the survival of CCA cells. As shown in FIGS. 15A-15D, the FXR agonist OCA inhibits CCA cell proliferation in a dose-dependent manner and inhibits CCA cell migration (these are associated with reduced metabolism of mitochondria in CCA cells). However, it does not induce apoptosis. FIG. 15A shows CCA cells treated with 10 or 25 μM OCA for 48 hours compared to untreated control cells (n = 4) performed using CFSE cell proliferation dye staining by flow cytometry. That is, it shows proliferation of EGI1) (n = 5) (unpaired t-test). This assay was performed in at least 3 separate tests. MRNA expression of proliferation markers (ie Ki67, PCNA, cyclin D1 and cyclin D3) in CCA cells (ie EGI1) treated with OCA (25 μM) for 3-6-12 hours when compared to untreated control cells. N = 5-6, unpaired t-test for untreated controls. FIG. 15B shows the results of the migration assay in CCA cells (ie EGI1). Representative microscopic images of wound healing assays (ie untreated controls or treated with OCA) at indicated time points and conditions and corresponding quantifications (n = 6 for each condition) (unpaired t-test). The assay was performed in two separate tests. Representative microscopic images of the transwell migration chamber at 24 hours in untreated cells and cells treated with OCA. This assay was performed once. FIG. 15C shows the rate of oxygen consumption (OCR) by Seahorse using a mitochondrial stress test kit in CCA cells (ie EGI1) untreated or treated with OCA (25 μM) after 3 hours of pretreatment. Bar graph of metabolic parameters calculated after OCR measurement. N = 11-12 for each group (unpaired t-test). This assay was performed in at least 3 separate tests. FIG. 15D represents a flow cytometry-based apoptosis assay by Annexin V and propidium iodide staining in untreated or treated with 10 or 25 μM OCA (ie EGI1). Quantification of representative histograms and corresponding pool data from two separate tests, total n = 6-7 (unpaired t-test). This assay was performed in three separate tests.

In contrast, activation of TGR5 with compound 4 stimulated CCA cell proliferation and migration. These phenomena were associated with increased mitochondrial energy metabolism (ie, increased basal respiration, proton leak and ATP production-related respiration) when compared to controls. As shown in FIGS. 16A-16C, compound 4, which is a TGR5 agonist, slightly stimulates CCA cell proliferation, migration and mitochondrial metabolism. FIG. 16A shows CCA cells (ie, EGI1) treated with 10 or 25 μM Compound 4 for 48 hours compared to untreated control cells analyzed using CFSE cell proliferation dye staining by flow cytometry. Shows proliferation. n = 5-6 (unpaired t-test). This assay was performed in three separate tests. MRNA expression of proliferation markers (ie Ki67, PCNA, cyclin D1 and cyclin D3) in CCA cells (ie EGI1) treated with compound 4 (25 μM) for 3-6-12 hours when compared to untreated control cells. n = 6 (unpaired t-test or Mann-Whitney test for untreated controls). FIG. 16B represents a migration assay in CCA cells (ie EGI1). Representative microscopic images and corresponding quantifications of wound healing assays (ie untreated controls or treated with Compound 4) at the indicated time points and conditions. Pool data (unpaired t-test) from two separate experiments (total n = 9 and 6). This assay was performed in at least 3 separate tests. Representative microscopic images of transwell migration chambers at 24 hours in untreated or treated with Compound 4 and corresponding quantifications (n = 4 and 2). This assay was performed in three separate tests. FIG. 16C shows the rate of oxygen consumption (OCR) by Seahorse using a mitochondrial stress test kit in CCA cells (ie, EGI1) untreated or treated with compound 4 (25 μM) after 3 hours of pretreatment. .. Bar graph of metabolic parameters calculated after OCR measurement. N = 11-12 for each group (unpaired t-test). This assay was performed in at least 3 separate tests.

Claims (7)

A composition for treating or preventing bile duct cancer in a subject in need thereof , comprising a FXR agonist having a daily dose of 1 to 100 mg, wherein the FXR agonist is compound 2 :

Or a composition which is a pharmaceutically acceptable salt or amino acid conjugate thereof. The composition according to claim 1 , wherein the FXR agonist is a glycine conjugate of compound 2. The composition according to claim 1 , wherein the FXR agonist is a taurine conjugate of compound 2. The composition according to claim 1 , wherein the FXR agonist is a sarcosine conjugate of compound 2. A pharmaceutical composition comprising a FXR agonist having a daily dose of 1 to 100 mg and a pharmaceutically acceptable additive for treating or preventing bile duct cancer in a subject in need thereof. The FXR agonist is compound 2 :

Or a pharmaceutical composition which is a pharmaceutically acceptable salt or amino acid conjugate thereof. A kit for treating or preventing bile duct cancer in a subject in need thereof , in Compound 2:

Or a kit comprising a pharmaceutically acceptable salt or amino acid conjugate thereof, wherein Compound 2 or the pharmaceutically acceptable salt or amino acid conjugate thereof is administered in a total daily dose of 1 to 100 mg . The use of a 1-100 mg FXR agonist in the manufacture of a pharmaceutical product for the treatment or prevention of bile duct cancer in a subject in need thereof, wherein the FXR agonist is compound 2 :

Or its pharmaceutically acceptable salt or amino acid conjugate , use.

Images