WO2013037482A1

WO2013037482A1 - Farnesoid x receptor agonists for cancer treatment and prevention

Info

Publication number: WO2013037482A1
Application number: PCT/EP2012/003814
Authority: WO
Inventors: Ulrich Deuschle; Claus Kremoser
Original assignee: Phenex Pharmaceuticals Ag
Priority date: 2011-09-15
Filing date: 2012-09-11
Publication date: 2013-03-21

Abstract

Provided is a Farnesoid X Receptor (FXR) agonist for use in the treatment or prevention of certain cancers, metastases, precancerogenic lesions or angiogenesis in the context of cancer in a patient, wherein the Farnesoid X Receptor (FXR) agonist is administered in a therapeutically effective amount to the patient. Also provided is an FXR agonist for use in inducing NDRG2 gene expression in certain tissues of a patient, wherein the FXR agonist is administered in a therapeutically effective amount to the patient. Also provided is an FXR agonist for use in reducing the rate of proliferation, migration, metastasis or angiogenesis of certain tumors.

Description

Farnesoid X Receptor Agonists for Cancer Treatment and Prevention

The present invention relates to an FXR agonist for use in the treatment or prevention of cancers, metastases, precancerogenic lesions or angiogenesis in the context of cancer in a patient. The invention also relates to an FXR agonist for use in increasing the expression of the NDRG2 gene in a patient.

Nuclear receptors constitute a superfamily of transcriptional regulatory proteins that share structural and functional properties and function as receptors for, e.g., steroids, retinoids, vitamin D and thyroid hormones (R. M. Evans, Science 1988, 240, 889). Most of these proteins bind to cis-acting DNA response elements in the promoters of their target genes and modulate gene expression in response to ligands for the receptors.

Nuclear receptors can be classified based on their DNA binding properties (C. K. Glass, Endocr. Rev. 1994, 15, 391). For example, class I nuclear receptors including the glucocorticoid, estrogen, androgen, progestin, and mineralocorticoid receptors do bind as homodimers to inverted repeat type hormone response elements (HREs). Class II receptors, including those activated by retinoic acid, thyroid hormone, vitamin D and fatty acids (for example peroxisome proliferator activated receptors (PPAR^'s), FXR and the liver X receptors (LXR^'s)), do bind to their recognition elements as a heterodimeric complex together with a retinoid X receptor (e.g. A. A. Levin et al., Nature 1992, 355, 359 and R. A. Heyman et al., Cell 1992, 68, 397).

The Farnesoid X Receptor (FXR, NR1H4) is a member of the nuclear hormone receptor superfamily, predominantly expressed in tissues exposed to high levels of bile acids, such as the entire gastrointestinal tract, the liver and the gallbladder. FXR mRNA can also be detected in tissues such as the adrenals, kidneys and adipose tissues (B. Goodwin et al., Mol. Cell 2000, 6, 517). FXR senses bile acids as endogenous ligands (D. J. Parks et al., Science 1999, 284, 1365), is a master regulator of bile acid homeostasis and prevents bile acid-induced liver toxicity by regulating directly and indirectly (e.g. via SHP, NR0B2) the expression of numerous genes involved in bile acid synthesis, conjugation, and transportation. Activation of FXR by synthetic derivatives of the natural bile acid ligands, such as 6-Ethyl-Chenodeoxycholic Acid (6-ECDCA), or by synthetic non-steroidal agonists like GW4064, results in beneficial metabolic adjustments in different mouse models such as glucose lowering, insulin sensitisation, triglyceride and cholesterol lowering (B. Carious et al., J. Biol. Chem. 2006, 281 , 11039; Y. Zhang et al., Proc. Natl. Acad. Sci. USA 2006, 103, 1006. K. Ma et al., J. Clin. Invest. 2006, 1 16, 1 102). Activation of FXR results in hepatoprotection in mouse models of non alcoholic fatty liver disease possibly mediated via a reduction of lipid accumulation, fibrosis and inflammation (S. Fiorucci et al., J. Pharmacol. Exp. Ther. 2005, 314, 584; S. Zhang et al., J. Hepatol. 2009, 51 , 380). In the intestine, FXR controls the expression of FGF15 in the mouse and FGF19 in humans. FGF19 injected into mice results in insulin sensitization, body weight and lipid lowering. Therefore FGF15 (FGF19 in humans) mediates an endocrine signaling of intestinal FXR activation to all those tissues expressing FGFR4/beta-Klotho, the receptor for FGF15/19 (H. Kurosu et al., J. Biol. Chem. 2007, 282, 26687).

FXR has therefore emerged as an interesting pharmacological target with broad therapeutic potential through elucidation of its integral role in biological pathways such as bile homeostasis, cholesterol and triglyceride homeostasis, gluconeogenesis, insulin sensitivity and secretion, the modulation of anti-inflammatory, anti-fibrotic and anti-steatotic protective mechanisms in the liver, intestine and kidney, as covered in a recent review (Y. D. Wang et al., Cell Res. 2008, 18, 1087).

Analysis of FXR knock-out mice linked the expression of functional FXR to the protection against hepatocellular carcinoma formation (F. Yang et al., Cancer Res. 2007, 67, 863 and I. Kim et al., Carcinogenesis 2007, 28, 940) and the protection against intestinal tumor formation (S. Modica et al., Cancer Res. 2008, 68, 9589 and R. R. Maran et al., J. Pharmacol. Exp. Ther. 2009, 328, 469). A recent report showed that Taurocholate reduced the formation of intestinal adenoma in the APC ^{in +} mouse model presumably through FXR activation (D. L. H. Smith et al., Carcinogenesis 2010, 31 , 1 100). Of clinical importance is the tumor-stage dependent reduction of FXR mRNA in human colon carcinoma (A. de Gottardi et al., Dig. Dis. Sci. 2004, 49, 982). The expression of FXR was recently linked to the more benign phenotype of No-dysplasia in Barretts oesophagus patients, while the expression was gradually reduced during neoplastic progression from dysplasia to adenocarcinoma in patients with Barretts oesophagus (A. van de Winkel et al., Histopathology 2011 , 58, 246).

Using genome-wide Chromatin Immunoprecipitation followed by deep sequencing (ChipSeq) two groups have identified numerous genes containing in vivo FXR binding sites in liver and intestine of the mouse (A. . Thomas AM et al., Hepatology 2010, 51 , 1410; H. K. Chong et al., Nucleic Acids Res. 2010, 38, 6007). Since the presence and expression of FXR protects against tumor formation in intestine and liver, one can speculate that some of the genes which are directly or indirectly regulated by FXR should be involved in this effect. The orphan receptor small heterodimer partner SHP (NR0B2) is transcriptionally upregulated as a direct target of FXR in the mouse liver and involved in a negative feed-back regulation of bile acid synthesis via repression of Cyp7a1 transcription (T. T. Lu et al., Mol. Cell 2000, 6, 507). SHP mutant mice do also show liver tumor formation beyond 12 months of age, suggesting a tumor suppressing activity in mouse liver (Y. Zhang et al., Hepatology 2008, 48, 289) similar to what is found in FXR mutant mice. Of clinical significance is the epigenetic silencing of the SHP gene in human liver tumor isolates and established HCC derived cell lines (N. He et al., Gastroenterology 2008, 134, 793). Interestingly, Adenovirus mediated expression of SHP in HepG2 cells reduces their tumor growth rate in nude mice compared to HepG2 cells carrying a control Adenovirus.

In recent years, N-myc downregulated gene 2 (human gene NDRG2, mouse gene Ndrg2) was reported as a candidate tumor suppressor in human liver cancer metastasis (D. C. Lee et al., Cancer Res. 2008, 68, 4210). Furthermore, reduced NDRG2 expression was published for high-risk adenomas and colorectal carcinoma (A. Lorentzen et al., BMC Cancer 2007, 7, 192; Y. J. Kim et al., Carcinogenesis 2009, 30, 598; A. Piepoli et al., BMC Med. Genomics 2009, 2, 1 1 ; D. Chu et al., Mol. Cancer Ther. 201 1 , 10, 47), glioblastoma (M. Tepel et al., Int. J. Cancer. 2008, 123, 2080), thyroid cancer (H. Zhao et al., BMC Cancer 2008, 8, 303), and breast cancer (J. Zheng et al., Asian Pac. J. Cancer Prev. 2010, 1 1 , 1817). Prior to our invention, the use of FXR agonists for the treatment of malignancies was claimed in US2008/2991 18 and US2009/131409, based in part upon the unexpected finding that FXR agonists can induce reversion-inducing-cysteine rich-protein with Kazal motifs (RECK). RECK was originally isolated as a gene that induces reversion of v-Ki-ras transformed NIH3T3 cells. The RECK gene has been implicated in the suppression of malignancies through inhibition of matrix metalloproteases (MMPs). No anti-tumor effect in vivo was shown in the two above listed patent applications. Interestingly, when comparing the expression of RECK in normal liver tissue with expression in tissue of stage 1 , stage 2, stage 3a and stage 4 HCC, the expression of RECK is only found to be reduced in stage 1 but not in other stages of HCC, suggesting that RECK may play a role in early (stage 1) hepatocarcinogenesis but not in later stages (see Fig. 9 of EXAMPLE 9). In contrast NDRG2 expression was found reduced in all HCC stages compatible with an important role of NDRG2 in hepatocarcinognesis of all stages. Interestingly small heterodimer partner SHP (NR0B2) was also found to be downregulated in all stages of HCC investigated (Fig. 9 of EXAMPLE 9). Increasing the expression of SHP via FXR agonists may therefore contribute to the anti-tumor activity of FXR agonists.

The problem underlying the present invention is the provision of FXR agonists for use in the treatment or prevention of cancers, metastases, precancerogenic lesions or angiogenesis in the context of cancer in a patient. Said problem has been solved by FXR agonists which induce the expression of NDRG2 in a patient.

The invention is based on the unexpected finding that potent agonists of FXR can induce the expression of NDRG2 in human hepatoma cells and in the liver of mice. While FXR agonists according to CLAIM 10 of this invention have been already described as FXR agonists in WO201 1/020615, it was not shown in this patent application that FXR could induce NDRG2 i.e. in human hepatoma cells and that this constitutes the basis for the development of FXR agonists as medicaments for the treatment of tumors that develop in an FXR mediated fashion.That FXR is involved in the transcriptional control of NDRG2 is also based on the finding that the expression of Ndrg2 mRNA is reduced significantly in livers of FXR-KO mice compared to wild type mice. The Ndrg2 mRNA reduction in FXR-KO mouse livers is comparable to the reduction of Shp mRNA levels. The invention is further based on the unexpected finding that agonists of FXR can reduce the growth rate and metastasis of SK- Hep-1 cells (human hepatocellular carcinoma cells) orthotopically transplanted into the livers of immunodeficient nude mice. Further the invention is based on the unexpected finding that agonists of FXR can even more efficiently reduce the growth rate and metastasis of SK-GI-18 cells (SK-Hep-1 derivative that contains a stably integrated FXR cDNA under control of the human CMV IE promoter and stably expresses human FXR (isoform 3 accession Nr. NM_005123)) when orthotopically transplanted into the livers of immunodeficient nude mice (see EXAMPLE 10).

Bile acids, conjugated bile acids and modified bile acids are known to activate FXR and bile acids and conjugates have been shown to induce apoptosis in various cancer cell lines (B.W. Katona et al., J. Biol. Chem. 2009, 284, 3354; N.S. Horowitz et al., Gynecol. Oncol. 2007, 107, 344; R. Pelliciari et al., J. Med. Chem. 2004, 47, 4559; S. Fiorucci et al., Gastroenterol. 2003, 124 (4 suppl. 1), A698}. However, the dosis necessary to induce apoptosis in such cells in vitro is very high (>100 μΜ) whereas the activation of FXR by CDCA does occur with an EC₅₀ of around 5-10 μΜ and 6-ECDCA has an EC₅0 of around 100-200 nM. The induction of NDRG2 by bile acids and bile acid derivatives such as CDCA and 6-ECDCA does occur in cells expressing FXR at a much lower concentration and this constitutes a completely novel mechanism for anti-tumor activity of bile acids. Certain bile acids such as lithocholic acid are considered to be pro-tumorigenic especially in the colon (C. Degirolamo et al., Trends Mol. Med. 201 1 , 17, 564) suggesting that bile acids and particularly secondary bile acids can have pro-tumorigenic activity although causing apoptosis of tumor cells at high concentration (above 100 μΜ).

Cafestol, a diterpenoid, has been demonstrated to be a FXR agonist (M.L. Ricketts et al., Mol. Endocrinol. 2007, 21 , 1603). Cafestol has also been reported to induce apoptosis in renal carcinoma Caki cells (M.J. Choi et al., Chem. -Biol. Interact. 201 1 , 190, 102) and there are multiple activities and mechanisms discussed for the anti-tumorigenic effects of coffee on the liver (K.S. Tao et al., Med. Hypotheses 2008, 71 , 730). However no indication is given as to whether cafestol or other incredients of coffee would be able to induce the expression of NDRG2 in cells expressing FXR to excert an anti-tumorigenic activity, which is the basis of our invention. In an embodiment in combination with any one of the above or below embodiments an FXR agonist for use in treating at least one malignancy in a patient is provided, wherein the FXR agonist is administered in a therapeutically effective amount to the patient. In a preferred embodiment, FXR agonist induces the expression of the N-myc downstream regulated gene 2 (NDRG2, Acc.Nr. NM_201535) and/or SHP (NR0B2, Acc.Nr. NM_021969) in certain tissues in a patient.

In an embodiment in combination with any one of the above or below embodiments at least one FXR agonist for use in increasing NDRG2 gene expression in a cell is provided, wherein the at least one FXR agonist is administered to a cell in an effective amount. In more preferred embodiments, FXR agonist induces expression of the NDRG2 gene in the cell.

In a further embodiment in combination with any one of the above or below embodiments at least one FXR agonist for use in modulating at least one of the following features or properties of a tumor or its surrounding tissue by administering an effective amount of the at least one FXR agonist to this area is provided. These features or properties that can be altered upon FXR agonist treatment is selected from proliferative activity, invasive activity, metastatic activity, migration activity and angiogenic activity of the tumor or its surrounding tissue. In some embodiments the FXR agonist induces expression of the NDRG2 gene in the cell.

In a further embodiment in combination with any one of the above or below embodiments methods for identifying a FXR agonist by incubating a test agent with a cell; determining at least one of the following parameters in the presence or absence of the test agent: the expression of the NDRG2 gene, the expression of NR0B2 (small heterodimer partner) are provided.

In a further embodiment in combination with any one of the above or below embodiments provided are methods for identifying a FXR modulator by incubating a test agent with a cell and determining at least one of the following parameters in the presence or absence of the test agent: the proliferative activity of the cell, the invasive activity of the cell, the metastatic activity of the cell, and the angiogenic activity of the cell; and selecting a FXR modulator which modulates at least one of the following features: the proliferative activity of the cell, the invasive activity of the cell, the metastatic activity of the cell, and the angiogenic activity of the cell.

In a further embodiment in combination with any one of the above or below embodiments methods for diagnosing the risk that a patient will develop at least one malignancy by measuring at least one of the level of expression of a FXR gene in at least one tissue of the patient and/or the level of FXR activity in at least one tissue of the patient are provided. FXR is known to bind to IR-1 type Sequences within genes regulated by FXR. IR-1 type elements are identified in the first introns of the NDRG2 genes of humans, mouse and rat (FIG. 2). When cloned into pGL4 (Promega), the human IR1 sequence was found to be functional in luciferase reporter gene assays in vitro (FIG. 2). A mutated version of the NDRG2 IR-1 type element (FIG. 1) has lost functionality in this assay (FIG. 2).

The Ndrg2 gene was upregulated in C57BL/6 wild-type mice when supplementing the food (High Fat Diet) with 30 mg/kg/day of the potent FXR agonist Px20350 (WO 2008/025540, FIG. 3). Ndrg2 mRNA was much lower in livers of FXR-KO mice when compared to wild-type mice (FIG. 4A) which is quite similar to the relative expression of Shp (FIG. 4B).

The expression of NDRG2 mRNA was upregulated by the non-steroidal FXR agonist Px20350 in a dose dependent fashion in HepG2 cells (FIG. 5A). This upregulation was pronounced when stably expressing human FXR in the stable HepG2 derivative HepG2-FXR5 (FIG. 5A). In HepG2-FXR5 cells, NDRG2 mRNA was also upregulated by the synthetic steroidal FXR agonist 6-ECDCA and with a lower potency by the natural bile acid CDCA (FIG. 5B potencies are: Px20350 EC50 66 nM, 6-EDCA 280 nM CDCA 33 μΜ). An upregulation of SHP mRNA was detected with non-steroidal and steroidal FXR agonists in both cell lines as well.

SK-Hep-1 cells are hepatoma cells, that do express rather low levels of FXR. A stable SK- Hep-1 derivative (named SK-GI-18) was generated by stably transfecting a human FXR cDNA under control of a CMV promoter. While the levels of NDRG2 and the induction of NDRG2 by Px20350 was rather low in SK-Hep-1 cells, in SK-GI-18 cells, the relative levels of NDRG2 mRNA was increased (when normalized to TATA box binding protein mRNA) and the induciblity of NDRG2 mRNA by Px20350was increased (FIG. 6).

While SK-Hep-1 cells do not change growth rate in vitro in response to the FXR agonist Px20350, SK-GI-18 cells that overexpress FXR do decrease their in vitro growth rate in a dose dependent fashion (FIG. 7).

The in vitro migration potential of SK-GI-18 cells was reduced more pronounced by 0.5 μΜ Px20350 in the growth medium when compared to the parent cell line SK-Hep-1 suggesting that activation of FXR is responsible for the reduced migration potential in both cell lines (FIG. 8).

The expression of FXR, NDRG2, SHP and RECK was tested in a commercially obtained cDNA panel derived from liver specimens of healthy controls, hepatocellular carcinoma from stage I, II, IMA and IV and non-malignant liver diseases such as liver cirrhosis (Origene Rockville, USA). The qPCR results show that FXR, NDRG2 and SHP are all significantly reduced in all tumor stages (FIG. 9). NDRG2 is reduced in a stage dependent fashion (expression in stage I is higher than the expression in stage IV). In contrast, RECK is reduced significantly only in stage I hepatocellular carcinoma patients but not in more severe stages. In the US 200802991 18 patent application, RECK is described as a FXR modulated gene linked to a possible anti-malignant activity of FXR modulators. The finding that RECK expression is not reduced in the more severe hepatocellular carcinoma stages II, IIIA and IV suggests that RECK could play only a significant role in very early stages of hepatocellular carcinoma. This is in contrast to NDRG2 and SHP, which are reduced in all HCC stages in a stage-dependent fashion (FIG. 9).

SK-Hep1 cells, orthotopically transplanted into the livers of immunodeficient nude mice do aggressively form primary tumors in the liver and metastases primarily in lymph nodes during a 56 day period (FIG.10A). Treatment by daily oral gavage with Sorafenib (100mg/kg/d), a pan Receptor Tyrosine Kinase Inhibitor clinically approved for late stage unresectable hepatocellular carcinoma, did reduce growth of SK-Hep-1 tumor cells in the primary tumor and metastases sites. Likewise, oral gavaging either one of two non-steroidal FXR agonists Px20606 and Px21256 at 10mg/kg/d did also significantly reduce primary tumor growth and metastases in lymph nodes (FIG- 10A).

SK-GI-18 cells that overexpress human FXR do form primary tumors and metastases with a slightly reduced rate. Sorafenib treatment did reduce tumor growth and metastases as efficient as with SK-Hep-1 cells. Primary tumors and also metastases induced with orthotopically implanted SK-GI-18 cells were even more efficiently suppressed in growth after gavaging Px20606 or Px21256 (each at 10mg/kg/d, FIG.10B).

Hong and colleagues performed genome wide expression analysis on RNA prepared from colonic mucosa samples of healthy controls and patients with non-FAP and non-hereditary nonpolyposis colorectal cancer (Y. Hong et ah, Clin. Cancer Res. 2007, 13, 1 107) and deposited these data in the NCBI Gene Expression Omnibus databank with the DataSet Record GDS2609. Analysis of these published data for expression of FXR (NR1 H4) shows that FXR is also significantly reduced in patients with non-FAP and non-heriditary nonpolyposis colorectal cancer compared to healthy controls (FIG.1 1 ). Thus, loss of FXR expression may be causally linked to this form of colorectal cancer.

As used in the present specification, the following words and phrases are generally intended to have the meanings as set forth below, except to the extent that the context in which they are used indicates otherwise.

As used herein, the terms "treat", "treating", and "treatment" refer to any manner in which one or more of the symptoms of a disease or disorder are beneficially altered so as to prevent or delay the onset, retard the progression, or ameliorate the symptoms of a disease or disorder. As used herein the term "tumor" can include a solid tumor or a cancer of hematopoietic origin. In some embodiments 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 include but are not limited to hepatocellular carcinoma, colorectal cancer, 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.

As used herein the phrase "therapeutically effective amount" refers to the amount sufficient to provide a therapeutic outcome regarding at least one symptom of a disease or condition.

As used herein, the term "farnesoid X receptor" or "FXR" refers to all mammalian forms of such receptor including, for example, alternative splice isoforms and naturally occurring isoforms (see e.g. R. M. Huber et al., Gene 2002, 290, 35). Representative FXR species include, without limitation the rat (GenBank Accession No. NM_21745), mouse (Genbank Accession No. NM_09108), and human (GenBank Accession No. NM_05123) forms of the receptor.

As used herein, the term "agonist" refers to an agent that triggers a response that is at least one response triggered by binding of an endogenous ligand of the receptor to the receptor. In certain embodiments, the agonist may act directly or indirectly on a second agent that itself modulates the activity of the receptor. In certain embodiments, the agonist may act indirectly by modulating the activity of one or more agent(s) that modulate the amount of FXR mRNA or FXR protein in certain cells of a patient. In certain embodiments, the at least one response of the receptor is an activity of the receptor that can be measured with assays including but not limited to physiological, pharmacological, and biochemical assays. Exemplary assays include but are not limited to assays that measure the binding of an agent to the receptor, the binding of the receptor to a substrate such as but not limited to a nuclear receptor and a regulatory element of a target gene, the effect on gene expression assayed at the mRNA or resultant protein level, and the effect on an activity of proteins regulated either directly or indirectly by the receptor. For example, FXR activity may be measured by monitoring expression of a NDRG2 gene and or expression of NR0B2.

As used herein, the term "agent" or "active agent" refers to a substance including, but not limited to a chemical compound, such as a small molecule or a complex organic compound, a protein, such as an antibody or antibody fragment or a protein comprising an antibody fragment, or a genetic construct which acts at the DNA or mRNA level in an organism.

As used herein, the term "expression" of a polynucleotide or gene refers to production of a RNA transcript. Because an RNA transcript encoded by a gene is translated into a protein the level of expression of a gene may be measured by directly assaying the level of mRNA produced or indirectly by assaying the level of protein produced. As used herein, the term "NDRG2" refers to all mammalian forms of c-myc downregulated gene 2 species, including, without limitation the mouse (Genbank Accession No. NM_013864) and human (GenBank Accession No. NM_201535) forms.

As used herein, 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.

As used herein, the term "metastatic" 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.

As used herein, the term "angiogenic" refers to the process by which new blood vessels form from pre-existing blood vessels. A malignancy may exhibit angiogenic properties in that it induces, promotes, or stimulates new blood vessels to form from pre-existing blood vessels.

As used herein, the term "coadministering" refers to a dosage regimen for a first agent that overlaps with the dosage regimen of a second agent, or to simultaneous administration of the first agent and the second agent. A dosage regimen is characterized by dosage amount, frequency, and duration. Two dosage regimens overlap if between a first and a second administration of a first agent the second agent is administered.

As used herein, the phrase "effective amount" refers to the amount sufficient to increase or reduce a specified activity, function, or feature.

As used herein, the term "SHP" refers to small heterodimer partner. Representative SHP species include, without limitation the rat (GenBank Accession No. NM_57133), mouse (Genbank Accession No. NM_1 1850), and human (GenBank Accession No. NM_21969) forms of SHP.

As used herein, the term "modulating" and "modulate" refers to changing or altering an activity, function, or feature. The term "modulator" refers to an agent which modulates an activity, function, or feature. For example, an agent may modulate an activity by increasing or decreasing the activity compared to the effects on the activity in the absence of the agent. In certain embodiments, a modulator that increases an activity, function, or feature is an agonist.

As used herein, the term "regulatory element" refers to cis-acting polynucleotide and/or transacting polynucleotide sequences which may affect the expression of coding sequences with which they are associated. For example, a regulatory element of a FXR target gene can include, but is not limited to, an inverted (IR-1) response element, in which consensus receptor-binding hexamers are separated by one nucleotide. An example of a IR-1 response element includes but is not limited to AGGTCAnTGACCT (SEQ ID No:5).

As used herein, the term "transcription unit" refers to the coding sequences of a gene and one or more regulatory elements of the gene, functionally linked to the coding sequences. In some embodiments, the transcription unit can be a sequence endogeneous to a cell, heterologous to a cell, or an artificial sequence.

"Px20350" is 4-(((6-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)-2- (trifluoromethyl)pyridin-3-yl)(methyl)amino)methyl)benzoic acid; "Px20606" is 4-(2-(2-chloro-4- ((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)phenyl)cyclopropyl)benzoic acid; "Px21256" is 3-trans-(3-(2-chloro-4-((cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4- yl)methoxy)phenyl)-3-hydroxycyclobutyl)benzoic acid.

In an embodiment of the invention in combination with any one of the above or below embodiments at least one FXR agonist for use in treating at least one tumor in a patient is provided, wherein the at least one FXR agonist is administered to the patient in a therapeutically effective amount. In some embodiments the at least one FXR agonist induces expression of the N-myc downregulated gene 2 (NDRG2) gene in the patient. In some embodiments the at least one tumor is selected from hepatocellular carcinoma, colorectal cancer and renal cancer. In some embodiments the at least one tumor is characterized by reduced expression of the human NR1 H4 (FXR) gene. In some embodiments the at least one malignancy is selected from hepatocellular carcinoma, colorectal cancer, gastric cancer, renal cancer, esophageal cancer, prostate cancer, breast cancer, salivary gland cancer, ovarian cancer, uterine body cancer, bladder cancer, and lung cancer. In some embodiments the FXR agonist reduces at least one feature of the tumor, wherein the at least one feature of the tumor is selected from invasive activity, metastatic activity, and angiogenic activity of the malignancy. In some embodiments at least one of an agent selected from Sorafenib, Sunitinib, Erlotinib or Imatinib is coadministered. In some embodiments at least one of an agent selected from abarelix, aldeleukin, allopurinol, altretamine, amifostine, anastozole, bevacizumab, capecitabine, carboplatin, cisplatin, docetaxel, doxorubicin, erlotinib, exemestane, 5- fluorouracil, fulvestrant, gemcitabine, goserelin acetate, irinotecan, lapatinib ditosylate, letozole, leucovorin, levamisole, oxaliplatin, paclitaxel, panitumumab, pemetrexed disodium, profimer sodium, tamoxifen, topotecan, and trastuzumab is coadministered. In some embodiments of the invention, the FXR agonist does induce expression of the SHP gene in the patient.

In an embodiment in combination with any one of the above or below embodiments a method of modulating NDRG2 gene expression in a cell is provided by administering to a cell an effective amount of at least one FXR modulator, to thereby modulate NDRG2 gene expression in the cell. In some embodiments NDRG2 gene expression is induced and the at least one FXR modulator is a FXR agonist.

In a further embodiment in combination with any one of the above or below embodiments a method of modulating at least one feature of a cell by administering an effective amount of at least one FXR modulator to the cell is provided. In some embodiments the at least one feature of the cell is selected from invasive activity, proliferative activity, apoptotic activity, metastatic activity, and angiogenic activity of the cell. In some embodiments the at least one FXR modulator induces expression of the NDRG2 gene in the cell. In some embodiments the feature is reduced and the at least one FXR modulator is a FXR agonist.

In another embodiment in combination with any one of the above or below embodiments a method of regulating expression of a RECK gene in a cell by administering at least one FXR modulator to the cell is provided. In some embodiments the at least one FXR modulator regulates binding of FXR to a regulatory element of the NDRG2 transcription unit. In some embodiments the expression of the NDRG2 gene is induced and the at least one FXR modulator is a FXR agonist.

Also provided is a method of identifying a FXR modulator by incubating a test agent with a cell; determining at least one of the following in the presence or absence of the test agent: the expression of the NDRG2 gene, the expression of NR0B2; and selecting a FXR modulator which fulfills at least one of the following features: modulating the expression of the NDRG2 gene, modulating the expression of NR0B2. In some embodiments the FXR modulator is a FXR agonist and the FXR agonist fulfills at least one of the following features: inducing the expression of the NDRG2 gene, inducing the expression of the NR0B2 gene.

Also provided is a method of identifying a FXR modulator by incubating a test agent with a cell; determining at least one of the following in the presence or absence of the test agent: the invasive activity of the cell, the proliferative activity of the cell, the apoptotic activity of the cell, the migratory activity of the cell, the metastatic activity of the cell, and the angiogenic activity of the cell; and selecting a FXR modulator which modulates at least one of the following features: the invasive activity of the cell, the proliferative activity of the cell, the apoptotic activity of the cell, the migratory activity of the cell, the metastatic activity of the cell, and the angiogenic activity of the cell. In some embodiments a FXR agonist is identified, and at least one of the following: the invasive activity of the cell, the proliferative activity of the cell, the apoptotic activity of the cell, the migratory activity of the cell, the metastatic activity of the cell, and the angiogenic activity of the cell is reduced.

Also provided is a method of diagnosing the risk that a patient will develop at least one malignancy by measuring at least one of the level of expression of a FXR gene in at least one tissue of the patient and/or the level of FXR activity in at least one tissue of the patient.

In certain embodiments of the invention the FXR agonist is selected from small molecule compounds which act as FXR modulators that have been disclosed in the following publications: WO 2000/037077, WO 2003/015771 , WO 2004/048349, WO 2007/076260, WO 2007/092751 , WO 2007/140174, WO 2007/140183, WO 2008/025539, WO 2008/025540, WO 2008/051942, WO 2008/073825, WO 2008/157270, WO 2009/005998, WO 2009/012125, WO 2009/027264, WO 2009/080555, WO 2009/127321 , WO 2009/149795, WO 2008/025540, WO 2010/028981 , WO 2010/034649 and WO 2010/034657. Further small molecule FXR modulators have been recently reviewed (R. C. Buijsman et al., Curr. Med. Chem. 2005, 12, 1017, M. L: Crawley, Expert Opin. Ther. Pat. 2010, 20, 1047) each of which are hereby incorporated herein by reference.

In a preferred embodiment in combination with any of the above or below embodiments, the FXR agonist is selected from natural bile acids, preferably Chenodeoxycholic acid [CDCA] or taurine- or glycine-conjugated CDCA [tauro-CDCA or glyco-CDCA] and synthetic derivatives of natural bile acids, preferably 6-Ethyl-CDCA or taurine- or glycine-conjugated 6-Ethyl-CDCA, natural non-steroidal agonists, preferably Diterpenoids such as Cafestol and Kahweol, or synthetic non-steroidal FXR agonists.

In a further preferred embodiment in combination with any of the above or below embodiments, the FXR agonist is selected from a compound according to Formula (1 ), an enantiomer, diastereomer, tautomer, solvate, prodrug or pharmaceutical acceptable salt thereof

wherein

A is selected from

A-Z- is selected from

More preferably, the FXR agonist is selected from

4-(((6-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)-2-(trifluoromethyl)pyridin-3- yl)(methyl)amino)methyl)benzoic acid;

3- (2-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4- yl)methoxy)phenyl)cyclopropyl)benzoic acid;

4- (2-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4- yl)methoxy)phenyl)cyclopropyl)benzoic acid;

5-(2-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4- yl)methoxy)phenyl)cyclopropyl)-1-isopropyl-1 H-pyrazole-3-carboxylic acid;

6-(2-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4- yl)methoxy)phenyl)cyclopropyl)-1 -methyl- 1 H-indazole-3-carboxylic acid;

6-(2-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4- yl)methoxy)phenyl)cyclopropyl)-1 -isopropyl-1 H-indazole-3-carboxylic acid;

3- (3-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)phenyl)-3- hydroxycyclobutyl)benzoic acid;

5- (3-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)phenyl)-3- hydroxycyclobutyl)-1 -isopropyl-1 H-pyrazole-3-carboxylic acid;

6-(3-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)phenyl)-3- hydroxycyclobutyl)-1-methyl-1 H-indazole-3-carboxylic acid;

4- (3-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)phenyl)-3- hydroxycyclobutyl)benzoic acid;

3-(3-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)phenyl)-3- hydroxyazetidin-1 -yl)benzoic acid; or

5- (3-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)phenyl)-3- hydroxyazetidin-1 -yl)nicotinic acid.

Pharmaceutical compositions for use in the present invention are formulated to contain therapeutically effective amounts of at least one FXR modulator. The pharmaceutical compositions are useful, for example, in the treatment of at least one tumor. In some embodiments, the at least one FXR agonist is formulated into a suitable pharmaceutical preparation such as solutions, suspensions, tablets, dispersible tablets, pills, capsules, powders, sustained release formulations or elixirs, for oral administration or in sterile solutions or suspensions for parenteral administration, as well as transdermal patch preparation and dry powder inhalers. Typically the FXR agonist described above is formulated into pharmaceutical compositions using techniques and procedures well known in the art (see, e.g. Ansel Introduction to Pharmaceutical Dosage Forms, Fourth Edition 1985, 126).

In the compositions, effective concentrations of one or more FXR agonists or pharmaceutically acceptable derivatives is (are) mixed with a suitable pharmaceutical carrier or vehicle.

Pharmaceutically acceptable derivatives include acids, bases, enol ethers and esters, salts, esters, hydrates, solvates and prodrug forms. A suitable derivative is selected such that its pharmacokinetic properties are superior with respect to at least one characteristic to the corresponding parent agent. The FXR agonist may be derivatized prior to formulation.

The concentrations of the FXR agonist in the compositions are effective for delivery of an amount, upon administration, that treats one or more of the symptoms of at least one tumor.

Typically, by way of example and without limitation, the compositions are formulated for single dosage administration. To formulate a composition, the weight fraction of FXR agonist is dissolved, suspended, dispersed or otherwise mixed in a selected vehicle at an effective concentration such that the treated condition, a malignancy, is relieved or ameliorated. Pharmaceutical carriers or vehicles suitable for administration of the FXR agonist include any such carriers known to those skilled in the art to be suitable for the particular mode of administration.

The active FXR agonist is included in the pharmaceutically acceptable carrier in an amount sufficient to exert a therapeutically useful effect in the absence of certain undesirable side effects on the patient treated. The therapeutically effective concentration may be determined empirically by testing the agents in in vitro and in vivo systems described herein and in WO 2011/020615.

The concentration of active FXR agonist in the pharmaceutical composition will depend on absorption, inactivation and excretion rates of the active agent, the physicochemical characteristics of the agent, the dosage schedule, and amount administered as well as other factors known to those of skill in the art. For example, the amount that is delivered is sufficient to treat at least one tumor as described herein.

Typically a therapeutically effective dosage should produce a serum concentration of active agent of from about 0.1 ng/mL to about 50-100 g/mL The pharmaceutical compositions typically should provide a dosage of from about 0.001 mg to about 2000 mg of FXR agonist per kilogram of body weight per day. Pharmaceutical dosage unit forms are prepared to provide from about 1 mg to about 1000 mg, such as from about 10 to about 500 mg of the active agent or a combination of agents per dosage unit form.

The active agent may be administered at once, or may be divided into a number of smaller doses to be administered at intervals of time. It is understood that the precise dosage and duration of treatment is a function of the tumor being treated and may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data. It is to be noted that concentrations and dosage values may also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed methods.

Thus, effective concentrations or amounts of one or more FXR agonist or pharmaceutically acceptable derivatives thereof are mixed with a suitable pharmaceutical carrier or vehicle for systemic, topical or local administration to form pharmaceutical compositions. FXR agonists are included in an amount effective for treating at least one tumor. The concentration of active agent in the composition will depend on absorption, inactivation, excretion rates of the active agent, the dosage schedule, amount administered, particular formulation as well as other factors known to those of skill in the art.

The compositions are intended to be administered by a suitable route, including by way of example and without limitation orally, parenterally, rectally, topically and locally. For oral administration, capsules and tablets can be used. The compositions are in liquid, semi-liquid or solid form and are formulated in a manner suitable for each route of administration.

Solutions or suspensions used for parenteral, intradermal, subcutaneous, or topical application can include any of the following components, in any combination: a sterile diluent, including by way of example without limitation, water for injection, saline solution, fixed oil, polyethylene glycol, glycerine, propylene glycol or other synthetic solvent; antimicrobial agents, such as benzyl alcohol and methyl parabens; antioxidants, such as ascorbic acid and sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid (EDTA); buffers, such as acetates, citrates and phosphates; and agents for the adjustment of tonicity such as sodium chloride or dextrose. Parenteral preparations can be enclosed in ampoules, disposable syringes or single or multiple dose vials made of glass, plastic or other suitable material.

In instances in which the agents exhibit insufficient solubility, methods for solubilizing agents may be used. Such methods are known to those of skill in this art, and include, but are not limited to, using co-solvents, such as dimethylsulfoxide (DMSO), using surfactants, such as TWEEN^® 80, or dissolution in aqueous sodium bicarbonate. Pharmaceutically acceptable derivatives of the agents may also be used in formulating effective pharmaceutical compositions.

Upon mixing or addition of the agent(s), the resulting mixture may be a solution, suspension, emulsion or the like. The form of the resulting mixture depends upon a number of factors, including the intended mode of administration and the solubility of the agent in the selected carrier or vehicle. The effective concentration is sufficient for treating one or more symptoms of at least one malignancy and may be empirically determined.

The pharmaceutical compositions are provided for administration to humans and animals in unit dosage forms, such as tablets, capsules, pills, powders, granules, sterile parenteral solutions or suspensions, and oral solutions or suspensions, and oil-water emulsions containing suitable quantities of the agents or pharmaceutically acceptable derivatives thereof. The pharmaceutically therapeutically active agents and derivatives thereof are typically formulated and administered in unit-dosage forms or multiple-dosage forms. Unit-dose forms as used herein refers to physically discrete units suitable for human and animal subjects and packaged individually as is known in the art. Each unit-dose contains a predetermined quantity of the therapeutically active agent sufficient to produce the desired therapeutic effect, in association with the required pharmaceutical carrier, vehicle or diluent. Examples of unit-dose forms include ampoules and syringes and individually packaged tablets or capsules. Unit-dose forms may be administered in fractions or multiples thereof. A multiple-dose form is a plurality of identical unit-dosage forms packaged in a single container to be administered in segregated unit-dose form. Examples of multiple-dose forms include vials, bottles of tablets or capsules or bottles of pints or gallons. Hence, multiple dose form is a multiple of unit-doses which are not segregated in packaging.

The composition can contain along with the active agent, for example and without limitation: a diluent such as lactose, sucrose, dicalcium phosphate, or carboxymethylcellulose; a lubricant, such as magnesium stearate, calcium stearate and talc; and a binder such as starch, natural gums, such as gum acacia gelatin, glucose, molasses, polyvinylpyrrolidone, celluloses and derivatives thereof, povidone, crospovidones and other such binders known to those of skill in the art. Liquid pharmaceutically administrable compositions can, for example, be prepared by dissolving, dispersing, or otherwise mixing an active agent as defined above and optional pharmaceutical adjuvants in a carrier, such as, by way of example and without limitation, water, saline, aqueous dextrose, glycerol, glycols, ethanol, and the like, to thereby form a solution or suspension. If desired, the pharmaceutical composition to be administered may also contain minor amounts of nontoxic auxiliary substances such as wetting agents, emulsifying agents, or solubilizing agents, pH buffering agents and the like, such as, by way of example and without limitation, acetate, sodium citrate, cyclodextrin derivatives, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, and other such agents. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example, see Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 15th Edition, 1975. The composition or formulation to be administered will, in any event, contain a quantity of the active agent in an amount sufficient to alleviate the symptoms of the treated subject.

Dosage forms or compositions containing active agent in the range of 0.005% to 100% with the balance made up from non-toxic carrier may be prepared. For oral administration, a pharmaceutically acceptable non-toxic composition is formed by the incorporation of any of the normally employed excipients, such as, for example and without limitation, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, talcum, cellulose derivatives, sodium crosscarmellose, glucose, sucrose, magnesium carbonate or sodium saccharin. Such compositions include solutions, suspensions, tablets, capsules, powders and sustained release formulations, such as, but not limited to, implants and microencapsulated delivery systems, and biodegradable, biocompatible polymers, such as collagen, ethylene vinyl acetate, polyanhydrides, polyglycolic acid, polyorthoesters, polylactic acid and others. Methods for preparation of these compositions are known to those skilled in the art. The contemplated compositions may contain 0.001 %-100% active agent, such as 0.1 -85%, or such as 75-95%.

The active agents or pharmaceutically acceptable derivatives may be prepared with carriers that protect the agent against rapid elimination from the body, such as time release formulations or coatings. The compositions may include other active agents to obtain desired combinations of properties. FXR agonists or pharmaceutically acceptable derivatives thereof, may also be advantageously administered for therapeutic or prophylactic purposes together with another pharmacological agent known in the general art to be of value in treating at least one malignancy.

Oral pharmaceutical dosage forms include, by way of example and without limitation, solid, gel and liquid. Solid dosage forms include tablets, capsules, granules, and bulk powders. Oral tablets include compressed, chewable lozenges and tablets which may be enteric-coated, sugar-coated or film-coated. Capsules may be hard or soft gelatin capsules, while granules and powders may be provided in non-effervescent or effervescent form with the combination of other ingredients known to those skilled in the art.

In certain embodiments, the formulations are solid dosage forms, such as capsules or tablets. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or agents of a similar nature: a binder; a diluent; a disintegrating agent; a lubricant; a glidant; a sweetening agent; and a flavoring agent.

Examples of binders include, by way of example and without limitation, microcrystalline cellulose, gum tragacanth, glucose solution, acacia mucilage, gelatin solution, sucrose, and starch paste. Lubricants include, by way of example and without limitation, talc, starch, magnesium or calcium stearate, lycopodium and stearic acid. Diluents include, by way of example and without limitation, lactose, sucrose, starch, kaolin, salt, mannitol, and dicalcium phosphate. Glidants include, by way of example and without limitation, colloidal silicon dioxide. Disintegrating agents include, by way of example and without limitation, crosscarmellose sodium, sodium starch glycolate, alginic acid, corn starch, potato starch, bentonite, methylcellulose, agar and carboxymethylcellulose. Coloring agents include, by way of example and without limitation, any of the approved certified water soluble FD and C dyes, mixtures thereof; and water insoluble FD and C dyes suspended on alumina hydrate. Sweetening agents include, by way of example and without limitation, sucrose, lactose, mannitol and artificial sweetening agents such as saccharin, and any number of spray dried flavors. Flavoring agents include, by way of example and without limitation, natural flavors extracted from plants such as fruits and synthetic blends of agents which produce a pleasant sensation, such as, but not limited to peppermint and methyl salicylate. Wetting agents include, by way of example and without limitation, propylene glycol monostearate, sorbitan monooleate, diethylene glycol monolaurate, and polyoxyethylene laural ether. Emetic-coatings include, by way of example and without limitation, fatty acids, fats, waxes, shellac, ammoniated shellac and cellulose acetate phthalates. Film coatings include, by way of example and without limitation, hydroxyethylcellulose, sodium carboxymethylcellulose, polyethylene glycol 4000 and cellulose acetate phthalate.

If oral administration is desired, the agent could be provided in a composition that protects it from the acidic environment of the stomach. For example, the composition can be formulated in an enteric coating that maintains its integrity in the stomach and releases the active agent in the intestine. The composition may also be formulated in combination with an antacid or other such ingredient.

When the dosage unit form is a capsule, it can contain, in addition to material of the above type, a liquid carrier such as a fatty oil. In addition, dosage unit forms can contain various other materials which modify the physical form of the dosage unit, for example, coatings of sugar and other enteric agents. The agents can also be administered as a component of an elixir, suspension, syrup, wafer, sprinkle, chewing gum or the like. A syrup may contain, in addition to the active agents, sucrose as a sweetening agent and certain preservatives, dyes and colorings and flavors. The active materials can also be mixed with other active materials which do not impair the desired action, or with materials that supplement the desired action, such as antacids, H2 blockers, and diuretics.

Pharmaceutically acceptable carriers included in tablets are binders, lubricants, diluents, disintegrating agents, coloring agents, flavoring agents, and wetting agents. Enteric-coated tablets, because of the enteric-coating, resist the action of stomach acid and dissolve or disintegrate in the neutral or alkaline intestines. Sugar-coated tablets are compressed tablets to which different layers of pharmaceutically acceptable substances are applied. Film-coated tablets are compressed tablets which have been coated with a polymer or other suitable coating. Multiple compressed tablets are compressed tablets made by more than one compression cycle utilizing the pharmaceutically acceptable substances previously mentioned. Coloring agents may also be used in the above dosage forms. Flavoring and sweetening agents are used in compressed tablets, sugar-coated, multiple compressed and chewable tablets. Flavoring and sweetening agents are useful in the formation of chewable tablets and lozenges.

Liquid oral dosage forms include aqueous solutions, emulsions, suspensions, solutions and/or suspensions reconstituted from non-effervescent granules and effervescent preparations reconstituted from effervescent granules. Aqueous solutions include, for example, elixirs and syrups. Emulsions are either oil-in-water or water-in-oil.

Elixirs are clear, sweetened, hydroalcoholic preparations. Pharmaceutically acceptable carriers used in elixirs include solvents. Syrups are concentrated aqueous solutions of a sugar, for example, sucrose, and may contain a preservative. An emulsion is a two-phase system in which one liquid is dispersed in the form of small globules throughout another liquid. Pharmaceutically acceptable carriers used in emulsions are non-aqueous liquids, emulsifying agents and preservatives. Suspensions use pharmaceutically acceptable suspending agents and preservatives. Pharmaceutically acceptable substances used in non-effervescent granules, to be reconstituted into a liquid oral dosage form, include diluents, sweeteners and wetting agents. Pharmaceutically acceptable substances used in effervescent granules, to be reconstituted into a liquid oral dosage form, include organic acids and a source of carbon dioxide. Coloring and flavoring agents may be used in any of the above dosage forms.

Solvents, include by way of example and without limitation, glycerin, sorbitol, ethyl alcohol and syrup. Examples of preservatives include without limitation glycerin, methyl and propylparaben, benzoic add, sodium benzoate and alcohol. Non-aqueous liquids utilized in emulsions, include by way of example and without limitation, mineral oil and cottonseed oil. Emulsifying agents, include by way of example and without limitation, gelatin, acacia, tragacanth, bentonite, and surfactants such as polyoxyethylene sorbitan monooleate. Suspending agents include, by way of example and without limitation, sodium carboxymethylcellulose, pectin, tragacanth, Veegum and acacia. Diluents include, by way of example and without limitation, lactose and sucrose. Sweetening agents include, by way of example and without limitation, sucrose, syrups, glycerin and artificial sweetening agents such as saccharin. Wetting agents, include by way of example and without limitation, propylene glycol monostearate, sorbitan monooleate, diethylene glycol monolaurate, and polyoxyethylene lauryl ether. Organic acids include, by way of example and without limitation, citric and tartaric acid. Sources of carbon dioxide include, by way of example and without limitation, sodium bicarbonate and sodium carbonate. Coloring agents include, by way of example and without limitation, any of the approved certified water soluble FD and C dyes, and mixtures thereof. Flavoring agents include, by way of example and without limitation, natural flavors extracted from plants such fruits, and synthetic blends of agents which produce a pleasant taste sensation.

For a solid dosage form, the solution or suspension, in for example propylene carbonate, vegetable oils or triglycerides, is encapsulated in a gelatin capsule. Such solutions, and the preparation and encapsulation thereof, are disclosed in US 4328245, 4409239, and 4410545. For a liquid dosage form, the solution, e.g., for example, in a polyethylene glycol, may be diluted with a sufficient quantity of a pharmaceutically acceptable liquid carrier, e.g., water, to be easily measured for administration.

Alternatively, liquid or semi-solid oral formulations may be prepared by dissolving or dispersing the active agent or salt in vegetable oils, glycols, triglycerides, propylene glycol esters (e.g., propylene carbonate) and other such carriers, and encapsulating these solutions or suspensions in hard or soft gelatin capsule shells. Other useful formulations include those set forth in US Re 28819 and US 4358603. Briefly, such formulations include, but are not limited to, those containing a agent provided herein, a dialkylated mono- or poly-alkylene glycol, including, but not limited to, 1 ,2-dimethoxymethane, diglyme, triglyme, tetraglyme, polyethylene glycol-350-dimethyl ether, polyethylene glycol-550-dimethyl ether, polyethylene glycol-750-dimethyl ether wherein 350, 550 and 750 refer to the approximate average molecular weight of the polyethylene glycol, and one or more antioxidants, such as butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), propyl gallate, vitamin E, hydroquinone, hydroxycoumarins, ethanolamine, lecithin, cephalin, ascorbic acid, malic acid, sorbitol, phosphoric acid, thiodipropionic acid and its esters, and dithiocarbamates.

Other formulations include, but are not limited to, aqueous alcoholic solutions including a pharmaceutically acceptable acetal. Alcohols used in these formulations are any pharmaceutically acceptable water-miscible solvents having one or more hydroxyl groups, including, but not limited to, propylene glycol and ethanol. Acetals include, but are not limited to, di(lower alkyl) acetals of lower alkyl aldehydes such as acetaldehyde diethyl acetal.

Tablets and capsules formulations may be coated as known by those of skill in the art in order to modify or sustain dissolution of the active ingredient. Thus, for example and without limitation, they may be coated with a conventional enterically digestible coating, such as phenylsalicylate, waxes and cellulose acetate phthalate.

Parenteral administration, generally characterized by injection, either subcutaneously, intramuscularly or intravenously is also contemplated herein. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Suitable excipients, include by way of example and without limitation, water, saline, dextrose, glycerol or ethanol. In addition, if desired, the pharmaceutical compositions to be administered may also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers, and other such agents, such as for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate and cyclodextrins.

Implantation of a slow-release or sustained-release system, such that a constant level of dosage is maintained (see e.g., US 3710795) is also contemplated herein. Briefly, a FXR agonist is dispersed in a solid inner matrix, e.g., polymethylmethacrylate, polybutylmethacrylate, plasticized or unplasticized polyvinylchloride, plasticized nylon, plasticized polyethyleneterephthalate, natural rubber, polyisoprene, polyisobutylene, polybutadiene, polyethylene, ethylene-vinylacetate copolymers, silicone rubbers, polydimethylsiloxanes, silicone carbonate copolymers, hydrophilic polymers such as hydrogels of esters of acrylic and methacrylic acid, collagen, cross-linked polyvinylalcohol and cross- linked partially hydrolyzed polyvinyl acetate, that is surrounded by an outer polymeric membrane, e.g., polyethylene, polypropylene, ethylene/propylene copolymers, ethylene/ethyl acrylate copolymers, ethylene/vinylacetate copolymers, silicone rubbers, polydimethyl siloxanes, neoprene rubber, chlorinated polyethylene, polyvinylchloride, vinylchloride copolymers with vinyl acetate, vinylidene chloride, ethylene and propylene, ionomer polyethylene terephthalate, butyl rubber epichlorohydrin rubbers, ethylene/vinyl alcohol copolymer, ethylene/vinyl acetate/vinyl alcohol terpolymer, and ethylene/vinyloxyethanol copolymer, that is insoluble in body fluids. The agent diffuses through the outer polymeric membrane in a release rate controlling step. The percentage of active agent contained in such parenteral compositions is highly dependent on the specific nature thereof, as well as the activity of the agent and the needs of the subject.

Parenteral administration of FXR agonists includes intravenous, subcutaneous and intramuscular administrations. Preparations for parenteral administration include sterile solutions ready for injection, sterile dry soluble products, such as lyophilized powders, ready to be combined with a solvent just prior to use, including hypodermic tablets, sterile suspensions ready for injection, sterile dry insoluble, products ready to be combined with a vehicle just prior to use and sterile emulsions. The solutions may be either aqueous or nonaqueous.

If administered intravenously, suitable carriers include physiological saline or phosphate buffered saline (PBS), and solutions containing thickening and solubilizing agents, such as glucose, polyethylene glycol, and polypropylene glycol and mixtures thereof.

Pharmaceutically acceptable carriers used in parenteral preparations include aqueous vehicles, nonaqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifying agents, sequestering or chelating agents and other pharmaceutically acceptable substances.

Aqueous vehicles include, by way of example and without limitation, sodium chloride injection, Ringers injection, isotonic dextrose injection, sterile water injection, dextrose and lactated Ringers injection. Nonaqueous parenteral vehicles include, by way of example and without limitation, fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil and peanut oil. Antimicrobial agents in bacteriostatic or fungistatic concentrations must be added to parenteral preparations packaged in multiple-dose containers which include phenols or cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl para-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride and benzethonium chloride. Isotonic agents include, by way of example and without limitation, sodium chloride and dextrose. Buffers include phosphate and citrate. Antioxidants include sodium bisulfate. Local anesthetics include procaine hydrochloride. Suspending and dispersing agents include sodium carboxymethylcelluose, hydroxypropyl methylcellulose and polyvinylpyrrolidone. Emulsifying agents include Polysorbate 80 (TWEEN^® 80). A sequestering or chelating agent of metal ions include EDTA. Pharmaceutical carriers also include, by way of example and without limitation, ethyl alcohol, polyethylene glycol and propylene glycol for water miscible vehicles and sodium hydroxide, hydrochloric acid, citric acid or lactic acid for pH adjustment.

The concentration of the pharmaceutically active agent is adjusted so that an injection provides an effective amount to produce the desired pharmacological effect. The exact dose depends on the age, weight and condition of the patient or animal as is known in the art.

The unit-dose parenteral preparations are packaged in an ampoule, a vial or a syringe with a needle. Preparations for parenteral administration should be sterile, as is known and practiced in the art.

Illustratively, intravenous or intraarterial infusion of a sterile aqueous solution containing an active agent is an effective mode of administration. Another embodiment is a sterile aqueous or oily solution or suspension containing an active agent injected as necessary to produce the desired pharmacological effect.

Injectables are designed for local and systemic administration. Typically a therapeutically effective dosage is formulated to contain a concentration of at least about 0.1% w/w up to about 90% w/w or more, such as more than 1% w/w of the active agent to the treated tissue(s). The active agent may be administered at once, or may be divided into a number of smaller doses to be administered at intervals of time. It is understood that the precise dosage and duration of treatment is a function of the tissue being treated and may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data. It is to be noted that concentrations and dosage values may also vary with the age of the individual treated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the formulations, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed formulations.

The agent may be suspended in micronized or other suitable form or may be derivatized, e.g. to produce a more soluble active product or to produce a prodrug or other pharmaceutically acceptable derivative. The form of the resulting mixture depends upon a number of factors, including the intended mode of administration and the solubility of the agent in the selected carrier or vehicle. The effective concentration is sufficient for ameliorating the symptoms of the condition and may be empirically determined.

Lyophilized powders can be reconstituted for administration as solutions, emulsions, and other mixtures or formulated as solids or gels.

The sterile, lyophilized powder is prepared by dissolving a agent provided herein, or a pharmaceutically acceptable derivative thereof, in a suitable solvent. The solvent may contain an excipient which improves the stability or other pharmacological component of the powder or reconstituted solution, prepared from the powder. Excipients that may be used include, but are not limited to, dextrose, sorbital, fructose, corn syrup, xylitol, glycerin, glucose, sucrose or other suitable agent. The solvent may also contain a buffer, such as citrate, sodium or potassium phosphate or other such buffer known to those of skill in the art at, typically, about neutral pH. Subsequent sterile filtration of the solution followed by lyophilization under standard conditions known to those of skill in the art provides the desired formulation. Generally, the resulting solution will be apportioned into vials for lyophilization. Each vial will contain, by way of example and without limitation, a single dosage (10-1000 mg, such as 100- 500 mg) or multiple dosages of the agent. The lyophilized powder can be stored under appropriate conditions, such as at about 4°C. to room temperature. Reconstitution of this lyophilized powder with water for injection provides a formulation for use in parenteral administration. For reconstitution, about 1-50 mg, such as about 5-35 mg, for example, about 9-30 mg of lyophilized powder, is added per mL of sterile water or other suitable carrier. The precise amount depends upon the selected agent. Such amount can be empirically determined.

Topical mixtures are prepared as described for the local and systemic administration. The resulting mixture may be a solution, suspension, emulsions or the like and are formulated as creams, gels, ointments, emulsions, solutions, elixirs, lotions, suspensions, tinctures, pastes, foams, aerosols, irrigations, sprays, suppositories, bandages, dermal patches or any other formulations suitable for topical administration.

The agents or pharmaceutically acceptable derivatives thereof may be formulated as aerosols for topical application, such as by inhalation (see e.g., US 4044126, 4414209, and 4364923, which describe aerosols for delivery of a steroid useful for treatment of inflammatory diseases, particularly asthma). These formulations for administration to the respiratory tract can be in the form of an aerosol or solution for a nebulizer, or as a microfine powder for insufflation, alone or in combination with an inert carrier such as lactose. In such a case, the particles of the formulation will, by way of example and without limitation, have diameters of less than about 50 microns, such as less than about 10 microns.

The agents may be formulated for local or topical application, such as for topical application to the skin and mucous membranes, such as in the eye, in the form of gels, creams, and lotions and for application to the eye or for intracisternal or intraspinal application. Topical administration is contemplated for transdermal delivery and also for administration to the eyes or mucosa, or for inhalation therapies. Nasal solutions of the active agent alone or in combination with other pharmaceutically acceptable excipients can also be administered.

These solutions, particularly those intended for ophthalmic use, may be formulated, by way of example and without limitation, as about 0.01% to about 10% isotonic solutions, pH about 5-7, with appropriate salts.

Other routes of administration, such as transdermal patches, and rectal administration are also contemplated herein.

Transdermal patches, including iotophoretic and electrophoretic devices, are well known to those of skill in the art. For example, such patches are disclosed in US 6267983, 6261595, 6256533, 6167301, 6024975, 6010715, 5985317, 5983134, 5948433, and 5860957.

Pharmaceutical dosage forms for rectal administration are rectal suppositories, capsules and tablets for systemic effect. Rectal suppositories are used herein mean solid bodies for insertion into the rectum which melt or soften at body temperature releasing one or more pharmacologically or therapeutically active ingredients. Pharmaceutically acceptable substances utilized in rectal suppositories are bases or vehicles and agents to raise the melting point. Examples of bases include cocoa butter (theobroma oil), glycerin-gelatin, carbowax (polyoxyethylene glycol) and appropriate mixtures of mono-, di- and triglycerides of fatty acids. Combinations of the various bases may be used. Agents to raise the melting point of suppositories include spermaceti and wax. Rectal suppositories may be prepared either by the compressed method or by molding. The typical weight of a rectal suppository is, by way of example and without limitation, about 2 to 3 g.

Tablets and capsules for rectal administration are manufactured using the same pharmaceutically acceptable substance and by the same methods as for formulations for oral administration.

The FXR agonists, or pharmaceutically acceptable derivatives thereof, may also be formulated to be targeted to a particular tissue, receptor, or other area of the body of the subject to be treated. Many such targeting methods are well known to those of skill in the art. Such targeting methods are contemplated herein for use in the instant compositions. For non-limiting examples of targeting methods, see e.g., US 6316652, 6274552, 6271359, 6253872, 6139865, 6131570, 6120751 , 6071495, 6060082, 6048736, 6039975, 6004534, 5985307, 5972366, 5900252, 5840674, 5759542 and 5709874.

In some embodiments, liposomal suspensions, including tissue-targeted liposomes, such as tumor-targeted liposomes, may also be suitable as pharmaceutically acceptable carriers. These may be prepared according to methods known to those skilled in the art. For example, liposome formulations may be prepared as described in US 452281 1. Briefly, liposomes such as multilamellar vesicles (MLV's) may be formed by drying down egg phosphatidyl choline and brain phosphatidyl serine (7:3 molar ratio) on the inside of a flask. A solution of a agent provided herein in phosphate buffered saline lacking divalent cations (PBS) is added and the flask shaken until the lipid film is dispersed. The resulting vesicles are washed to remove unencapsulated agent, pelleted by centrifugation, and then resuspended in PBS.

The FXR agonists or pharmaceutically acceptable derivatives for use in the present invention may be packaged as articles of manufacture containing packaging material, a FXR agonist or pharmaceutically acceptable derivative thereof provided herein, which is effective for modulating the activity of FXR or for treatment, of one or more symptoms of at least one tumor within the packaging material, and a label that indicates that the FXR agonist or composition, or pharmaceutically acceptable derivative thereof, is used for modulating the activity of FXR for treatment of one or more symptoms of at least one tumor.

The articles of manufacture provided herein contain packaging materials. Packaging materials for use in packaging pharmaceutical products are well known to those of skill in the art. See e.g. US 5323907, 5052558 and 5033252. Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, inhalers, pumps, bags, vials, containers, syringes, bottles, and any packaging material suitable for a selected formulation and intended mode of administration and treatment.

Standard physiological, pharmacological and biochemical procedures are available for testing agents to identify those that possess biological activities that modulate the activity of FXR. Such assays include, for example, biochemical assays such as binding assays, fluorescence polarization assays, FRET based coactivator recruitment assays (see generally J. F. Glickman et al., J. Biomol. Screen. 2002, 7, 3), as well as cell based assays including the co-transfection assay, the use of LBD-Gal 4 chimeras, protein-protein interaction assays (see J. M. Lehmann et al., J. Biol Chem. 1997, 272, 3137), and gene expression assays.

Assays that do not require washing or liquid separation steps can be used for high throughput screening systems and include biochemical assays such as fluorescence polarization assays (see e.g. J. C. Owicki, J. Biomol. Screen. 2000, 5, 297) scintillation proximity assays (SPA) (see e.g. J. W. Carpenter et al., Methods Mol. Biol. 2002, 190, 31) and fluorescence resonance energy transfer energy transfer (FRET) or time resolved FRET based coactivator recruitment assays (R. Mukherjee et al., J. Steroid Biochem. Mol. Biol. 2002, 81 , 217; G. Zhou et al., Mol. Endocrinol. 1998, 12, 1594). Generally such assays can be preformed using either the full length receptor, or isolated ligand binding domain (LBD). In the case of FXR, the LBD comprises amino acids 187 to 472 of the full length sequence.

If a fluorescently labeled ligand is available, fluorescence polarization assays provide a way of detecting binding of agents to FXR by measuring changes in fluorescence polarization that occur as a result of the displacement of a trace amount of the label ligand by the agent. Additionally this approach can also be used to monitor the ligand dependent association of a fluorescently labeled coactivator peptide to FXR to detect ligand binding to FXR.

The ability of an agent to bind to a receptor, or heterodimer complex with RXR, can also be measured in a homogeneous assay format by assessing the degree to which the agent can compete off a radiolabeled ligand with known affinity for the receptor using a SPA. In this approach, the radioactivity emitted by a radiolabeled agent generates an optical signal when it is brought into close proximity to a scintillant such as a Ysi-copper containing bead, to which FXR is bound. If the radiolabeled agent is displaced from FXR the amount of light emitted from FXR bound scintillant decreases, and this can be readily detected using standard microplate liquid scintillation plate readers such as, for example, a Wallac MicroBeta reader.

This approach can also be exploited to measure the ligand dependent interaction of a co- activator peptide with FXR in order to characterize the agonist or antagonist activity of the agents disclosed herein. Typically the assay in this case involves the use a recombinant Glutathione-S-transferase (GST)-FXR LBD fusion protein and a synthetic biotinylated peptide sequenced derived from the receptor interacting domain of a co-activator peptide such as the steroid receptor coactivator 1 (SRC-1 ). Typically GST-LBD is labeled with a europium chelate (donor) via a europium-tagged anti-GST antibody, and the coactivator peptide is labeled with allophycocyanin via a streptavidin-biotin linkage.

In the presence of an agonist for FXR, the peptide is recruited to the GST-LBD bringing europium and allophycocyanin into close proximity to enable energy transfer from the europium chelate to the allophycocyanin. Upon excitation of the complex with light at 340 nm excitation energy absorbed by the europium chelate is transmitted to the allophycocyanin moiety resulting in emission at 665 nm. If the europium chelate is not brought in to close proximity to the allophycocyanin moiety there is little or no energy transfer and excitation of the europium chelate results in emission at 615 nm. Thus the intensity of light emitted at 665 nm gives an indication of the strength of the protein-protein interaction. The activity of a FXR antagonist can be measured by determining the ability of a agent to competitively inhibit (i.e., IC₅₀) the activity of an agonist for FXR. This approach is described in WO 201 1/020615.

In addition a variety of cell based assay methodologies may be successfully used in screening assays to identify and profile the specificity of agents described herein. These approaches include the co-transfection assay, translocation assays, and gene expression assays.

Three basic variants of the co-transfection assay strategy exist, co-transfection assays using full-length FXR, co-transfection assays using chimeric FXRs comprising the ligand binding domain of FXR fused to a heterologous DNA binding domain, and assays based around the use of the mammalian two hybrid assay system.

The basic co-transfection assay is based on the co-transfection into the cell of an expression plasmid to express FXR in the cell with a reporter plasmid comprising a reporter gene whose expression is under the control of DNA sequence that is capable of interacting with that nuclear receptor (See e.g. WO 201 1/020615). Treatment of the transfected cells with an agonist for FXR increases the transcriptional activity of that receptor, which is reflected by an increase in expression of the reporter gene, which may be measured by a variety of standard procedures.

For those receptors that function as heterodimers with RXR, such as FXR, the co-transfection assay typically includes the use of expression plasmids for both FXR and RXR. Typical co- transfection assays require access to full-length FXR and suitable response elements that provide sufficient screening sensitivity and specificity to FXR.

Genes encoding the following full-length previously described proteins, which are suitable for use in the co-transfection studies and profiling the agents described herein, include rat FXR (GenBank Accession No. NM_21745), mouse FXR (GenBank Accession No. NM_009108), human FXR (GenBank Accession No. NM_05123), human RXR alpha (GenBank Accession No. NM_02957).

Reporter plasmids may be constructed using standard molecular biological techniques by placing cDNA encoding for the reporter gene downstream from a suitable minimal promoter. For example luciferase reporter plasmids may be constructed by placing cDNA encoding firefly luciferase immediately down stream from the herpes virus thymidine kinase promoter (located at nucleotides residues -105 to +51 of the thymidine kinase nucleotide sequence) which is linked in turn to the various response elements.

Numerous methods of co-transfecting the expression and reporter plasmids are known to those of skill in the art and may be used for the co-transfectton assay to introduce the plasmids into a suitable cell type. Typically such a cell will not endogenously express FXRs that interact with the response elements used in the reporter plasmid.

Numerous reporter gene systems are known in the art and include, for example, alkaline phosphatase (J. Berger et al., Gene 1988, 66, 1 ; S. R. Kain, Methods Mol. Biol. 1997, 63, 49), β-galactosidase (See US 5070012 and I. Bronstein et al., J. Chemilum. Biolum. 1989, 4, 99), chloramphenicol acetyltransferase (See C. M. Gorman et al., Mol. Cell. Biol. 1982, 2, 1044), β- glucuronidase, peroxidase, β-lactamase (US 5741657 and US 5955604), catalytic antibodies, luciferases (US 5221623, US 5683888, US 5674713, US 5650289, US 5843746) and naturally fluorescent proteins (R. Y. Tsien, Annu. Rev. Biochem. 1998, 67, 509).

The use of chimeras comprising the LBD of FXR to a heterologous DNA binding domain (DBD) expands the versatility of cell based assays by directing activation of FXR in question to defined DNA binding elements recognized by defined DNA binding domain (see WO 95/18380). This assay expands the utility of cell based co-transfection assays in cases where the biological response or screening window using the native DNA binding domain is not satisfactory.

In general the methodology is similar to that used with the basic co-transfection assay, except that a chimeric construct is used in place of the full-length FXR. As with the full-length FXR, treatment of the transfected cells with an agonist for the FXR LBD increases the transcriptional activity of the heterologous DNA binding domain which is reflected by an increase in expression of the reporter gene as described above. Typically for such chimeric constructs, the DNA binding domains from defined FXRs, or from yeast or bacterially derived transcriptional regulators such as members of the GAL 4 and Lex A/Umud super families are used. A third cell based assay of utility for screening agents is a mammalian two-hybrid assay that measures the ability of the nuclear hormone receptor to interact with a cofactor in the presence of a ligand (see e.g. US 5667973, US 5283173 and US 5468614). The basic approach is to create three plasmid constructs that enable the interaction of FXR with the interacting protein to be coupled to a transcriptional readout within a living cell. The first construct is an expression plasmid for expressing a fusion protein comprising the interacting protein, or a portion of that protein containing the interacting domain, fused to a GAL4 DNA binding domain. The second expression plasmid comprises DNA encoding the FXR fused to a strong transcription activation domain such as VP16, and the third construct comprises the reporter plasmid comprising a reporter gene with a minimal promoter and GAL4 upstream activating sequences.

Once all three plasmids are introduced into a cell, the GAL4 DNA binding domain encoded in the first construct allows for specific binding of the fusion protein to GAL4 sites upstream of a minimal promoter. However because the GAL4 DNA binding domain typically has no strong transcriptional activation properties in isolation, expression of the reporter gene occurs only at a low level. In the presence of a ligand, the FXR-VP16 fusion protein can bind to the GAL4- interacting protein fusion protein bringing the strong transcriptional activator VP 16 in close proximity to the GAL4 binding sites and minimal promoter region of the reporter gene. This interaction significantly enhances the transcription of the reporter gene, which can be measured for various reporter genes as described above. Transcription of the reporter gene is thus driven by the interaction of the interacting protein and FXR in a ligand dependent fashion.

An agent can be tested for the ability to induce nuclear localization of a nuclear protein receptor, such as FXR. Upon binding of an agonist, FXR translocates from the cytoplasm to the nucleus. Microscopic techniques can be used to visualize and quantitate the amount of FXR located in the nucleus. In some embodiments, this assay can utilize a chimeric FXR fused to a fluorescent protein.

An agent can also be evaluated for its ability to increase or decrease the expression of genes known to be modulated by FXR in vivo, using Northern-blot, RT PCR or oligonucleotide microarray analysis to analyze RNA levels. Western-blot analysis can be used to measure expression of proteins encoded by FXR target genes. Expression of N-myc downregulated gene 2 (NDRG2) gene is modulated by FXR. Additional genes known to be regulated by the FXR in the liver include cholesterol 7 cc-hydroxylase (CYP7A1), the rate limiting enzyme in the conversion of cholesterol to bile acids, SHP, the bile salt export pump (BSEP, ABCB1 1), canalicular bile acid export protein, sodium taurocholate cotransporting polypeptide (NTCP, SLC10A1) and intestinal bile acid binding protein (l-BABP) and FGF19 in the ileum. Expression of a FXR target gene can be conveniently normalized to an internal control and the data plotted as fold induction relative to untreated or vehicle treated cells. Suitable internal control genes include but are not limited to TATA box binding protein (human TBP GenBank Acc. Nr. NM_003194 , mouse Tbp GeneBank Acc. Nr. NM_013684). A control agent, such as an agonist, may be included along with DMSO as high and low controls respectively for normalization of the assay data.

In another approach, an agent can be tested for FXR activity by evaluating its effect on an activity mediated by NDRG2. NDRG2 codes for an around 41 kD protein expression of which is downregulated in a number of human tumors including hepatocellular carcinoma, colorectal cancer, esophageal cancer and breast cancer. Forced overexpression of NDRG2 in certain tumor cells resulted in suppression of NFkB activity, downregulation of certain matrix metalloproteinases (MMPs) such as MMP-9 and MMP-2 as well as downregulation of CD24 all of which result in a reduction of tumor cell adhesion-, invasion- and metastasis-activity (see J. Zheng et al., BMC Cancer 2011 , 1 1 , 251 ; D. C. Lee et al., Cancer Res. 2008, 68, 4210; A. Kim et al., Carcinogenesis 2009, 30, 927).

It is known that NDRG2 inhibits the secretion and activities of multiple MMPs. The MMP may be MMP-9. The amount of MMPs secreted from a cell treated in the absence or presence of a agent can be measured using standard assays including but not limited to Western blot, enzyme linked immunosorbent assay, and gelatin zymography. Agonists of FXR may induce the expression of NDRG2, thereby leading to decreased secretion of MMPs.

The activities of MMPs affect the invasive, metastatic, and/or angiogenic activities of a cell, a group of cells, or a malignancy. Agonists of FXR may induce NDRG2 expression, thereby decreasing MMP secretion and MMP activity and decreasing the invasive, metastatic, and/or angiogenic activities of a cell.

The invasive and metastatic activities of cells can be measured using standard in vitro and in vivo methods. In some embodiments, invasion potential can be measured with assays that monitor the ability of cells to migrate through synthetic matrix. In some embodiments, metastatic potential can be measured in assays that monitor the dissemination of implants of test cells in animals.

Any agent which is a candidate for modulation of FXR may be tested by the methods described above. Generally, though not necessarily, agents are tested at several different concentrations and administered one or more times to optimize the chances that activation of the receptor will be detected and recognized if present. Typically assays are performed in triplicate, for example, and vary within experimental error by less than about 15%. Each experiment is typically repeated about three or more times with similar results. In some embodiments, the effects of agents and compositions on FXR activity can be evaluated in cells. Typically, the cells will express FXR either endogenously or heterogeneously by co-transfection. Cells that express FXR endogenously include, by way of example and without limitation: hepatocytes, including primary hepatocytes isolated from human, monkey, mouse, or rat, or hepatocyte cell lines, including HepG2, Huh7, or SK-Hep-1 cells; and intestinal cells including HT-29, CaCo2 and FHs 74 Int.

In some embodiments, the effects of agents and compositions on FXR gene expression can be evaluated in animals. After the administration of agents, various tissues can be harvested to determine the effect of agents on activities directly or indirectly regulated by FXR.

Provided herein are methods involving both in vitro and in vivo uses of an agent that modulates FXR activity. Such agents will typically exhibit FXR agonist, partial agonist, partial antagonist or antagonist activity in one of the in vitro assays described herein.

Methods of altering FXR activity, by contacting the receptor with at least one agent, are provided.

Compounds for use in treating at least one tumor are provided. The tumor may be characterized by proliferative activity, and the ability to invade surrounding tissues and to metastasize to other parts of the body. Invasion of tumor cells through the extracellular matrix propagates the tumor and is an important step in metastasis. MMPs are zinc dependent endopeptidases that play crucial roles in tissue remodeling processes including tissues invasion, metastasis, and angiogenesis. MMPs degrade components of the extracellular matrix, thereby expediting these processes and promoting tumor progression.

Exemplary tumors include and are not limited to hepatocellular carcinoma, colorectal cancer, 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.

Appropriate treatment for tumors 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 tumor 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.

In one type of staging system, cases are grouped into four stages, denoted by Roman numerals I to IV. In 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.

Another staging system is TNM staging which stands for the categories: Tumor, Nodes, and Metastases. In this system, 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.

These staging systems or variations of these staging systems or other suitable staging systems may be used to describe a tumor such as hepatocellular carcinoma. Few options only are available for the treatment of hepatocellular 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 hepatocellular cancer. Additional systemic treatments may be used to treat invasive and metastatic tumors.

The therapy of late stage hepatocellular carcinoma and late stage renal carcinomal has been shown efficacy to extend survival of tumor patients.

NDRG2 is widely expressed in normal tissues and nonneoplastic cell lines. Suppression of NDRG2 expression has been observed in many human tumor types.

Conversely, elevated NDRG2 expression has been shown to correlate with beneficial prognoses for several malignancies including hepatocellular carcinoma, colorectal cancer, and breast cancer. Restoration of NDRG2 expression in malignant cells can suppress their invasive, metastatic, and angiogenic activities through inhibition of matrix metalloproteases. Specifically, NDRG2 expression in tumor cells has been shown to suppress MMP-2 and CD24 expression.

Elevated MMP expression is observed in most human cancers and generally correlates with poor prognosis. Inhibitors of MMP activity have been tested in various experimental systems and shown some success in animal models of cancers.

Methods of inducing NDRG2 expression are provided herein through administering at least one FXR agonist.

Provided is a method for diagnosing the risk that a patient will develop at least one tumor. This method comprises measuring the level or expression of FXR and or the level of FXR activity in at least one tissue. Methods of measuring FXR expression include Northern-blot, RT PCR or oligonucleotide microarray analysis to analyze RNA levels and Western blot to measure protein levels. Methods of measuring FXR activity are described above. Measurement of FXR activity can be done by determining the level of direct of indirect target genes of FXR such as FGF19 induced by FXR in the human Ileum and secreted into the blood stream.

Administering at least one FXR agonist can potentiate the effects of known agents useful for the treatment of tumors. Contemplated herein is combination therapy using at least one FXR agonist or a pharmaceutically acceptable derivative thereof, in combination with one or more of the following: Sorafenib, Sunitinib, Erlotinib or Imatinib.

The FXR agonist, or pharmaceutically acceptable derivative thereof, is administered simultaneously with, prior to, or after administration of one or more of the above agents.

The invention is further illustrated by the following non-limiting examples.

EXAMPLES

Example 1

Using a Phenex proprietary software package (RePhex), IR1-type nuclear receptor response elements were identified to be present in the first introns of the NDRG2 genes of human, mouse and rat species.

Fig 1 : IR1 sites are predicted by RePhex to be located in the first introns of human, mouse and rat NDRG2 genes respectively. The location of the IR1 site in the first intron is depicted and the relevant sequences written in bold and underlined. The human sequence contains two IR- 1 type sequences in the first intron. The mutated version of the human NDRG2 gene that is used in Example 2 is shown below.

Example 2

The human IR-1 type sequence and a mutated version thereof were cloned into pGL4 (using the unique Xhol site) and both reporter plasmids and pGL2-2xFXRE (containing two copies of the IR-1 response elements from the human IBABP promoter) were cotransfected into HEK293 together with a human FXR isoform 3 expression plasmid (pTReX-DEST30-hFXR). After further growth in MEM supplemented with 8.6% FCS and Glutamine in presence of DMSO or 0.5 μΜ Px20350, luciferase activities were determined from extracts of cells and presented here as relative fold induction (Px20350 versus DMSO) corrected for internal Renilla Luciferase activities (cotransfected pCMV-RL Stratagene). FIG. 2: The wild-type but not a mutated NDRG2 IR-1 element is functional in luciferase reporter assays. Relative fold induction of Firefly luciferase activities (corrected by Renilla luciferase) of Px20350 versus DMSO treated cells (n = 4) are shown. For comparison, a pGL2 reporter plasmid containing tandem IR-1 type elements from the human IBABP promoter is shown. T-Test shows statistical significance (** = p<0.01 and *^** = p<0.001).

Example 3

Male C57BI/6 mice fed a Western diet, were administered vehicle or 30 mg/kg/d Px20350 mixed into the food for 4 weeks. After a four hours fasting period, mice were euthanized and real-time PCR analysis of liver RNA was performed.

FIG. 3 shows the effects of Px20350 on expression of the Ndrg2 gene in mice administered vehicle (grey bar) or 30 mg/kg/d Px20350 (black bar). Ndrg2 expression levels were calculated according to the comparative Ct method using normalization to Tbp as a house keeping transcript and the mean level of Ndrg2 expression in vehicle treated mice was defined as 1.0. Data are presented as the mean ± standard deviation (SD). 12 mice were tested in each group. The data show that Ndrg2 expression is induced by Px20350 (^* = p<0.05).

Example 4

Wildtype or FXR deficient mice on the C57BI/6 background were fed a standard chow diet. Mice were euthanized and real-time PCR analysis of liver RNA was performed.

FIG. 4 shows the relative expression of FXR candidate target genes in wt or FXR deficient mice (light grey bar) or FXR deficient (black bar) mice on the C57BI/6 background. Expression levels of Ndrg2, Nr0b2, Cyp7a1 and Cylophilin E mRNA^'s were normalized to Tbp and the mean level of Tbp expression in male C57BLJ6 mice was defined as 1.0. Data are presented as the mean ± SD. Group size were 5 mice each. The data show that the expression of Ndrg2 and Nr0b2 (Shp) mRNA^'s are reduced in FXR deficient mice compared to FXR expressing wt mice, while the expression of Cyp7a1 is increased. Cyclophilin E is not changed significantly.

Example 5

HepG2 and HepG2-FXR5 (overexpressing human FXR isoform 3) cells were plated in 96 well plates at a density of around 60%. After growth for 16 h in RPMI supplemented with 8.6% FCS, and Glutamine, Px20350 was titrated by three-fold dilutions with a top concentration of 10 μΜ and DMSO as a vehicle control in triplicates and grown for a further 16 h before RNA was isolated from these cells and q-RT-PCR performed and analyzed using the comparative Ct method.

FIG. 5A shows the dose-dependent relative induction of NDRG2 mRNA by the FXR agonist Px20350. The EC50 of induction by Px20350 is calculated to be around 25 nM for both cells lines (HepG2 and HepG2-FXR5) which is very close to the ECso determined in FXR Gal4- reporter assays in HEK293 cells. The absolute inducibility is much higher in the HepG2-FXR5 cell derivative which overproduces FXR compared to the parent HepG2 cell line.

FIG. 5B shows the dose-dependent relative induction of NDRG2 mRNA by the FXR agonists Px20350, 6-Ethyl-Chenodeoxycholic acid (6-ECDCA) and Chenodeoxycholic acid (CDCA) against DMSO as the vehicle. All three agonists do induce NDRG2 but with different potencies (the ECso^'s for Px20350, 6-ECDCA and CDCA are 0.07, 0.28 and 33 μΜ respectively).

Example 6

SK-Hep-1 human hepatocellular carcinoma cells were stably transfected with an expression vector (pTReX-DEST30-hFXR) to generate SK-GI-18 a SK-Hep-1 derivative that stably expresses human FXR isoform 3. SK-Hep-1 cells and SK-GI-18 cells were grown in 24-well plates in presence of DMSO or 0.5 μΜ Px20350 for 72 h. Total RNA was isolated from cells at around 60% confluency and NDRG2 and TBP abundancy determined by q-RT-PCR using the comparative Ct method.

FIG. 6: Increased expression and inducibility of NDRG2 mRNA by Px20350 in SK-GI-18 cells compared to parental SK-Hep-1 cells.

The diagram shows q-RT-PCR data for NDRG2 mRNA drawn as relative fold increase over the TBP corrected values for DMSO treated SK-Hep-1 and SK-GI-18 cells using the comparative Ct method. FXR (NR1 H4) mRNA was detectable in SK-GI-18 cells, but merely in SK-Hep-1 cells. The inducibility of NDRG2 by Px20350 was just below statistical significance in SK-Hep-1 but highly significant in SK-GI-18 cells (p<0.01). The absolute levels of NDRG2 are increased in SK-GI-18 cells compared to SK-Hep-1 parent cells contributing to the increased liability of detection of NDRG2 transcripts.

Example 7

SK-Hep-1 or SK-GI-18 cells (that stably express human FXR isoform 3) were seeded into 96 well plates at a density of 5000 cells per well (n = 12) and grown for 4 days in presence of DMSO or increasing amounts of Px20350, before relative cell numbers were determined using the CyQuant Direct Proliferation Assay (Invitrogen) according to the instruction of the manufacturer.

Fig. 7: Proliferation of SK-GI-18 cells is dose dependently reduced by Px20350, while the proliferation of SK-Hep-1 cells are merely influenced by Px20350 in vitro. Fluorescence data at 535 nm are presented as mean of 12 wells with SEM and plotted against the concentration of Px20350 in the medium. Values for DMSO are plotted at 0.001 μΜ. The calculated IC₅₀ for Px20350 is 25 nM in the SK-GI-18 cells line, which closely fits to the EC₅₀ of transcriptional activation of FXR by Px20350in luciferase reporter assays in HEK293 cells. No IC₅₀ could be determined for the SK-Hep-1 parent cell line.

Example 8

SK-Hep-1 or SK-GI-18 cells (that stably express human FXR isoform 3) were seeded at a density of 7000 cells per 96 well into an Oris Fibronectin Coated Plate (Platyplus) in RPMI medium containing 8.6% FCS and 20mM Alanyl-Glutamine (Sigma). After 14 h of attachment, the rubber stoppers were removed and cells were further grown for 72 h in presence of growth medium containing either DMSO as a vehicle or 1 μΜ Px20350 in DMSO, before migrated cells were quantified using the Cyquant Direct Cell Proliferation Assay (Invitrogen) and emission fluorescence was determined at 535 nm using and Envision multilabe reader (Perkin Elmer).

FIG. 8 shows the migration of SK-GI-18 and SK-Hep-1 cells in presence of DMSO or 1 μΜ Px20350 in the growth medium. The emission fluorescence at 535 nm, respresenting relative cell number (stained nuclei) was determined using an Envision multilabel reader (Perkin Elmer). Data are presented as mean with SEM from quadruplicate determinations. The data show that the migration of SK-GI-18 cells is reduced around 3-fold by 1 μΜ Px20350 (*^** = p<0.0001), while the migration of SK-Hep-1 cells is reduced by around 1.5-fold (^** = p<0.01 ).

Example 9

FXR (NR1 H4), NDRG2, SHP (NR0B2) and RECK mRNA levels were quantified in 8 normal, 34 HCC and 12 non-HCC liver disease (LD) cDNA^'s purchased from ORIGENE (Cat. Nr. LVRT501 ) by q-RT-PCR. The cDNA samples have been obtained from patient liver samples verified by pathologists prior to isolation of RNA and conversion to cDNA which were normalized against beta-actin by RT-PCR and arrayed onto 96 well plate.

FIG. 9: Decreased expression of FXR and FXR target genes in human HCC. A total of 34 HCC cDNA samples from different stages (7 samples stage I, 8 samples of stages II and 111 A, 12 non-HCC liver disease (LD) and 8 normal liver samples were studied. The mRNA expression for the indicated genes (FXR (NR1 H4), NDRG2, SHP (NR0B2) and RECK) was determined by Real Time PCR on an ABI HT 7900 Real Time PCR device and the data are expressed as mean+SEM. Statistical significance: * = p<0.05, ^** = p<0.01 , *** = p<0.001. All comparisons are between the normal livers and the respective stages of HCC or non-HCC liver disease.

Example 10

SK-Hep-1 cells and SK-GI-18 cells (a SK-Hep-1 derivative that stably expresses human FXR isoform 3) were grown in RPMI medium (Sigma) supplemented with 8.6% FCS (Sigma) and 20 mM Alanyl-Glutamine (Sigma). NMRI female nude mice (Charles River, Sulzfeld, Germany) were implanted with 5x106 cell (either SK-Hep-1 or SK-GI-18) by injection into one liver lobe at experimental day 0 (Experiments done at Oncotest GmbH, Freiburg, Germany according to Oncotest SOP). Starting with experimental day 3, the mice received a daily gavage of 100 plot either vehicle (0.5% KVP and 0.1% Tween® 80 in 0.15M NaCI) or 4 mg/mL of Px20606 or Px21256 in vehicle or 40mg/mL of Sorafenib in vehicle daily at 5 pm. The calculated dosages are 0, 10 or 100 mg/kg/day respectively. On experimental day 6 mice were injected via the tail- vein a fluorophore coupled antibody towards human CD10 and fluorescence emission was determined on experimental day 7 using a Kodak FX imaging system followed by x-ray imaging allowing quantification and localization of tumor load a very exact manner in the same animal over time. A total of 9 animals in each group were injected and one typical animal for each group was selected for presentation in Figures 10A and 10B. Imaging was performed on experimental days 7, 14, 21 , 28, 35 and 56. Data show that SK-Hep-1 cells are much more aggressive to form large primary tumors and metastases in lymph nodes, gut and bone (FIG. 10A vehicle) than SK-GI-18 cells (FIG. 10B vehicle) demonstrating that the expression of FXR in SK-Hep-1 cells does reduces the aggressiveness of tumor formation and metastatic spread of these tumor cells in NMRI nude mice.

Sorafenib does reduce primary tumor formation and metastasis of tumor cells in mice receiving SK-Hep-1 or SK-GI-18 cells comparably well. However the FXR agonists Px20606 and also Px21256 do reduce the tumor growth and metastasis more effectively in SK-GI-18 cells that do stably express human FXR isoform 3 compared to SK-Hep-1 cells that do express only very low to undetectable levels of endogenous FXR.

FIG. 10A shows the imaging results for one representative animal of a group of animals (n = 9) that received the parental SK-Hep-1 cells and which was daily gavaged with either vehicle, 10mg/kg/d of Px20606 or Px21256 or 10mg/kg/d of Sorafenib (Nexavar^®, a pan receptor tyrosine kinase inhibitor clinically used for treatment of lates stage HCC).

FIG. 10B shows the imaging results for one representative animal of a group of animals (n = 9) that received the parental SK-GI-18 cells (that stably express human FXR isoform 3) and which was daily gavaged with either vehicle, 10mg/kg/d of Px20606 or Px21256 or 10mg/kg/d of Sorafenib.

Example 11

Retrospective analysis of publically available expression data derived from RNA prepared from colonic mucosa samples of healthy controls (n = 10) and patients with non-FAP and non- hereditary nonpolyposis colorectal cancer (n =12; Y. Hong et al., Clin. Cancer Res. 2007, 13, 1 107). RNAs extracted from colonic mucosa specimens were analyzed using GeneChip U133- Plus 2.0 Array and the data deposited in the NCBI Gene Expression Omnibus databank with the DataSet Record GDS2609. The published average difference data (Affymetrix software) for FXR (NR1 H4) were retrieved and analyzed with Graph Pad Prism 4 software (FIG. 1 1 ).

FIG. 1 1 : FXR (NR1 H4) mRNA expression levels (in average difference units: AVD) in mucosa from patients with early onset colorectal cancer (patients mucosa) was significantly (p<0.0001 determined by Student's t-test) reduced compared to the expression in mucosa of healthy controls.

It will be readily apparent to those skilled in the relevant arts that other suitable modifications and adaptations to the methods and applications described herein are suitable and may be made without departing from the scope of the invention or any embodiment thereof. While the invention has been described in connection with certain embodiments, it is not intended to limit the invention to the particular forms set forth, but on the contrary, it is intended to cover such alternatives, modifications and equivalents as may be included within the spirit and scope of the invention as defined by the following claims.

Claims

1. An FXR (Farnesoid X Receptor, NR1 H4) agonist for use in inducing the expression of N- myc downstream-regulated gene 2 (NDRG2) in cells of a patient.

2. The FXR agonist of claim 1 for use in inducing the expression of NDRG2 in cells of a patient and thereby preventing or reducing neoplastic transformation, growth, metastasis and angiogenesis in the context of cancer or precancerogenic lesions.

3. The FXR agonist of claims 1 or 2 for use in the treatment of cancers, metastasis, angiogenesis in the context of cancer or precancerogenic lesions in which NDRG2 is downregulated compared to healthy tissue.

4. At least one FXR agonist of claim 3, for use in treating a patient harbouring at least one tumor, wherein the at least one FRX agonist is administered to the patient in a therapeutically effective amount in a pharmaceutical formulation.

5. The at least one FXR agonist of claim 4, wherein the at least one tumor is selected from hepatocellular carcinoma, hepatocellular adenoma, cholangiocarcinoma, colorectal cancer, colorectal adenoma, ileal adenoma, renal cancer, oesophageal cancer, gastric cancer, breast cancer or Barett^'s esophagus.

6. The at least one FXR agonist of claims 4 or 5, wherein the FXR agonist is selected from natural bile acids, preferably Chenodeoxycholic acid [CDCA] or taurine- or glycine-conjugated CDCA [tauro-CDCA or glyco-CDCA] and synthetic derivatives of natural bile acids, preferably 6-Ethyl-CDCA or taurine- or glycine-conjugated 6-Ethyl-CDCA.

7. The at least one FXR agonist of claims 4 or 5, wherein the FXR agonist is selected from natural non-steroidal agonists, preferably Diterpenoids such as Cafestol and Kahweol.

8. The at least one FXR agonist of claims 4 or 5, wherein the FXR agonist is selected from synthetic non-steroidal FXR agonists.

9. The at least one FXR agonist of claims 4 or 5, wherein the FXR agonist is selected from a compound according to Formula (1 ), an enantiomer, diastereomer, tautomer, solvate, prodrug or pharmaceutical acceptable salt thereof

wherein

A is selected from

A-Z- is selected from

10. The at least one FXR agonist of claim 9, wherein the FXR agonist is selected from

3-(2-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4- yl)methoxy)phenyl)cyclopropyl)benzoic acid; 4- (2-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4- yl)methoxy)phenyl)cyclopropyl)benzoic acid;

5- (2-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4- yl)methoxy)phenyl)cyclopropyl)-1 -isopropyl-1 H-pyrazole-3-carboxylic acid;

6-(2-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4- yl)methoxy)phenyl)cyclopropyl)-1-methyl-1 H-indazole-3-carboxylic acid;

6- (2-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4- yl)methoxy)phenyl)cyclopropyl)-1 -isopropyl-1 H-indazole-3-carboxylic acid;

3-(3-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)phenyl)-3- hydroxycyclobutyl)benzoic acid;

6- (3-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)phenyl)-3- hydroxycyclobutyl)-1 -methyl- 1 H-indazole-3-carboxylic acid;

4-(3-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)phenyl)-3- hydroxycyclobutyl)benzoic acid;

3-(3-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)phenyl)-3- hydroxyazetidin-1-yl)benzoic acid; or

5-(3-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)phenyl)-3- hydroxyazetidin-1 -yl)nicotinic acid.

1 1. The at least one FXR agonist of one of claims 4 to 10, wherein the FXR agonist reduces at least one feature of the tumor, wherein the at least one feature of the tumor is selected from proliferative activity, invasive activity, metastatic activity, apoptotic activity and angiogenic activity.

12. The at least one FXR agonist of one of claims 4 to H, wherein the at least one FXR agonist is coadministered together with a receptor tyrosine kinase inhibitor selected from sorafenib, regorafinib, sunitinib, erlotinib or imatinib.

13. The at least one FXR agonist of one of claims 4 to H, wherein the at least one FXR agonist is coadministered with at least one of an agent selected from abarelix, aldeleukin, allopurinol, altretamine, amifostine, anastozole, bevacizumab, capecitabine, carboplatin, cisplatin, docetaxei, doxorubicin, eriotinib, exemestane, 5-flurouracil, fulvestrant, gemcitabine, goserelin acetate, irinotecan, lapatinib ditosylate, letozole, leucovorin, levamisole, oxaliplatin, paclitaxel, panitumumab, pemetrexed disodium, profimer sodium, tamoxifen, topotecan, and trastuzumab.

14. The FXR modulator of one of claims 1 to 1 1 , wherein the FXR modulator induces expression of the small heterodimer partner (SHP, NR0B2) gene in addition to NDRG2 in tissues of the patient.