Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
  • A61K31/575—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of three or more carbon atoms, e.g. cholane, cholestane, ergosterol, sitosterol
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
  • A61K47/542—Carboxylic acids, e.g. a fatty acid or an amino acid
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents

Definitions

• Cancer is characterized primarily by an increase in the number of abnormal cells derived from a given normal tissue, invasion of adjacent tissues by the abnormal cells, or lymphatic or blood-borne spread of malignant cells to regional lymph nodes and to distant sites (metastasis).
• Clinical data and molecular biologic studies indicate that cancer is a multistep process that begins with minor pre-neoplastic changes, which may under certain conditions progress to neoplasia.
• HCC hepatocellular carcinoma
• CCA cholangiocarcinoma
• HCC is the most common form of primary liver cancer, and develops within the hepatocyte. HCC occurs mostly in men and patients that suffer from cirrhosis. HCC is the one of the most common cancers worldwide and the third most common cause of cancer-related deaths. The disease is often diagnosed late in the course of clinical manifestation. As a result, only 10- 15% of patients are candidates for curative surgery. For the majority of HCC patients, systemic chemotherapies or supportive therapies are the mainstay treatment options.
• cholangiocellular carcinoma or bile duct cancer which mainly develops in the small bile duct epithelial cells (i.e. cholangiocytes) within the liver.
• This type of cancer is more common among women.
• the incidence of cholangiocarcinoma (CCA) is increasing worldwide and already represents the second most common primary liver cancer.
• CCA patients show poor outcome due to late diagnosis and the refractory nature of these tumors.
• doxorubicin a high dosage of tamoxifen in combination doxorubicin or EA-PFL (etoposide, adriamycin, cisplatin, fluorouracil and leucovorin), is an effective example.
• the remission rate of these drugs can achieve levels between 15 and 30%.
• the patients of hepatocellular carcinoma usually develop complication of liver cirrhosis and other organ damage.
• Cholangiocarcinoma is a relatively rare neoplasm that is classified as an
• adenocarcinoma a cancer that forms glands or secretes significant amounts of mucins. It has an annual incidence rate of 1-2 cases per 100,000 in the Western world, but rates of cholangiocarcinoma have been rising worldwide over the past few decades.
• Cholangiocarcinoma is considered to be an incurable and rapidly lethal cancer unless both the primary tumor and any metastases can be fully removed by surgery. No potentially curative treatment exists except surgery, but most people have advanced stage disease at presentation and are inoperable at the time of diagnosis. People with cholangiocarcinoma are generally managed, though not cured, with chemotherapy, radiation therapy, and other palliative care measures.
• the present invention addresses these needs. Therefore, it is the object of the present invention to provide improved methods of treating or preventing cancer such as hepatocellular carcinoma and cholangiocarcinoma.
• Figure 1 A is a bar graph showing the effects of Compound 1-Na and control diet on the number of hepatic tumors in multidrug resistance protein 2 (Mdr2 "/_ ) knockout mice. *p ⁇ 0.01 vs. control.
• Figure IB is a bar graph showing the effects of Compound 1-Na and control diet on the percent reduction of tumors (>5mm diameter) in Mdr2 _/" mice.
• Figure 2A is a bar graph showing the effects of OCA (Compound 2) and control diet on the number of hepatic tumors Mdr2 _/" knockout mice.
• Figure 2B is a bar graph showing the effects of OCA and control diet on the percent reduction of tumors (>5mm diameter) in Mdr2 _/" mice.
• Figure 3 A is a bar graph showing the effects of Compound 1-Na and control diet on the number of hepatic tumors in Farnesoid X Receptor (FXR "/_ ) mice.
• Figure 3B is a bar graph showing the effects of Compound 1-Na and control diet on the percent reduction of tumors (>5mm diameter) in FXR _/" mice.
• Figure 4 is a bar graph showing the effects of Compound 1-Na and control diet on the percent reduction on the number of hepatic tumors in FXR _/" mice.
• Figure 5 A is a bar graph showing the effects of Compound 1-Na and control diet on the percent reduction on liver/body weight ratio in Mdr2 _/" mice. *p ⁇ 0.01 vs. control.
• Figure 5B is a bar graph showing the effects of Compound 1-Na and control diet on the percent reduction on liver/body weight ratio in FXR _/" mice.
• Figure 6 A is a bar graph showing the effect of Compound 1-Na and control diet on alanine transaminase (ALT) levels in Mdr2 _/" and FXR _/" mice. *p ⁇ 0.01 vs. control.
• Figure 6B is a bar graph showing the effect of Compound 1-Na and control diet on aspartate transaminase (AST) levels in Mdr2 _/" and FXR _/" mice. *p ⁇ 0.01 vs. control.
• Figure 7 A is a bar graph showing the effect of Compound 1-Na and control diet on ileal gene expression of fibroblast growth factor 15 (Fgfl5) in Mdr2 _/" and FXR _/” mice. *p ⁇ 0.01 vs. control.
• Figure 7B is a bar graph showing the effect of Compound 1-Na and control diet on ileal gene expression of small heterodimer partner (Shp) in Mdr2 _/" and FXR _/" mice.
• Showp small heterodimer partner
• control Figure 8 is a bar graph showing the effect of Compound 1-Na and control diet on the downregulation of cholesterol 7 alpha-hydroxylase (cyp7al) in Mdr2 _/" and FXR _/" mice.
• Figure 9 A is a bar graph showing the effect of Compound 1-Na and control diet on hepatic gene expression of small heterodimer partner (Shp) in Mdr2 _/" and FXR _/" mice. *p ⁇ 0.01 vs. control.
• Figure 9B is a bar graph showing the effect of Compound 1-Na and control diet on hepatic gene expression of bile salt export pump (Bsep) in Mdr2 _/" and FXR _/" mice. *p ⁇ 0.01 vs. control
• Figure 1 OA is a bar graph showing the effect of Compound 1-Na and control diet on the total serum bile acids in Mdr2 _/" mice. *p ⁇ 0.01 vs. control.
• Figure 10B is a bar graph showing the effect of Compound 1-Na3and control diet on the total serum bile acids in FXR _/" mice.
• Figure 11 A shows FXR mRNA microarray expression in whole tissue of CCA tumors compared to surrounding human tissue.
• Figure 1 IB shows FXR mRNA microarray expression in whole tissue of CCA tumors grouped upon tumor differentiation grade.
• Figure 11C shows FXR mRNA expression (qPCR) in CCA tumors compared to normal human liver tissue and surrounding human liver tissue.
• Figure 1 ID shows FXR mRNA expression (qPCR) in CCA tumors compared to matched- surrounding human liver tissue.
• Figure 12A shows TGR mRNA microarray expression in whole tissue of CCA tumors compared to surrounding human tissue.
• Figure 12B shows TGR5 mRNA microarray expression in whole tissue of CCA tumors upon clinico-pathological parameters: anatomical location and perineural invasion.
• Figure 12C shows TGR5 mRNA expression (qPCR) in CCA tumors compared to surrounding human liver tissue.
• Figure 12D shows TGR5 mRNA expression (qPCR) in CCA tumors compared to matched- surrounding human liver tissue.
• Figure 13 A shows FXR mRNA expression (qPCR) in normal human cholangiocytes and CCA cell lines.
• Figure 13B shows TGR5 mRNA expression (qPCR) in normal human cholangiocytes and CCA cell lines.
• Figure 14A shows representative MRI and liver images of untreated control mice, OCA- treated mice and Compound 4-treated mice.
• Figure 14B is a bar-graph showing tumor volume fold-change quantified by MRI.
• Figurel4C shows mRNA expression levels of proliferation (i.e. Ki67 and PCNA), biliary (i.e. CK19) and epithelial (i.e. ZO-1) markers in liver orthotopic CCA tumors.
• proliferation i.e. Ki67 and PCNA
• biliary i.e. CK19
• epithelial i.e. ZO-1
• Figure 14D shows representative immunohistochemistry images of proliferation markers (i.e. Ki67 and PCNA) in liver orthotopic CCA tumors of mice untreated or treated with OCA or Compound 4.
• proliferation markers i.e. Ki67 and PCNA
• Figure 15A shows mRNA expression levels of proliferation markers upon OCA treatment and proliferation of CCA cells (i.e. EGIl) treated with OCA at 10 or 25 ⁇ for 48 h compared to non-treated control cells, performed by flow cytometry using CFSE cell proliferation dye staining.
• Figure 15B shows migration assays in CCA cells (i.e. EGIl) and representative microscope images of wound-healing assay, corresponding quantification and representative microscope images of transwell migration chambers at 24 h.
• FIG. 15C shows Seahorse Oxygen Consumption Rate (OCR) using mitochondrial stress test kit in CCA cells (i.e. EGIl) and a bar-graph of metabolic parameters calculated upon OCR measurements.
• OCR Seahorse Oxygen Consumption Rate
• Figure 15D shows flow cytometry -based apoptosis assays by Annexin V and Propidium Iodide staining in CCA cells (i.e. EGIl) non-treated or treated with 10 or 25 ⁇ of OCA and representative histograms and corresponding quantification of pooled data.
• Figure 16A shows mRNA expression levels of proliferation markers upon Compound 4 treatment and proliferation of CCA cells (i.e. EGIl) treated with Compound 3 at 10 or 25 ⁇ for 48 h compared to non-treated control cells.
• CCA cells i.e. EGIl
• Figure 16B shows migration assays in CCA cells (i.e. EGIl), representative microscope images of wound-healing assay and corresponding quantification, and representative microscope images of transwell migration chambers at 24 h in non-treated and Compound 4- treated cells and corresponding quantification.
• FIG 16C shows Seahorse Oxygen Consumption Rate (OCR) using mitochondrial stress test kit in CCA cells (i.e. EGIl) non-treated or treated with Compound 4 (25 ⁇ ) and a bar-graph of metabolic parameters calculated upon OCR measurements.
• Figure 17 shows mRNA expression levels of proliferation (i.e. Cdc25a, cyclin Dl and cyclin D3) markers in liver orthotopic CCA tumors.
• the present application relates to methods of treating or preventing cancer in a subject in need thereof comprising administering a therapeutically effective amount of a Farnesoid X Receptor (FXR) agonist.
• FXR Farnesoid X Receptor
• the FXR agonist is Compound 1 or Compound
• the cancer is selected from the group consisting of hepatocellular carcinoma, pancreatic cancer, kidney cancer, prostate cancer, esophageal cancer,
• the cancer is hepatocellular carcinoma.
• the cancer is pancreatic cancer.
• the cancer is kidney cancer.
• the cancer is prostate cancer. In one
• 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
• the cancer is salivary gland cancer. In one embodiment, the cancer is ovarian cancer. In one embodiment, the cancer is uterine body cancer. In one embodiment, the cancer is lung cancer.
• the FXR agonist is Compound 1 or a pharmaceutically acceptable salt thereof.
• the FXR agonist is the sodium salt of Compound 1 (i.e., Compound 1-Na).
• the FXR agonist is the ⁇ , ⁇ -diethaneamine salt of Compound 1 (i.e. Compound 1-DEA).
• the FXR agonist is Compound 2 or a pharmaceutically acceptable salt or amino acid conjugate thereof.
• the FXR agonist is the glycine conjugate of Compound 2.
• the FXR agonist is the taurine conjugate of Compound 2.
• the FXR agonist is the sarcosine conjugate of Compound 2.
• the present invention further relates to the use of Compound 1 or a pharmaceutical iy acceptable salt thereof or Compound 2 or a pharmaceutically acceptable salt or amino acid conjugate thereof in the manufacture of a medicament for treating or preventing cancer in a subject in need thereof.
• the present invention further relates to Compound 1 or a pharmaceutically acceptable salt thereof and Compound 2 or a pharmaceutically acceptable salt or amino acid conjugate thereof for use in treating or preventing cancer in a subject in need thereof.
• the present invention also relates a pharmaceutical composition
• a pharmaceutical composition comprising
• the present invention further relates to a kit for treating or preventing cancer in a subject in need thereof comprising Compound 1 or a pharmaceutically acceptable salt thereof and Compound 2 or a pharmaceutically acceptable salt or amino acid conjugate thereof.
• the present invention is based at least in part on the discovery that Compounds 1 and 2 were effective in treating cancer in animal models predictive of cancer. As described in the examples below, the inventors have discovered that a compound of the invention suppressed tumor growth in murine models of spontaneous hepatocarcinogenesis.
• cancer refers to any of the diseases characterized by the presence of cancerous tissue in a subject.
• cancer tissue refers to a tissue that comprises malignant neoplastic cells, exhibits an abnormal growth of cells and/or hyperproliferative cells.
• Cancerous tissue can be a primary malignant tumor, arising from a tissue or organ of origin, or it can be a metastatic malignant tumor, growing in a body tissue which was not the source of the original tumor.
• tumor can include a solid tumor or a cancer of
• the tumor may be characterized by its ability to invade surrounding tissues, to metastasize to other parts of the body, and/or by its angiogenic activity.
• Exemplary tumors result from hepatocellular carcinoma, gastric cancer, renal cancer, prostate cancer, adrenal cancer, pancreatic cancer, breast cancer, bladder cancer, salivary gland cancer, ovarian cancer, uterine body cancer, and lung cancer.
• the term "invasive" refers to the process by which a cell, a group of cells, or a malignancy spreads from a site to adjacent sites.
• metal refers to the process by which a cell, a group of cells, or a malignancy spreads from a site to sites not adjacent to the first site.
• hepatocellular carcinoma As used herein, “hepatocellular carcinoma”, “HCC”, and “malignant hepatoma” are used interchangeably and refer to primary and secondary (metastasized) tumors that originate from the liver tissue.
• the term “refractory hepatocellular carcinoma” as used herein refers to hepatocellular carcinoma that fails to respond favorably to an antineoplastic treatment.
• a hepatocellular carcinoma refractory to a treatment refers to a hepatocellular carcinoma that fails to respond favorably to, or resistant to the treatment, or alternatively, recurs or relapses after responding favorably to the treatment.
• cholangiocarcinoma or “CCA” or “cholangiocellular carcinoma” or a bile duct cancer is a form of cancer that is composed of mutated epithelial cells (or cells showing characteristics of epithelial differentiation) that originate in the bile ducts which drain bile from the liver into the small intestine.
• a proportion of CCAs arises under cholestatic liver conditions.
• intrahepatic accumulation of bile acids does not induce carcinogenesis directly they may facilitate the cocarcinogenic effect by promoting
• Compound 1-Na or "1 -Na” which is also known as 6a-ethyl-3a,7a,23-trihydrox 24-nor-5p-cholan-23-sulphate sodium” are used interchangeably and refer to the sodium salt of Compound 1.
• Compound 1-DEA or “1-DEA” which is also known as 6a-ethyl-3o,7a,23-trihydroxy-24-nor-5p-cholan-23-sulphate N,N-diethylethaneamiiie” are used interchangeably and refer to the ⁇ , ⁇ -diethylethaneamine salt of Compound 1.
• the structures of Compound 1 -Na and Compound 1 -DEA are provided below.
• OCA obeticholic acid
• 6-ECDCA 6-alpha-ethyl chenodeoxycholic acid
• 6a-ethyl-3a,7a-dihydroxy-5P-cholan-24-oic acid OCA
• OCA obeticholic acid
• 6-ECDCA 6-alpha-ethyl chenodeoxycholic acid
• 6a-ethyl-3a,7a-dihydroxy-5P-cholan-24-oic acid 6a-ethyl-3a,7a-dihydroxy-5P-cholan-24-oic acid
• TGR5 modulator which is also known as 6a-ethyl-23(S)-methyl-3a,7a,12a-trihydroxy-5P-cholan-24-oic acid.
• Compound 4 described in U.S. Patent No. 8, 114,862 is a TGR5 modulator.
• TGR5 modulator is an agonist.
• TGR5 modulator means any compound that interacts with the TGR5 receptor. The interaction is not limited to a compound acting as an antagonist, agonist, partial agonist, or inverse agonist of the TGR5 receptor.
• agonist means a compound that enhances the activity of another molecule or receptor site.
• An agonist by classical definition, whether a orthosteric, allosteric, inverse or a co-agonist has a property to bind to the receptor, alter its receptor state and result in a biological action. Consequently, agonism is defined as a property of an agonist or a ligand to produce a biological action.
• a TGR5 agonist is a receptor ligand or compound that binds to TGR5 and increases the concentration of cyclic adenosine
• cAMP monophosphate
• a "compound of the invention” as used herein encompasses Compounds 1, 1 -Na, 1-DEA, 2, and 3, or a pharmaceutically acceptable salt or amino acid conjugate thereof.
• treating refers to any indicia of success in the treatment or amelioration of the cancer. Treating can include, for example, reducing or alleviating the severity of one or more symptoms of the cancer, or it can include reducing the frequency with which symptoms of a disease, defect, disorder, or adverse condition, and the like, are experienced by a patient. “Treating” can also refer to reducing or eliminating a condition of a part of the body, such as a cell, tissue or bodily fluid, e.g., blood.
• the term “preventing” refers to the partial or complete prevention of the cancer in an individual or in a population, or in a part of the body, such as a cell, tissue or bodily fluid (e.g., blood).
• prevention does not establish a requirement for complete prevention of a disease or condition in the entirety of the treated population of individuals or cells, tissues or fluids of individuals.
• treat or prevent is used herein to refer to a method that results in some level of treatment or amelioration of the cancer, and contemplates a range of results directed to that end, including but not restricted to prevention of the cancer entirely.
• terapéuticaally effective amount refers to an effective amount comprising an amount sufficient to cause a tumor to shrink and/or to decrease the growth rate of the tumor (such as to suppress tumor growth) or to prevent or delay other unwanted cell proliferation in cancer.
• an effective amount is an amount sufficient to delay the development of cancer.
• an effective amount is an amount sufficient to prevent or delay recurrence.
• An effective amount can be administered in one or more administrations.
• the effective amount of the drug or composition may: (i) reduce the number of tumor cells; (ii) reduce tumor size; (iii) inhibit, retard, slow to some extent and preferably stop cancer cell infiltration into peripheral organs; (iv) inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay occurrence and/or recurrence of tumor, and/or (vii) relieve to some extent one or more of the symptoms associated with cancer.
• a regimen can include periods of active administration and periods of rest as known in the art. Active administration periods include administration of a compound of the invention in a defined course of time, including, for example, the number of and timing of dosages of the compositions. In some regimens, one or more rest periods can be included where no compound is actively administered, and in certain instances, includes time periods where the efficacy of such compounds can be minimal.
• combination therapy refers to a compound of the invention can be administered in conjunction with another therapeutic agent.
• “In conjunction with” refers to administration of one treatment modality in addition to another treatment modality, such as administration of a compound of the invention as described herein in addition to
• administering refers to administration of one treatment modality before, during, or after delivery of a second treatment modality to the subject.
• pharmaceutically acceptable refers to a material that is not biologically or otherwise undesirable, e.g., the material may be incorporated into a
• compositions administered to a patient without causing any significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the composition in which it is contained.
• Pharmaceutically acceptable carriers or excipients have met the required standards of toxicological and manufacturing testing and/or are included on the Inactive Ingredient Guide prepared by the U.S. Food and Drug administration.
• the present invention is based at least in part based on the discovery that a compound of the invention is effective in treating cancer in murine models predictive of cancer in humans. Accordingly, the present application relates to methods of treating or preventing cancer in a subject in need thereof comprising administering therapeutically effective amount of a FXR agonist selected from the group consisting of Compounds 1, 1-Na, 1-DEA, 2, and 3 :
• 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.
• exemplary cancers are selected from the group consisting of hepatocellular carcinoma, cholangiocarcinoma, pancreatic cancer, prostate cancer, esophageal cancer, breast cancer, gastric cancer, renal cancer, salivary gland cancer, ovarian cancer, uterine body cancer, bladder cancer, and lung cancer.
• the appropriate treatment regimen for cancer depends on the type of cell from which the tumor derived, the stage and severity of the malignancy, and the genetic abnormality that contributes to the tumor.
• Cancer staging systems describe the extent of cancer progression. In general, the staging systems describe how far the cancer has spread and puts patients with similar prognosis and treatment in the same staging group. In general, there are poorer prognoses for tumors that have become invasive or metastasized.
• cases are grouped into four stages, denoted by Roman numerals I to IV.
• Stage I cancers are often localized and are usually curable.
• Stage II and IIIA cancers are usually more advanced and may have invaded the surrounding tissues and spread to lymph nodes.
• Stage IV cancers include metastatic cancers that have spread to sites outside of lymph nodes.
• TNM staging which stands for the categories: Tumor, Nodes, and Metastases.
• malignancies are described according to the severity of the individual categories. For example, T classifies the extent of a primary tumor from 0 to 4 with 0 representing a malignancy that does not have invasive activity and 4 representing a malignancy that has invaded other organs by extension from the original site.
• N classifies the extent of lymph node involvement with 0 representing a malignancy with no lymph node involvement and 4 representing a malignancy with extensive lymph node involvement.
• M classifies the extent of metastasis from 0 to 1 with 0 representing a malignancy with no metastases and 1 representing a malignancy with metastases.
• staging systems or variations of these staging systems or other suitable staging systems may be used to describe a tumor. Few options are available for the treatment of cancer depending on the stage and features of the cancer. Treatments include surgery, treatment with sorafenib, and targeted therapies. In general, surgery is the first line of treatment for early stage localized cancer. Additional systemic treatments may be used to treat invasive and metastatic tumors.
• a method for treating hepatocellular carcinoma comprises treating a subj ect in need thereof having hepatocellular carcinoma with a therapeutically effective amount of a compound of the invention. That is, the present invention is directed to the use a compound of the invention for the manufacture of medicaments for treating hepatocellular carcinoma patients identified or diagnosed as having hepatocellular carcinoma.
• the treatment method optionally comprises a step of diagnosing or identifying a patient as having hepatocellular carcinoma. The identified patient is then treated with or administered with a therapeutically effective amount of a compound of the invention.
• Hepatocellular carcinoma can be diagnosed in any conventional diagnostic methods known in the art including ultrasound, CT scan, MRI, alpha- fetoprotein testing, des-gamma carboxyprothrombin screening, and biopsy.
• the present invention also provides a method of treating refractory hepatocellular carcinoma comprising treating a patient identified as having refractory hepatocellular carcinoma with a therapeutically effective amount of a compound of the invention.
• the patient has a hepatocellular carcinoma that is refractory to a treatment comprising one or more drugs selected from the group consisting of sorafenib, regorafenib, anthracyclines (e.g., doxorubicin, daunorubicin, epirubicin, idarubicin), platinum agents (e.g., cisplatin, carboplatin, oxaliplatin, picoplatin), 5-FU and capecitabine.
• drugs selected from the group consisting of sorafenib, regorafenib, anthracyclines (e.g., doxorubicin, daunorubicin, epirubicin, idarubicin), platinum agents (e.g.,
• the present invention is also directed to the use of a compound of the invention for the manufacture of medicaments for treating refractory hepatocellular carcinoma, e.g., a hepatocellular carcinoma refractory to one or more drugs selected from sorafenib, regorafenib, anthracyclines (e.g., doxorubicin, daunorubicin, epirubicin, idarubicin), platinum agents (cisplatin, carboplatin, oxaliplatin, picoplatin), 5-FU and capecitabine.
• drugs selected from sorafenib, regorafenib, anthracyclines (e.g., doxorubicin, daunorubicin, epirubicin, idarubicin), platinum agents (cisplatin, carboplatin, oxaliplatin, picoplatin), 5-FU and capecitabine.
• recurring hepatocellular carcinoma This can be accomplished by monitoring the patient's cancer's response to the initial treatment which, e.g., may include 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.
• the response, lack of response or relapse of the cancer to the initial treatment can be determined by any suitable method practiced in the art. For example, this can be accomplished by the assessment of tumor size and number.
• An increase in tumor size or, alternatively, tumor number indicates that the tumor is not responding to the chemotherapy or that a relapse has occurred.
• the determination can be done according to the "RECIST" criteria as described in detail in Therasse et al, J. Natl. Cancer Inst. 92:205-216 (2000).
• a method for preventing or delaying the onset of hepatocellular carcinoma (or hepatocellular carcinoma), or preventing or delaying the recurrence of hepatocellular carcinoma which comprises treating a patient in need of the prevention or delay with a prophylactically effective amount of a compound of the invention.
• hepatocellular carcinoma patients with a family history of hepatocellular carcinoma can also be identified for the application of the present method of preventing or delaying the onset of hepatocellular carcinoma.
• hepatocellular carcinoma patients who have been treated and are in remission or in a stable or progression free-state may be treated with a prophylactically effective amount of a compound of the invention to effectively prevent or delay the recurrence or relapse of hepatocellular carcinoma.
• a method for treating cholangiocarcinoma comprises treating a subject in need thereof having cholangiocarcinoma with a therapeutically effective amount of a compound of the invention. That is, the present invention is directed to the use a compound of the invention for the manufacture of medicaments for treating cholangiocarcinoma patients identified or diagnosed as having cholangiocarcinoma.
• the treatment method optionally comprises a step of diagnosing or identifying a patient as having cholangiocarcinoma. The identified patient is then treated with or administered with a therapeutically effective amount of a compound of the invention.
• Cholangiocarcinoma can be diagnosed in any conventional diagnostic methods known in the art including ultrasound, CT scan, MRI, carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA19-9) screening, and biopsy.
• a method for preventing or delaying the onset of cholangiocarcinoma, or preventing or delaying the recurrence of cholangiocarcinoma which comprises treating a patient in need of the prevention or delay with a prophylactically effective amount of a compound of the invention.
• cholangiocarcinoma patients who have been treated and are in remission or in a stable or progression free-state may be treated with a prophylactically effective amount of a compound of the invention to effectively prevent or delay the recurrence or relapse of
• a method for inhibiting proliferation and migration of CCA cells with an FXR agonist.
• a method for inhibiting proliferation and migration of CCA cells with a therapeutically effective amount of a compound of the invention.
• a method for inhibiting progression of CCA which comprises treating a patient in need 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.
• esophageal cancer by administering a therapeutically effective amount of a compound of the invention.
• 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.
• a method of treating renal cancer by administering a therapeutically effective amount of a compound of the invention.
• a method of treating salivary gland cancer by administering a therapeutically effective amount of a compound of the invention.
• 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 uterine body cancer by administering a therapeutically effective amount of a compound of the invention.
• a method of treating bladder cancer by administering a therapeutically effective amount of a compound of the invention.
• a method of treating lung cancer by administering a therapeutically effective amount of a compound of the invention.
• a compound of the invention is useful for the treatment of a cancer described herein, such compounds can be co-administered with a second agent.
• the second agent useful in methods of treating cancers provided herein can include any known class of anti-cancer agents such as, for example, radiation therapy, operations, alkylating agents, antimetabolites, anthracyclines, campothecins, vinca alkaloids, taxanes or platinums, as well as other antineoplastic agents known in the art.
• anti-cancer agent and antineoplastic agent classifications are known in the art and used in accordance with their plain and ordinary meaning.
• anti-cancer agents include but are not limited to: ABRAXA E;
• abiraterone ace-11; aclarubicin; acivicin; acodazole hydrochloride; acronine; actinomycin; acylfulvene; adecypenol; adozelesin; adriamycin; aldesleukin; all trans-retinoic acid (ATRA); altretamine; ambamustine; ambomycin; ametantrone acetate; amidox; amifostine;
• aminoglutethimide aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole; andrographolide; antarelix; anthramycin; aphidicolin glycinate; apurinic acid; ara-CDP-DL-
• PTBA arginine deaminase
• ARRY-162 ARRY-300
• ARRY- 142266 ARRY- 142266
• AS703026 AS703026
• axinastatin 2 axinastatin 3; azasetron; azatoxin; azatyrosine; azacitidine; AZD8330; azetepa; azotomycin; balanol; batimastat; BAY 11-7082; BAY 43-9006; BAY 869766; bendamustine; benzochlorins; benzodepa; benzoylstaurosporine; beta-alethine; betaclamycin B; betulinic acid; b-FGF inhibitor; bicalutamide; bisantrene; bisaziridinylspermine; bisnafide; bisnafide dimesylate; bistratene A; bisantrene hydrochloride; bleomycin; bleomycin sulfate; busulfan; bizelesin; breflate; bortezomib; brequinar sodium; bropirimine; budotitane
• CARN 700 caracemide; carbetimer; carboplatin; carmustine; carubicin hydrochloride;
• chloroquinoxaline sulfonamide cicaprost; chlorambucil; Chlorofusin; cirolemycin; cisplatin;
• collismycin B combretastatin A4; combretastatin analogue; conagenin; crambescidin 816; crisnatol; crisnatol mesylate; cryptophycin 8; cryptophycin A derivatives; curacin A;
• cyclopentanthraquinones cycloplatam; cypemycin; cyclophosphamide; cytarabine; cytarabine ocfosfate; cytolytic factor; cytostatin; dacarbazine; dactinomycin; daunorubicin; daunorubicin hydrochloride; decarbazine; dacliximab; dasatinib; decitabine; dehydrodidemnin B;
• dezaguanine dezaguanine mesylate; diaziquone; didemnin B; didox; diethylnorspermine; dihydro 5 azacytidine; dihydrotaxol; 9-dioxamycin; diphenyl spiromustine; docosanol;
• dolasetron docetaxel; doxorubicin; doxorubicin hydrochloride; doxifluridine; droloxifene; droloxifene citrate; dromostanolone propionate; dronabinol; duazomycin; duocarmycin SA; ebselen; ecomustine; edelfosine; edrecolomab; edatrexate; eflornithine hydrochloride;
• eflornithine eflornithine
• elemene emitefur
• elsamitrucin enloplatin
• enpromate epipropidine
• epirubicin epirubicin hydrochloride
• epristeride erbulozole
• eribulin esorubicin hydrochloride
• estramustine estramustine phosphate sodium; etanidazole; etoposide; etoposide phosphate; etoprine; exemestane; fadrozole; fadrozole hydrochloride; trasrabine; fenretinide; filgrastim; finasteride; flavopiridol; flezelastine; fluasterone; floxuridine; fludarabine phosphate;
• fludarabine fluorodaunorubicin hydrochloride; forfenimex; formestane; fluorouracil;
• floxouridine flurocitabine; fosquidone; fostriecin sodium; fostriecin; fotemustine; gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix; gelatinase inhibitors; gemcitabine;
• geldanamycin gossyphol; GDC-0973; GSK1120212/trametinib; herceptin; hydroxyurea; hepsulfam; heregulin; hexamethylene bisacetamide; hypericin; ibandronic acid; ibrutinib; idarubicin; idarubicin hydrochloride; ifosfamide; canfosfamide; ilmofosine; iproplatin;
• idoxifene idramantone; ilmofosine; ilomastat; imidazoacridones; imatinib (e.g., GLEEVEC); imiquimod; iniparib (BSI 201); iobenguane; iododoxorubicin; ipomeanol; irinotecan;
• irinotecan hydrochloride irsogladine; isobengazole; isohomohalicondrin B; itasetron;
• interleukin IL-2 including recombinant interleukin II; or rlL.sub.2
• interferon alfa-2a interferon alfa-2b
• interferon alfa-nl interferon alfa-n3
• interferon beta-la interferon beta-la
• interferon gamma- lb jasplakinolide
• kahalalide F lamellarin N triacetate; lanreotide; leinamycin;
• lenograstim lentinan sulfate; leptolstatin; letrozole; leuprorelin; levamisole; lenalidomide; lenvatinib; liarozole; lissoclinamide 7; lobaplatin; lombricine; lometrexol; lonidamine;
• losoxantrone lovastatin; loxoribine; lurtotecan; lutetium texaphyrin; lysofylline; lanreotide acetate; lapatinib; letrozole; leucovorin; leuprolide acetate; liarozole hydrochloride;
• lometrexol sodium lomustine; losoxantrone hydrochloride; pomalidomide; LY294002;
• miltefosine miltefosine; mirimostim; mitoguazone; mitolactol; mitonafide; mitoxantrone; mofarotene; molgramostim; mopidamol; mycaperoxide B; myriaporone; maytansine; mechlorethamine hydrochloride; megestrol acetate; melengestrol acetate; melphalan; mercaptopurine;
• methotrexate methotrexate sodium; metoprine; meturedepa; mitindomide; mitocarcin;
• mitocromin mitogillin; mitomalcin; mitomycin; mitosper; mitotane; mitoxantrone hydrochloride; mycophenolic acid; nafarelin; nagrestip; napavin; naphterpin; nartograstim; nedaplatin; nemorubicin; neridronic acid; nilutamide; nisamycin; nitric oxide modulators; nitroxide antioxidant; nitrullyn; nocodazole; nogalamycin; oblimersen (GENASENSE);
• octreotide okicenone; olaparib (LYNPARZA); oligonucleotides; onapristone; ondansetron; oracin; oral cytokine inducer; ormaplatin; oxisuran; oxaloplatin; osaterone; oxaliplatin;
• oxaunomycin palauamine; palmitoylrhizoxin; pamidronic acid; panaxytriol; panomifene; parabactin; PARP (polyADP ribose polymerase) inhibitors; pazelliptine; pegaspargase;
• peldesine pentosan polysulfate sodium; pentostatin; pentrozole; perflubron; perfosfamide; perillyl alcohol; phenazinomycin; phenylacetate; phosphatase inhibitors; picibanil; pilocarpine hydrochloride; pirarubicin; piritrexim; placetin A; placetin B; porfiromycin; prednisone;
• prostaglandin J2 pyrazoloacridine; paclitaxel; PD035901; PD 184352; PD318026; PD98059; peliomycin; pentamustine; peplomycin sulfate; PKC412; pipobroman; piposulfan;
• piroxantrone hydrochloride piroxantrone hydrochloride; plicamycin; plomestane; podophyllotoxin; polyphenol E;
• porfimer sodium porfiromycin; prednimustine; procarbazine; procarbazine hydrochloride; puromycin; puromycin hydrochloride; pyrazofurin; raltitrexed; ramosetron; retelliptine demethylated; rhizoxin; rituximab; RII retinamide; rogletimide; rohitukine; romurtide;
• Spongistatin 9 squalamine; stipiamide; stromelysin inhibitors; sulfinosine; suradista; suramin; swainsonine; SB239063; selumetinib/AZD6244; sirazene; SP600125; sparfosate sodium; sparsomycin; spirogermanium hydrochloride; spiroplatin; streptonigrin; streptozocin;
• tropisetron turosteride; tyrphostins; talisomycin; TAK-733; taxotere; tegafur; teloxantrone hydrochloride; teroxirone; testolactone; thiamiprine; thioguanine; thiotepa; tiazofurin; tirapazamine; toremifene citrate; trastuzumab; trestolone acetate; triciribine phosphate;
• trimetrexate trimetrexate glucuronate; triptorelin; tubulozole hydrochloride; tumor necrosis factor-related apoptosis-inducing ligand (TRAIL); UBC inhibitors; ubenimex; U0126; uracil mustard; uredepa; vapreotide; variolin B; velaresol; veliparib (ABT-888); veramine;
• TRAIL tumor necrosis factor-related apoptosis-inducing ligand
• Erbulozole e.g., R-55104
• Mivobulin isethionate e.g., CI-980
• NSC-639829 e.g., NSC-639829
• Discodermolide e.g., NVP-XX-A-296; ABT-751 (Abbott; e.g., E-7010); Altorhyrtin A; Altorhyrtin C; Cemadotin hydrochloride (e.g., LU-103793 and NSC-D-669356); CEP 9722; Epothilone A; Epothilone B; Epothilone C; Epothilone D; Epothilone E; Epothilone F;
• SAH-49960 (Lilly/Novartis); SDZ-268970 (Lilly/Novartis); AM-97 (Armad/Kyowa Hakko); AM-132 (Armad); AM-138 (Armad/Kyowa Hakko); IDN-5005 (Indena); Cryptophycin 52 (e.g., LY-355703); AC-7739 (Ajinomoto; e.g., AVE-8063A and CS-39.HC1); AC-7700 (Ajinomoto; e.g., AVE-8062; AVE-8062A; CS-39-L-Ser.HCl; and RPR-258062A);
• DDE-261 and WHI-261 H10 (Kansas State University); H16 (Kansas State University); Oncocidin Al (e.g., BTO-9
• deoxyadenosine sertraline; pitavastatin; clofazimine; 5-nonyloxytryptamine; vemurafenib; dabrafenib; gefitinib (IRESSA); erlotinib (TARCEVA); cetuximab (ERBITUX); lapatinib
• TYKERB panitumumab
• VECTIBIX vandetanib
• CAPRELSA afatinib/BIBW2992
• CI- 1033/canertinib neratinib/HKI-272
• CP-724714 TAK-285
• AST-1306 ARRY334543;
• Demethoxygeldanamycin (17-AAG); 20-epi-l,25 dihydroxyvitamin D3; 5 ethynyluracil; and BMS-599626.
• One embodiment is a method for treating patients with hepatocellular carcinoma (optionally refractory) by administering a compound of the invention in combination with capecitabine and/or PLX4032 (Plexxikon).
• hepatocellular carcinoma in another embodiment is a method for treating hepatocellular carcinoma (optionally refractory) by administering a compound of the invention in combination with capecitabine, xeloda, and/or CPT-11.
• hepatocellular carcinoma in another embodiment is a method for treating hepatocellular carcinoma (optionally refractory) by administering a compound of the invention in combination with capecitabine, xeloda, and/or CPT-11.
• a method for treating patients with hepatocellular carcinoma (optionally refractory) or patients with unresectable or metastatic hepatocellular carcinoma by administering a compound of the invention in combination with capecitabine and irinotecan.
• In another embodiment is a method for treating patients with unresectable or metastatic hepatocellular carcinoma by administering a compound of the invention in combination with interferon alpha or capecitabin.
• in another embodiment is a method for treating patients with pancreatic cancer by administering a compound of the invention in combination with gemcitabine.
• One of the embodiments is a method for treating patients with CCA by administering a compound of the invention in combination with another agent or agents or combination thereof.
• One of the embodiments is a method for treating patients with CCA by administering a compound of the invention in combination with gemcitabine, cisplatin or combination thereof.
• a “pharmaceutical composition” is a formulation containing a compound of the invention in a form suitable for administration to a subject.
• the pharmaceutical composition is in bulk or in unit dosage form. It can be advantageous to formulate compositions in dosage unit form for ease of administration and uniformity of dosage.
• the specification for the dosage unit forms are dictated by and directly dependent on the unique characteristics of the active reagent and the particular therapeutic effect to be achieved.
• Possible formulations include those suitable for oral, sublingual, buccal, parenteral (e.g., subcutaneous, intramuscular, or intravenous), rectal, topical including transdermal, intranasal and inhalation administration. Most suitable means of administration for a particular patient will depend on the nature and severity of the disease being treated or the nature of the therapy being used and on the nature of the active compound, but where possible, oral administration may be used for the prevention and treatment cancer.
• Formulations suitable for oral administration may be provided as discrete units, such as tablets, capsules, cachets, lozenges, each containing a predetermined amount of the active compound; as powders or granules; as solutions or suspensions in aqueous or non-aqueous liquids; or as oil-in-water or water-in-oil emulsions.
• Formulations suitable for sublingual or buccal administration include lozenges comprising a compound of the invention and typically a flavored base, such as sugar and acacia or tragacanth and pastilles comprising the active compound in an inert base, such as gelatin and glycerin or sucrose acacia.
• a flavored base such as sugar and acacia or tragacanth
• pastilles comprising the active compound in an inert base, such as gelatin and glycerin or sucrose acacia.
• Formulations suitable for parenteral administration typically comprise sterile aqueous solutions containing a predetermined concentration of the active compound; the solution may be isotonic with the blood of the intended recipient. Additional formulations suitable for parenteral administration include formulations containing physiologically suitable co-solvents and/or complexing agents such as surfactants and cyclodextrins. Oil-in-water emulsions are also suitable formulations for parenteral formulations. Although such solutions may be administered intravenously, they may also be administered by subcutaneous or intramuscular injection.
• Formulations suitable for rectal administration may be provided as unit-dose suppositories comprising a compound of the invention in one or more solid carriers forming the suppository base, for example, cocoa butter.
• Formulations suitable for topical or intranasal application include ointments, creams, lotions, pastes, gels, sprays, aerosols and oils.
• Suitable carriers for such formulations include petroleum jelly, lanolin, polyethylene glycols, alcohols, and combinations thereof.
• Formulations of the invention may be prepared by any suitable method, typically by uniformly and intimately admixing a compound of the invention with liquids or finely divided solid carriers or both, in the required proportions and then, if necessary, shaping the resulting mixture into the desired shape.
• a tablet may be prepared by compressing an intimate mixture comprising a powder or granules of the active ingredient and one or more optional ingredients, such as a binder, lubricant, inert diluent, or surface active dispersing agent, or by molding an intimate mixture of powdered active ingredient and inert liquid diluent.
• Suitable formulations for administration by inhalation include fine particle dusts or mists which may be generated by means of various types of metered dose pressurized aerosols, nebulizers, or insufflators.
• the particle size of the powder or droplets is typically in the range about 0.5-10 ⁇ or 1-5 ⁇ , to ensure delivery into the bronchial tree.
• a particle size in the range about 10-500 ⁇ may be used to ensure retention in the nasal cavity.
• Metered dose inhalers are pressurized aerosol dispensers, typically containing a suspension or solution formulation of a compound of the invention in a liquefied propellant. During use, these devices discharge the formulation through a valve adapted to deliver a metered volume, typically from about 10 to 150 ⁇ , to produce a fine particle spray containing the active ingredient.
• Suitable propellants include certain chlorofluorocarbon compounds, for example, dichlorodifluoromethane, trichlorofluorom ethane,
• the formulation may additionally contain one or more co-solvents, for example, ethanol surfactants, such as oleic acid or sorbitan trioleate, anti-oxidants and suitable flavoring agents.
• co-solvents for example, ethanol surfactants, such as oleic acid or sorbitan trioleate, anti-oxidants and suitable flavoring agents.
• Nebulizers are commercially available devices that transform solutions or suspensions of the active ingredient into a therapeutic aerosol mist either by means of acceleration of a compressed gas typically air or oxygen, through a narrow venturi orifice, or by means of ultrasonic agitation.
• Suitable formulations for use in nebulizers consist of the active ingredient in a liquid carrier and comprise up to about 40% w/w of the formulation, and may comprise less than about 20% w/w.
• the carrier is typically water or a dilute aqueous alcoholic solution, preferably made isotonic with body fluids by the addition of, for example, sodium chloride.
• Optional additives include preservatives if the formulation is not prepared sterile, for example, methyl hydroxy-benzoate, anti-oxidants, flavoring agents, volatile oils, buffering agents and surfactants.
• Suitable formulations for administration by insufflation include finely comminuted powders which may be delivered by means of an insufflator or taken into the nasal cavity in the manner of a snuff.
• the powder is contained in capsules or cartridges, typically made of gelatin or plastic, which are either pierced or opened in situ and the powder delivered by air drawn through the device upon inhalation or by means of a manually- operated pump.
• the powder employed in the insufflator consists either solely of the active ingredient or of a powder blend comprising the active ingredient, a suitable powder diluent, such as lactose, and an optional surfactant.
• the active ingredient typically comprises from about 0.1 to 100% w/w of the formulation.
• the present invention provides a pharmaceutical composition
• a pharmaceutical composition comprising, as active ingredient, a compound of the invention together, and/or in admixture, with at least one pharmaceutical carrier or diluent.
• the carrier is pharmaceutically acceptable and must be compatible with, i.e. not have a deleterious effect upon, the other ingredients in the composition.
• the carrier may be a solid or liquid and is preferably formulated as a unit dose formulation, for example, a tablet which may contain from about 0.05 to 95% by weight of the active ingredient. If desired, other physiologically active ingredients may also be incorporated in the pharmaceutical
• compositions of the invention are provided.
• formulations of the present invention may include other agents known to those skilled in the art of pharmacy, having regard for the type of formulation in issue.
• formulations suitable for oral administration may include flavoring agents and formulations suitable for intranasal administration may include perfumes.
• a pharmaceutical composition is administered in a dosage form which comprises a compound of the invention in a daily total amount of from about 0.1-1500 mg, 0.2-1200 mg, 0.3-1000 mg, 0.4-800 mg, 0.5-600 mg, 0.6-500 mg, 0.7-400 mg, 0.8-300 mg, 1-200 mg, 1-100 mg, 1-50 mg, 1-30 mg, 4-26 mg, 5-25 mg, or 5-10mg.
• a compound of the invention can be used in combination with other hepatocellular carcinoma treating drugs, such as anticancer chemotherapeutic drugs, hormones, biological response modifier(s), and other angiogenesis inhibitors; or in combination with
• Compound 1 can be prepared by methods known in the art ⁇ e.g., those described in U.S. Patent No. 7,932,244).
• Compound 1 can be prepared by a process as shown in Scheme 1 and disclosed in WO 2014/066819.
• Step 1 is the esterification of Compound 2 to obtain Compound 4.
• Step 2 is a reaction to form Compound 5 from Compound 4.
• Step 3 is the protection of the hydroxy group at the C3 position of Compound 5 to afford Compound 6.
• Step 4 is the oxidative cleavage of Compound 6 to afford Compound 7.
• Step 5 is the reduction of Compound 7 to afford Compound 8.
• Step 6 is the sulfonation of Compound 8 to afford the sodium salt of
• Compound 1 (1-Na).
• the sodium salt of Compound 1 can be converted to its free acid form (i.e., Compound 1) or other salt forms (e.g., Compound 1-DEA or the ⁇ , ⁇ -diethaneamine salt of Compound 1) according to procedures known in the art.
• Compound 2 can be prepared by the conventional methods (e.g., those described in U.S. Publication No. 2009/0062526, U.S. Patent No. 7,138,390, and WO 2006/122977), such as by a 6-step synthesis followed produce Compound 1 (obeticholic acid, or OCA) as shown in Scheme 2 below.
• conventional methods e.g., those described in U.S. Publication No. 2009/0062526, U.S. Patent No. 7,138,390, and WO 2006/122977
• OCA obeticholic acid
• Step 1 is the esterification of the C-24 carboxylic acid of 7-keto lithocholic acid (KLCA) using methanol in the presence of acidic catalyst and heat to produce the methyl ester Compound 7.
• Step 2 is silylenol ether formation from Compound a using a strong base followed by treatment with chlorosilane to produce Compound 8.
• Step 3 is an aldol condensation reaction of the silylenol ether of compound 8 and acetaldehyde to produce Compound 10.
• Step 4 is saponification of the C-24 methyl ester of Compound 10 to produce Compound 11.
• Step 5 is the hydrogenation of the 6-ethylidene moiety of Compound 11 to produce compound 12.
• Step 6 is the selective reduction of the 7- keto group of Compound 12 to a 7a-hydroxy group to produce Compound 1.
• Example 3 Hepatocarcinogenesis in Mdr2 _ " and FXR _ " mice
• the multidrug resistance protein 2 (Abcb4) is a member of the superfamily of ATP- binding cassette (ABC) transporters.
• the multidrug resistance protein 2 gene knockout mice (Mdr2 "/_ ) provides an in-vivo model of spontaneous hepatocarcinogenesis (Katzenellengoben, et al. Mol. Cancer Res. 2007, 5, 11, 1159-1170). Mice lacking the Abc4 protein encoded by the multidrug resistance-2 gene develop chronic periductular inflammation and cholestatic liver disease resulting in the development of hepatocellular carcinoma.
• the farnesoid X receptor protein is a nuclear receptor that functions as a bile acid sensor controlling bile acid homeostasis. This receptor is highly expressed in the liver and other organs. FXR knockout (FXR " " ) mice develop hepatocellular adenoma and carcinoma after 15 months of age (Yang, et al. Cancer Res., 2007, 67, 863).
• OCA is a FXR agonist while Compound 1-Na is a dual FXR/TGR5 agonist.
• the potency of Compound 1-Na for FXR is about 10 fold greater than OCA.
• mice were randomly divided into three experimental groups. The mice were fed a specific rodent diet or a diet supplemented with OCA, Compound 1-Na, or control diet for 15 months. All mice were housed under pathogen-free conditions in a temperature-controlled room (23°C) on a 12-hour light/dark cycle and drank water ad libitum. The Ethical Committee of the University of Bari approved this experimental set-up, which also was certified by the Italian Ministry of Health in accordance with internationally accepted guidelines for animal care. After 16 months, the mice were sacrificed and serum, liver and intestine were collected. The total number of hepatic tumors was counted and the diameter of each tumor was measured.
• Figures 1 A and 2 A show the effect of Compound 1-Na, OCA, and control on the reduction of the number of tumors in Mdr2 _/" mice.
• Compound 1-Na clearly prevented hepatocellular carcinoma development in this study compared to control.
• Mdr2 _/" control mice and OCA treated mice displayed clearly identifiable liver tumors while minute tumors were found in Compound 1-Na group.
• Figures IB and 2B show the effects of Compound 1-Na, OCA, and control on percent reduction of tumors having a diameter of >5mm.
• Figures 5 A and 5B describe the effect of Compound 1-Na and control on the percent ratio of liver and body weight. Consistent with previously generatd data, the Compound 1-Na treated Mdr2 _/" mice exhibited a significant reduction in LW/BW ratio compared to control and OCA treated Mdr2 _/" mice. No difference in LW/BW ratio was observed in the FXR _/" groups.
• ALT alanine aminotransferase
• AST aspartate aminotransferase
• Cholesterol 7 alpha-hydroxylase (cyp7al) is the rate limiting enzyme in the classical biosynthetic pathways which convert cholesterol into bile acids. Both Compound 1-Na and OCA inhibited Cyp7al gene expression in Mdr2 _/" mice only ( Figure 8).
• the bile salt export pump (Bsep) is a membrane protein that uses energy of ATP hydrolysis to actively transport bile acid salts. As indicated in Figures 9A and 9B, Compound 1-Na administration induced hepatic Bsep activation in Mdr2 _/" mice.
• OCA did not promote hepatic Bsep induction suggesting that in contrast to Compound 1-Na, which efficently activates FXR in intestine and liver, OCA is less likely to have hepatic activity in Mdr2 _/" mice.
• FXR and TGR5 expression was determined in CCA human biopsies and controls by using 2 approaches (mRNA microarray and qPCR) and 2 different cohorts of patients (Copenhagen and San Diego), as well as in different human CCA cell lines compared to normal human cholangiocytes (NHC) (by qPCR).
• the growth of an orthotopic model of human CCA in immunodeficient mice was evaluated by magnetic resonance imaging (MRI) under chronic administration of the specific FXR or TGR5 agonists (OCA or Compound 4, respectively; 0,03% in chow for 2 months; Intercept Pharmaceuticals).
• MRI magnetic resonance imaging
• FXR is downregulated and TGR5 upregulated in human CCA tissue samples compared to normal surrounding liver tissue and normal intrahepatic bile ducts in both Copenhagen and San Diego cohorts by mRNA microarray and qPCR, respectively.
• FXR Activation of FXR inhibits, whereas TGR5 may promote, CCA progression through the regulation of proliferation, migration and mitochondrial energetic metabolism. Regulation of FXR and TGR5 activities represent a potential therapeutic strategy for CCA. In vitro, FXR was also found downregulated and TGR5 upregulated in different human CCA cell lines compared to NHC.
• TGR5 expression is increased in CCA tumors, an is higher in perihilar than in intrahepatic CCAs, and correlates with perineural invasion (Figure 12A).
• OCA obeticholic acid
• Figure 14A shows representative MRI and liver images of untreated control mice, OCA-treated mice and Compound 4-treated mice.
• Representative immunohistochemistry images of proliferation markers (i.e. Ki67 and PCNA) in liver orthotopic CCA tumors of mice untreated or treated with OCA or Compound 4 are shown in Figure 14D. In contrast, no effects on CCA tumor growth were observed in animals treated chronically with the TGR5 agonist Compound 4.
• Figure 17 shows effects of OCA and Compound 4 in liver xenograph tumoral expression of proliferation markers.
• FXR activation did not affect the survival of CCA cells.
• FIG. 15A-15D the FXR agonist OCA inhibits CCA cell proliferation in a dose-dependent manner and inhibits CCA cell migration, associated with decreased mitochondrial metabolism in CCA cells, without inducing apoptosis.
• mRNA expression of proliferation markers i.e. Ki67, PCNA, cyclin Dl and cyclin D3 in CCA cells (i.e.
• FIG. 16B represents migration assays in CCA cells (i.e. EGIl). Representative microscope images of wound-healing assay at the indicated time-points and conditions (i.e. non-treated control or Compound 4-treated) and corresponding

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