WO2016049070A1

WO2016049070A1 - Methods of treating liver disease

Info

Publication number: WO2016049070A1
Application number: PCT/US2015/051531
Authority: WO
Inventors: William J. Watkins
Original assignee: Gilead Sciences, Inc.
Priority date: 2014-09-24
Filing date: 2015-09-22
Publication date: 2016-03-31

Abstract

The present disclosure relates to a method of preventing and/or treating liver disease comprising administering a FXR agonist in combination with a LOXL2 inhibitor, to a patient in need thereof.

Description

METHODS OF TREATING LIVER DISEASE

FIELD

The present disclosure relates to a method of preventing and/or treating liver disease.

BACKGROUND

Liver disease is generally classified as acute or chronic based upon the duration of the disease. Liver disease may be caused by infection, injury, exposure to drugs or toxic compounds, alcohol, impurities in foods, and the abnormal build-up of normal substances in the blood, an autoimmune process, a genetic defect (such as

haemochromatosis), or unknown cause(s).

Liver disease is a leading cause of death world wide. In particular, it has been seen that a diet high in fat damages the liver in ways that are surprisingly similar to hepatitis. The American Liver Foundation estimates that more than 20 percent of the population has non-alcoholic fatty liver disease (NAFLD). It is suggested that obesity, unhealthy diets, and sedentary lifestyles may contribute to the high prevalence of NAFLD. When left untreated, NAFLD can progess to non-alcoholic steatohepatitis (NASH), causing serious adverse effects. Once NASH is developed, it would cause the liver to swell and scar (i.e. cirrhosis) over time.

Although preliminary reports suggest positive lifestyle changes could prevent or reverse liver damage, there are no effective medical treatments for NAFLD.

Accordingly, there remains a need to provide new effective pharmaceutical agents to treat liver diseases.

SUMMARY

Disclosed herein is a method of treating and/or preventing liver disease in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a farnesoid X receptor (FXR) agonist in combination with a therapeutically effective amount of a lysyl oxidase-like 2 (LOXL2) inhibitor. The liver disease can be any liver disease, including, but not limited to, chronic and/or metabolic liver diseases. In one embodiment, the liver disease is nonalcoholic fatty liver disease (NAFLD). In certain embodiments, provided herein is a method of treating and/or preventing nonalcoholic steatohepatitis (NASH) in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a FXR agonist in combination with a therapeutically effective amount of a LOXL2 inhibitor.

In other embodiments, provided herein is a method of treating and/or preventing liver fibrosis in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a FXR agonist in combination with a therapeutically effective amount of a LOXL2 inhibitor.

In still other embodiments, provided herein is a method of treating and/or preventing primary sclerosing cholangitis (PSC) in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a FXR agonist in combination with a therapeutically effective amount of a LOXL2 inhibitor.

In the methods provided herein, the FXR agonist and the LOXL2 inhibitor can be coadministered. In such embdiments, the FXR agonist and the LOXL2 inhibitor can be administered together as a single pharmaceutical composition, or separately in more than one pharmaceutical composition. Accordingly, also provided herein is a pharmaceutical composition comprising a therapeutically effective amount of an FXR agonist and a therapeutically effective amount of a LOXL2 inhibitor.

DETAILED DESCRIPTION

Definitions and General Parameters

As used in the present specification, the following terms 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 term "about" used in the context of quantitative

measurements means the indicated amount ± 10%, or alternatively the indicated amount ± 5% or ± 1%.

As used herein, a "FXR agonist" refers to any agent that is capable of binding and activating farnesoid X receptor (FXR) which may be referred to as bile acid receptor (BAR) or NR1H4 (nuclear receptor subfamily 1, group H, member 4) receptor. FXR agonist may act as agonists or partial agonists of FXR. The agent may be a chemical compound or biological molecule (e.g., a protein or antibody). The activity of a FXR agonist may be measured by several different methods, e.g. in an in vitro assay using the fluorescence resonance energy transfer (FRET) cell free assay as described in Pellicciari, et al. Journal of Medicinal Chemistry, 2002 vol. 15, No. 45:3569-72.

As used herein, a "LOXL2 inhibitor" any agent that is capable of inactivating lysyl oxidase-like 2 (LOXL2) protein. The agent may be a chemical compound or biological molecule (e.g., a protein or antibody). The LOXL2 protein activity may be measured by several different methods (see, e.g., U.S. 2009/0053224 and U.S.

201 1/0044907, both of which are incorporated herein by reference in their entirety). In certain embodiments, the LOXL2 inhibitor is an anti-LOXL2 antibody and antigen binding fragments thereof that bind to and/or inhibit LOXL2. In othe embodiments, the LOXL2 inhibitor is the anti-LOXL2 antibody descrbed in U.S. 8,461,303, U.S.

2012/0309020, U.S. 2013/0324705, and U.S. 2014/0079707, each of which are incorporated herein by reference in their entirety. The term "antibody" is used herein refers to a population of immunoglobulin molecules and/or immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antibody combining site or paratope. Thus, reference to an "antibody" also includes reference to any of the antigen binding fragments of antibodies. The term "antibody" also includes molecules which have been engineered through the use of molecular biological technique to include only portions of the native molecule as long as those molecules have the ability to bind a particular antigen or sequence of amino acids with the required specificity. Such alternative antibody molecules include classically known portions of the antibody molecules, single chain antibodies, and single chain binding molecules. Thus, it is used in the broadest sense and specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, diabodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments including but not limited to single chain binding polypeptides, so long as they exhibit the desired biological activity. An antibody can be a humanized antibody. Humanized forms of non-human (e.g., murine) antibodies include, for example, chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, scFv, Fab, Fab', F(ab')2, single chain binding polypeptide, VH, VL, or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Chimeric antibodies include those in which the heavy and light chain variable regions are combined with human constant regions (Fc). Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. A humanized antibody can also contain at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al, Nature, 321 :522-525 (1986); Riechmann et al, Nature, 332:323-329(1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992)). Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as "import" or "donor" residues, which are typically taken from an "import" or "donor" variable domain. Humanization can be essentially performed following the method of Winter and co-workers (Jones et al., Nature, 321 :522 525 (1986); Riechmann et al., Nature, 332:323 327 (1988)); Verhoeyen et al. Science, 239: 1534 1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such "humanized" antibodies include chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

"Antigen binding fragments" comprise a portion of an intact antibody, and can include the antigen binding or variable region of an intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, Fv fragments, scFv fragments, diabodies, linear antibodies (Zapata et al, Protein Eng. 8(10): 1057-1062 (1995)), single-chain antibody molecules, single chain binding polypeptides, and multispecific antibodies formed from antibody fragments. Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, a designation reflecting the ability to crystallize readily. Pepsin treatment yields an F(ab')2 fragment that has two antigen combining sites and is still capable of cross-linking antigen.

The term "binding" refers to a direct association between two molecules, due to, for example, covalent, electrostatic, hydrophobic, and ionic and/or hydrogen-bond interactions, including interactions such as salt bridges and water bridges. The term "specific binding" is applicable to a situation in which an antibody or antigen binding fragment thereof does not show any significant binding to molecules other than its epitope. In one embodiment, an antibody or antigen binding fragment thereof specifically binds to a human LOX or to human LOXL2 with a dissociation constant Kd equal to or lower than about 100 nM, lower than about 10 nM, lower than about 1 nM, lower than about 0.5 nM, lower than about 0.1 nM, lower than about 0.01 nM, or lower than about 0.005 nM measured at a temperature of about 4°C, 25°C, 37°C or 42 °C.

"Homology" or "identity" or "similarity" refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology and identity can each be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When an equivalent position in the compared sequences is occupied by the same base or amino acid, then the molecules are identical at that position; when the equivalent site occupied by the same or a similar amino acid residue (e.g., similar in steric and/or electronic nature), then the molecules can be referred to as homologous (similar) at that position. Expression as a percentage of homology/similarity or identity refers to a function of the number of identical or similar amino acids at positions shared by the compared sequences. A sequence which is "unrelated" or "non-homologous" shares less than 40% identity, though preferably less than 25% identity with a sequence of the present invention. In comparing two sequences, the absence of residues (amino acids or nucleic acids) or presence of extra residues also decreases the identity and homology/similarity.

"Homology" describes a mathematically based comparison of sequence similarities which is used to identify genes or proteins with similar functions or motifs. The nucleic acid (nucleotide, oligonucleotide) and amino acid (protein) sequences of the present invention may be used as a "query sequence" to perform a search against public databases to, for example, identify other family members, related sequences or homologs. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to nucleic acid molecules of the invention. BLAST amino acid searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al, (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and BLAST) can be used (see, ncbi.nlm.nih.gov).

"Identity" means the percentage of identical nucleotide or amino acid residues at corresponding positions in two or more sequences when the sequences are aligned to maximize sequence matching, i.e., taking into account gaps and insertions. Identity can be readily calculated by known methods, including but not limited to those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988). Methods to determine identity are designed to give the largest match between the sequences tested. Moreover, methods to determine identity are codified in publicly available computer programs. Computer program methods to determine identity between two sequences include, but are not limited to, the GCG program package (Devereux, J., et al, Nucleic Acids Research 12(1): 387 (1984)), BLASTP, BLASTN, and FASTA (Altschul, S. F. et al, J. Molec. Biol. 215: 403-410 (1990) and Altschul et al. Nuc. Acids Res. 25: 3389- 3402 (1997)). The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al, NCBI NLM NIH Bethesda, Md. 20894;

Altschul, S., et al, J. Mol. Biol. 215: 403-410 (1990). The well known Smith Waterman algorithm may also be used to determine identity.

The term "pharmaceutically acceptable salt" refers to salts of pharmaceutical compounds e.g. compound of formula (I) that retain the biological effectiveness and properties of the underlying compound, and which are not biologically or otherwise undesirable. There are acid addition salts and base addition salts. Pharmaceutically acceptable acid addition salts may be prepared from inorganic and organic acids.

Acids and bases useful for reaction with an underlying compound to form pharmaceutically acceptable salts (acid addition or base addition salts respectively) are known to one of skill in the art. Similarly, methods of preparing pharmaceutically acceptable salts from an underlying compound (upon disclosure) are known to one of skill in the art and are disclosed in for example, Berge, at al. Journal of Pharmaceutical Science, Jan. 1977 vol. 66, No. l, and other sources.

As used herein, "pharmaceutically acceptable carrier" includes excipients or agents such as solvents, diluents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like that are not deleterious to the disclosed compound or use thereof. The use of such carriers and agents to prepare compositions of pharmaceutically active substances is well known in the art (see, e.g., Remington's Pharmaceutical Sciences, Mace Publishing Co., Philadelphia, PA 17th Ed. (1985); and Modern Pharmaceutics, Marcel Dekker, Inc. 3rd Ed. (G.S. Banker & C.T. Rhodes, Eds.).

The terms "therapeutically effective amount" and "effective amount" are used interchangibly and refer to an amount of a compound that is sufficient to effect treatment as defined below, when administered to a patient (e.g., a human) in need of such treatment in one or more doses. The therapeutically effective amount will vary depending upon the patient, the disease being treated, the weight and/or age of the patient, the severity of the disease, or the manner of administration as determined by a qualified prescriber or care giver.

The term "treatment" or "treating" means administering a compound or pharmaceutically acceptable salt of formula (I) for the purpose of: (i) delaying the onset of a disease, that is, causing the clinical symptoms of the disease not to develop or delaying the development thereof; (ii) inhibiting the disease, that is, arresting the development of clinical symptoms; and/or (iii) relieving the disease, that is, causing the regression of clinical symptoms or the severity thereof.

Liver Diseases Liver diseases are acute or chronic damages to the liver based in the duration of the disease. The liver damage may be caused by infection, injury, exposure to drugs or toxic compounds such as alcohol or impurities in foods, an abnormal build-up of normal substances in the blood, an autoimmune process, a genetic defect (such as

haemochromatosis), or other unknown causes. Exemplary liver diseases include, but are not limited to, cirrhosis, liver fibrosis, non-alcoholic fatty liver disease ( AFLD), nonalcoholic steatohepatitis (NASH), hepatic ischemia reperfusion injury, primary biliary cirrhosis (PBC), and hepatitis, including both viral and alcoholic hepatitis.

Non-alcoholic fatty liver disease (NAFLD) is the build up of extra fat in liver cells that is not caused by alcohol. NAFLD may cause the liver to swell (i.e.

steatohepatitis), which in turn may cause scarring (i.e. cirrhosis) over time and may lead to liver cancer or liver failure. NAFLD is characterized by the accumulation of fat in hepatocyes and is often associated with some aspects of metabolic syndrome (e.g. type 2 diabetes mellitus, insulin resistance, hyperlipidemia, hypertension). The frequency of this disease has become increasingly common due to consumption of carbohydrate-rich and high fat diets. A subset (-20%) of NAFLD patients develop nonalcoholic steatohepatitis (NASH).

NASH, a subtype of fatty liver disease, is the more severe form of NAFLD. It is characterized by macrovesicular steatosis, balloon degeneration of hepatocytes, and/or inflammation ultimately leading to hepatic scarring (i.e. fibrosis). Patients diagnosed with NASH progress to advanced stage liver fibrosis and eventually cirrhosis. The current treatment for cirrhotic NASH patients with end-stage disease is liver transplant.

A study has shown that a significant proportion of diagnosed NASH patients (39%) have not had a liver biopsy to confirm the diagnosis. A greater proportion of diagnosed NASH patients have metabolic syndrome parameters than what is reported in the literature (type-II diabetes mellitus 54%, Obesity 71%, metabolic syndrome 59%). 82% of physicians use a lower threshold value to define significant alcohol consumption compared with practice guideline recommendations. 88% of physicians prescribe some form of pharmacologic treatment for NASH (Vit E: prescribed to 53% of NASH patients, statins: 57%, metformin: 50%). Therefore, the vast majority of patients are prescribed medications despite a lack of a confirmed diagnosis or significant data to support the intervention and alcohol thresholds to exclude NASH are lower than expected. Another common liver disease is primary sclerosing cholangitis (PSC). It is a chronic or long-term liver disease that slowly damages the bile ducts inside and outside the liver. In patients with PSC, bile accumulates in the liver due to blocked bile ducts, where it gradually damages liver cells and causes cirrhosis, or scarring of the liver. Currently, there is no effective treatment to cure PSC. Many patients having PSC ultimately need a liver transplant due to liver failure, typically about 10 years after being diagnosed with the disease. PSC may also lead to bile duct cancer.

Liver fibrosis is the excessive accumulation of extracellular matrix proteins, including collagen, that occurs in most types of chronic liver diseases. Advanced liver fibrosis results in cirrhosis, liver failure, and portal hypertension and often requires liver transplantation.

Methods

Disclosed herein is a method of treating and/or preventing liver disease in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a FXR agonist in combination with a therapeutically effective amount of a LOXL2 inhibitor. The presence of active liver disease can be detected by the existence of elevated enzyme levels in the blood. Specifically, blood levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), above clinically accepted normal ranges, are known to be indicative of on-going liver damage. Routine monitoring of liver disease patients for blood levels of ALT and AST is used clinically to measure progress of the liver disease while on medical treatment. Reduction of elevated ALT and AST to within the accepted normal range is taken as clinical evidence reflecting a reduction in the severity of the patients on-going liver damage.

In certain embodiments, the liver disease is a chronic liver disease. Chronic liver diseases involve the progressive destruction and regeneration of the liver parenchyma, leading to fibrosis and cirrhosis. In general, chronic liver diseases can be caused by viruses (such as hepatitis B, hepatitis C, cytomegalovirus (CMV), or Epstein Barr Virus (EBV)), toxic agents or drugs (such as alcohol, methotrexate, or nitrofurantoin), a metabolic disease (such as non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), haemochromatosis, or Wilson's Disease), an autoimmune disease (ssuch as Autoimmune Chronic Hepatitis, Primary Biliary Cirrhosis, or Primary Sclerosing Cholangitis), or other causes (such as right heart failure). In one embodiment, provided herein is a method for reducing the level of cirrhosis. In one embodiment, cirrhosis is characterized pathologically by loss of the normal microscopic lobular architecture, with fibrosis and nodular regeneration.

Methods for measuring the extent of cirrhosis are well known in the art. In one embodiment, the level of cirrhosis is reduced by about 5% to about 100%. In one embodiment, the level of cirrhosis is reduced by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% in the subject.

In certain embodiments, the liver disease is a metabolic liver disease. In one embodiment, the liver disease is non-alcoholic fatty liver disease (NAFLD). NAFLD is associated with insulin resistance and metabolic syndrome (obesity, combined hyperlipidemia, diabetes mellitus (type II) and high blood pressure). NAFLD is considered to cover a spectrum of disease activity, and begins as fatty accumulation in the liver (hepatic steatosis).

It has been shown that both obesity and insulin resistance probably play a strong role in the disease process of NAFLD. In addition to a poor diet, NAFLD has several other known causes. For example, NAFLD can be caused by certain medications, such as amiodarone, antiviral drugs (e.g., nucleoside analogues), aspirin (rarely as part of Reye's syndrome in children), corticosteroids, methotrexate, tamoxifen, or tetracycline. NAFLD has also been linked to the consumption of soft drinks through the presence of high fructose corn syrup which may cause increased deposition of fat in the abdomen, although the consumption of sucrose shows a similar effect (likely due to its breakdown into fructose). Genetics has also been known to play a role, as two genetic mutations for this susceptibility have been identified.

If left untreated, NAFLD can develop into non-alcoholic steatohepatitis (NASH), which is the most extreme form of NAFLD, a state in which steatosis is combined with inflammation and fibrosis. NASH is regarded as a major cause of cirrhosis of the liver of unknown cause. Accordingly, provided herein is a method of treating and/or preventing nonalcoholic steatohepatitis (NASH) in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a FXR agonist in combination with a therapeutically effective amount of a LOXL2 inhibitor.

Also provided herein is a method of treating and/or preventing liver fibrosis in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a FXR agonist in combination with a therapeutically effective amount of a LOXL2 inhibitor. Liver fibrosis is the excessive accumulation of extracellular matrix proteins including collagen that occurs in most types of chronic liver diseases. In certain embodiments, advanced liver fibrosis results in cirrhosis and liver failure. Methods for measuring liver histologies, such as changes in the extent of fibrosis, lobular hepatitis, and periportal bridging necrosis, are well known in the art.

In one embodiment, the level of liver fibrosis, which is the formation of fibrous tissue, fibroid or fibrous degeneration, is reduced by more that about 90%. In one embodiment, the level of fibrosis, which is the formation of fibrous tissue, fibroid or fibrous degeneration, is reduced by at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 10%, at least about 5% or at least about 2%.

In one embodiment, the compounds provided herein reduce the level of fibrogenesis in the liver. Liver fibrogenesis is the process leading to the deposition of an excess of extracellular matrix components in the liver known as fibrosis. It is observed in a number of conditions such as chronic viral hepatitis B and C, alcoholic liver disease, drug-induced liver disease, hemochromatosis, auto-immune hepatitis, Wilson disease, primary biliary cirrhosis, sclerosing cholangitis, liver schistosomiasis and others. In one embodiment, the level of fibrogenesis is reduced by more that about 90%. In one embodiment, the level of fibrogenesis is reduced by at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least 40%, at least about 30%, at least about 20%, at least about 10%, at least about 5% or at least 2%.

FXR Agonist Farnesoid X receptors are known to be expressed in liver, intestine, kidney and adrenal tissues. Activated FXR translocates to the cell nucleus and forms a heterodimer with retinoid X receptor ("RXR"). The dimer binds to hormone response elements on DNA causing, among other effects, a suppression of cholesterol 7 alpha-hydroxylase ("CYP7A1"), the rate-limiting enzyme in bile acid synthesis from cholesterol, and stimulation of intestinal bile acid binding protein ("ΓΒΑΒΡ"). Both CYP7A1 and IBABP are involved in homeostatis of bile acid and cholesterol. A FXR agonist for use in the methods and pharmaceutical compositions disclosed herein may be any chemical compound or biological molecule (e.g., a protein or antibody) capable of binding and activating FXR. For example, GW4064 (3-(2,6-Dichlorophenyl)-4-(3'-carboxy-2- chlorostilben-4-yl)oxymethyl-5-is- opropylisoxazole) and bile acids such as

chenodeoxycholic acid ("CDCA"), lithocholic acid (LCA) and deoxycholic acid (DCA) are known to act as agonists of FXR. Additional examples of FXR agonist may be found in U.S. Publication Nos. 20100184809, 20100210660, 2012 0232116, 20140221659, 20140039007, 20140187633, 20140134262, and 20140057886. All publications cited in the application are incorporated by reference in their entirety.

It is suggested that FXR is a nuclear bile acid sensor that modulates the synthetic output of bile acids in the liver and their recycling in the intestine (by regulating bile acid binding proteins). Beyond bile acid physiology, FXR may be involved in the regulation of many diverse physiological processes which are relevant in the etiology and for the treatment of diseases as diverse as cholesterol gallstones, metabolic disorders such as Type II Diabetes, dyslipidemias or obesity, chronic inflammatory diseases such as inflammatory bowel diseases or chronic intrahepatic forms of cholestasis and many others diseases (see Claudel et al., Arteriosclerosis, Thrombosis, and Vascular Biology 2005 vol. 25, No. 10, 2020-2030; Westin et al, Mini Review Medicinal Chemistry 2005 vol. 5, No. 8, 719-727).

FXR regulates a complex pattern of response genes in the liver. The gene products have impact on diverse physiological processes. In the course of functional analysis of FXR, the first regulatory network that was analyzed was the regulation of bile acid synthesis. While the LXRs induce the key enzyme of the conversion of cholesterol into bile acids, Cyp7Al, via the induction of the regulatory nuclear receptor LRH-1, FXR represses the induction of Cyp7Al via the upregulation of mRNA encoding SHP, a further nuclear receptor that is dominant repressive over LRH- 1. Since FXR binds the end products of this pathway, primary bile acids such as cholic acid (CA) or

chenodeoxycholic acid (CDCA), this can be regarded as an example of feedback inhibition on the gene expression level (Goodwin et al., Molecular Cell 2000, vol. 6 No. 3, 517-526; Lu et al, Molecular Cell 2000, vol. 6, No. 3, 507-515). Parallel to the repression of bile acid synthesis via SHP, FXR induces a range of so-called ABC (for ATP-binding cassette) transporters that are responsible for the export of toxic bile acids from the hepatocyte cytosol into the canaliculi, the small bile duct ramifications where the bile originates. This hepatoprotective function of FXR became first apparent with the analysis of FXR knockout mice (Sinai et al, Cell 2000, vol. 102, No. 6, 731-744) where under- or overexpression of several ABC-transporters in the liver was shown. Further detailed analysis revealed that the major bile salt excretory pump BSEP or ABCB11 (Ananthanarayananet al, Journal of Biological Chemisty 2001, vol. 276, No. 31, 28857-28865; Plass et al, Hepatology 2002, vol. 35 No. 3, 589-96) as well as the key enzyme which mediates lipid transfer from lipoproteins to phospholipids, PLTP (Urizar et al., Journal of Biological Chemisty 2000, vol. 275, No. 50, 39313-39317), and the two key canalicular membrane transporters for phospholipids, MRP-2 (ABCC4) (Kast et al, Journal of Biological Chemisty 2002, vol. 277, No.4, 2908-2915) and MDR-3 (ABCB4) (Huang et al, Journal of Biological Chemisty 2003, vol. 278, No. 51, 51085-51090) are direct targets for ligand-directed transcriptional activation by FXR (see Miyata, Journal of Pharmacology and Experimental Therapeutics 2005, vol. 312, No. 2, 1 '59-766; Rizzo et al., Current Drug Targets - Immune, Endocrine & Metabolic Disorders 2005, vol. 5, No. 3, 289-303.).

LOXL2 Inhibitors

A LOXL2 inhibitor for use in the methods and pharmaceutical compositions described herein may be any agent that is capable of inactivating lysyl oxidase-like 2

(LOXL2) protein. The agent may be a chemical compound or biological molecule (e.g., a protein or antibody). Such inhibitors are readily identified by known methods (see, e.g., U.S. 8,461,303, U.S. 2009/0053224 and U.S. 2011/0044907, which are hereby incorporated herein by reference in their entirety).

In certain embodiments, the LOXL2 inhibitor is an anti-LOXL2 antibody (see, e.g., U.S. 8,461,303, U.S. 2012/0309020, U.S. 2013/0324705, and U.S. 2014/0079707, which are incorporated herein by reference in their entirety). The anti-LOXL2 antibody can be a monoclonal antibody (including full length monoclonal antibody), polyclonal antibody, human antibody, humanized antibody, chimeric antibody, diabody, multispecific antibody (e.g., bispecific antibody), or an antibody fragment including, but not limited to, a single chain binding polypeptide, so long as it exhibits the desired biological activity.

In certain embodiments, the anti-LOXL2 antibody is a monoclonal anti-LOXL2 antibody, or antigen-binding fragment thereof. In other embodiments, the anti-LOXL2 antibody is a polyclonal anti-LOXL2 antibody, or antigen-binding fragment thereof. Such antibodies are known in the art or are available from commercial sources. In one embodiment, the anti-LOXL2 antibodies or antigen binding fragment thereof specifically binds to an epitope having an amino acid sequence set forth as SEQ ID NO: 1. In some embodiments, the anti-LOXL2 antibody is an isolated antibody or antigen binding fragment thereof, comprising the complementarity determining regions (CDRs), CDRl, CDR2, and CDR3, of a heavy chain variable region comprising the amino acid sequence set forth as SEQ ID NO: 2, 3, 4, or 5, and the CDRs, CDRl, CDR2, and CDR3, of a light chain variable region comprising the amino acid sequence set forth as SEQ ID NO: 6, 7, or 8, wherein the isolated antibody or antigen binding fragment thereof specifically binds a lysyl oxidase-like 2(LOXL2) protein. In other embodiments, CDRl, CDR2, and CDR3 of the heavy chain variable region comprise the amino acid sequences set forth as SEQ ID NOs: 9, 10, and 11, respectively, and the CDRl, CDR2, and CDR3 of the light chain variable region comprise the amino acid sequences set forth as SEQ ID NOs: 12, 13, and 14, respectively. In some other embodiments, the anti-LOXL2 antibody has a heavy chain variable region comprising the amino acid sequence set forth as SEQ ID NO: 2, 3, 4, or 5, and a light chain variable region comprising the amino acid sequence set forth as SEQ ID NO: 6, 7, or 8, wherein the isolated antibody or antigen binding fragment thereof specifically binds a lysyl oxidase-like 2 (LOXL2) protein. In further embodiment, the LOXL2 inhibitor is anti-LOXL2 antibody having the heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 4 and the light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 7. In further additional embodiments, the LOXL2 inhibitor is an anti-LOXL2 antibody comprising the sequences having about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 4. In some additional embodiments, the LOXL2 inhibitor is an anti-LOXL2 antibody comprising the sequences having about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 7. In certain embodiments, the isolated antibody or antigen binding fragment is humanized.

In additional embodiment, the LOXL2 inhibitor is anti-LOXL2 antibody AB0023 having the heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 15 and the light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 16. The methods of generating AB0023 and other anti-LOXL2 antibodies are generally disclosed in the '303 patent. In certain embodiments, the isolated antibody or antigen binding fragment is humanized. In further additional embodiments, the LOXL2 inhibitor is an anti-LOXL2 antibody comprising the sequences having about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 15. In some additional embodiments, the LOXL2 inhibitor is an anti-LOXL2 antibody comprising the sequences having about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 16.

Dosing and Administration

While it is possible for an active ingredient to be administered alone, it may be preferable to present them as pharmaceutical formulations or pharmaceutical compositions as described below. The formulations, both for veterinary and for human use, of the disclosure comprise at least one of the active ingredients, together with one or more acceptable carriers therefor and optionally other therapeutic ingredients. The carrier(s) must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and physiologically innocuous to the recipient thereof.

Each of the active ingredients can be formulated with conventional carriers and excipients, which will be selected in accord with ordinary practice. Tablets can contain excipients, glidants, fillers, binders and the like. Aqueous formulations are prepared in sterile form, and when intended for delivery by other than oral administration generally will be isotonic. All formulations will optionally contain excipients such as those set forth in the Handbook of Pharmaceutical Excipients (1986). Excipients include ascorbic acid and other antioxidants, chelating agents such as EDTA, carbohydrates such as dextrin, hydroxyalkylcellulose, hydroxyalkylmethylcellulose, stearic acid and the like. The pH of the formulations ranges from about 3 to about 11, but is ordinarily about 7 to 10. The therapeutically effective amount of active ingredient can be readily determined by a skilled clinician using conventional dose escalation studies. Typically, the active ingredient will be administered in a dose from 0.01 milligrams to 2 grams. In one embodiment, the dosage will be from about 10 milligrams to 450 milligrams. In another embodiment, the dosage will be from about 25 to about 250 milligrams. In another embodiment, the dosage will be about 50 or 100 milligrams. In one

embodiment, the dosage will be about 100 milligrams. It is contemplated that the active ingredient may be administered once, twice or three times a day. Also, the active ingredient may be administered once or twice a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, or once every six weeks.

The pharmaceutical composition for the active ingredient can include those suitable for the foregoing administration routes. The formulations can conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Techniques and formulations generally are found in Remington's Pharmaceutical Sciences (Mack Publishing Co., Easton, PA). Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

Formulations suitable for oral administration can be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous or nonaqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient may also be administered as a bolus, electuary or paste. In certain embodiments, the active ingredient may be administered as a subcutaneous injection.

A tablet can be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets can be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, or surface active agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered active ingredient moistened with an inert liquid diluent. The tablets may optionally be coated or scored and optionally are formulated so as to provide slow or controlled release of the active ingredient therefrom.

The active ingredient can be administered by any route appropriate to the condition. Suitable routes include oral, rectal, nasal, topical (including buccal and sublingual), vaginal and parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural), and the like. It will be appreciated that the preferred route may vary with for example the condition of the recipient. In certain embodiments, the active ingredients are orally bioavailable and can therefore be dosed orally. In one embodiment, the patient is human. When used in combination in the methods disclosed herein, the FXR agonist and the LOXL2 inhibitor can be administered together in a single pharmaceutical composition, or serperatly (either concurrently or sequentially) in more than one pharmaceutical composition. In certain embodiments, the FXR agonist and the LOXL2 inhibitor are administered together. In other embodiments, the FXR agonist and the LOXL2 inhibitor are administered separately. In some aspects, the FXR agonist is administered prior to the LOXL2 inhibitor. In some aspects, the LOXL2 inhibitor is administered prior to the FXR agonist. When administered separately, the FXR agonist and the LOXL2 inhibitor can be administered to the patient by the same or different routes of delivery. For example, the FXR agonist may be administered orally and the LOXL2 inhibitor may be administered subcutaneously.

Pharmaceutical Compositions

The pharmaceutical compositions of the disclosure provide for an effective amount of an FXR agonist, with or without, an effective amount of a LOXL2 inhibitor.

When used for oral use for example, tablets, troches, lozenges, aqueous or oil suspensions, dispersible powders or granules, emulsions, hard or soft capsules, syrups or elixirs may be prepared. Compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents including sweetening agents, flavoring agents, coloring agents and preserving agents, in order to provide a palatable preparation. Tablets containing the active ingredient in admixture with non-toxic pharmaceutically acceptable excipient which are suitable for manufacture of tablets are acceptable. These excipients may be, for example, inert diluents, such as, for example, calcium or sodium carbonate, lactose, lactose monohydrate, croscarmellose sodium, povidone, calcium or sodium phosphate; granulating and disintegrating agents, such as, for example, maize starch, or alginic acid; binding agents, such as, for example, cellulose, microcrystalline cellulose, starch, gelatin or acacia; and lubricating agents, such as, for example, magnesium stearate, stearic acid or talc. Tablets may be uncoated or may be coated by known techniques including microencapsulation to delay disintegration and adsorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as, for example, glyceryl monostearate or glyceryl distearate alone or with a wax may be employed.

Formulations for oral use may be also presented as hard gelatin capsules where the active ingredient is mixed with an inert solid diluent, for example calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, such as, for example, peanut oil, liquid paraffin or olive oil. Aqueous suspensions of the disclosure contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients include a suspending agent, such as, for example, sodium carboxymethylcellulose, methylcellulose, hydroxypropyl methylcelluose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia, and dispersing or wetting agents such as, for example, a naturally occurring phosphatide (e.g., lecithin), a condensation product of an alkylene oxide with a fatty acid (e.g., polyoxyethylene stearate), a condensation product of ethylene oxide with a long chain aliphatic alcohol (e.g., heptadecaethyleneoxycetanol), a condensation product of ethylene oxide with a partial ester derived from a fatty acid and a hexitol anhydride (e.g., polyoxyethylene sorbitan monooleate). The aqueous suspension may also contain one or more preservatives such as, for example, ethyl or n- propyl p-hydroxy-benzoate, one or more coloring agents, one or more flavoring agents and one or more sweetening agents, such as, for example, sucrose or saccharin.

Oil suspensions may be formulated by suspending the active ingredient in a vegetable oil, such as, for example, arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as, for example, liquid paraffin. The oral suspensions may contain a thickening agent, such as, for example, beeswax, hard paraffin or cetyl alcohol.

Sweetening agents, such as, for example, those set forth above, and flavoring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an antioxidant such as, for example, ascorbic acid.

Dispersible powders and granules of the disclosure suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, a suspending agent, and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those disclosed above. Additional excipients, for example sweetening, flavoring and coloring agents, may also be present.

The pharmaceutical compositions of the disclosure may also be in the form of oil- in- water emulsions. The oily phase may be a vegetable oil, such as, for example, olive oil or arachis oil, a mineral oil, such as, for example, liquid paraffin, or a mixture of these. Suitable emulsifying agents include naturally-occurring gums, such as, for example, gum acacia and gum tragacanth, naturally occurring phosphatides, such as, for example, soybean lecithin, esters or partial esters derived from fatty acids and hexitol anhydrides, such as, for example, sorbitan monooleate, and condensation products of these partial esters with ethylene oxide, such as, for example, polyoxyethylene sorbitan monooleate. The emulsion may also contain sweetening and flavoring agents. Syrups and elixirs may be formulated with sweetening agents, such as, for example, glycerol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative, a flavoring or a coloring agent.

The pharmaceutical compositions of the disclosure may be in the form of a sterile injectable preparation, such as, for example, a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, such as, for example, a solution in 1,3-butane-diol or prepared as a lyophilized powder. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile fixed oils may conventionally be employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as, for example, oleic acid may likewise be used in the preparation of injectables. The amount of active ingredient that may be combined with the carrier material to produce a single dosage form will vary depending upon the host treated and the particular mode of administration, such as oral administration or subcutaneous injection. For example, a time-release formulation intended for oral administration to humans may contain approximately 1 to 1000 mg of active material compounded with an appropriate and convenient amount of carrier material which may vary from about 5 to about 95% of the total compositions (weigh weight). The pharmaceutical composition can be prepared to provide easily measurable amounts for administration. For example, an aqueous solution intended for intravenous infusion may contain from about 3 to 500 μg of the active ingredient per milliliter of solution in order that infusion of a suitable volume at a rate of about 30 mL/hr can occur. When formulated for subcutaneous administration, the formulation is typically administered about twice a month over a period of from about two to about four months.

Formulations suitable for parenteral administration include aqueous and non- aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.

The formulations can be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injection, immediately prior to use. Extemporaneous injection solutions and suspensions are prepared from sterile powders, granules and tablets of the kind previously described. Preferred unit dosage formulations are those containing a daily dose or unit daily sub- dose, as herein above recited, or an appropriate fraction thereof, of the active ingredient.

In one embodiment, the FXR agonist and LOXL2 inhibitor may be administered together in a combination formulation or in seperate pharmaceutical compositions, where each inhibitor may be formulated in any suitable dosage form. In certain embodiments, the methods provided herein comprise administering separately a pharmaceutical composition comprising an FXR agonist and a pharmaceutically acceptable carrier or excipient and a pharmaceutical composition comprising a LOXL2 inhibitor and a pharmaceutically acceptable carrier or excipient. Combination formulations according to the present disclosure comprise an FXR agonist and a LOXL2 inhibitor together with one or more pharmaceutically acceptable carriers or excipients and optionally other therapeutic agents. Combination formulations containing the active ingredient may be in any form suitable for the intended method of administration. Below is a listing of sequences described throughout the specification.

SEQ ID Sequence

NO:

8 DIVMTQTPLSLSVTPGQPASISCRSSKSLLHSNGNTYLYWYLQKPG

QSPQFLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC

MQHLEYPYTFGGGTKVEIK

9 GYAFTYYLIE

10 VINPGSGGTNY EKFKG

11 NWMNFDY

12 RSSKSLLHSNGNTYLY

13 RMSNLAS

14 MQHLEYPYT

15 MEWSRVFIFLLSVTAGVHSQVQLQQSGAELVRPGTSVKVSCKASGYAFTYY LIEWVKQRPGQGLEWIGVINPGSGGTNYNEKFKGKATLTADKSSSTAYMQ LSSLTSDDSAVYFCARNWMNFDYWGQGTTLTVSS

16 MRCLAEFLGLLVLWIPGAIGDIVMTQAAPSVSVTPGESVSISCRSSKSLLHSN GNTYLYWFLQRPGQSPQFLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEA EDVGVYYCMQHLEYPYTFGGGTKLEIK

*CDRs are bolded.

Claims

What is claimed is:

1. A method of treating and/or preventing liver disease in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of an FXR agonist and a

therapeutically effective amount of a LOXL2 inhibitor.

2. The method of claim 1, wherein the liver disease is selected from chronic liver disease, metabolic liver disease, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), liver fibrosis, or primary sclerosing cholangitis (PSC).

3. The method of any one of claims 1-2, wherein the LOXL2 inhibitor is an anti-LOXL2 antibody.

4. The method of any one of claims 1-3, wherein the anti-LOXL2 antibody is a monoclonal anti-LOXL2 antibody or antigen-binding fragment thereof.

5. The method of any one of claims 1-3, wherein the anti-LOXL2 antibody is a polyclonal anti- LOXL2 antibody or antigen-binding fragment thereof.

6. The method of any one of claims 1-5, wherein the anti-LOXL2 antibody is an isolated antibody or antigen binding fragment thereof, comprising the complementarity determining regions (CDRs), CDRl, CDR2, and CDR3, of a heavy chain variable region comprising the amino acid sequence set forth as SEQ ID NO: 2, 3, 4, or 5, and the CDRs, CDRl, CDR2, and CDR3, of a light chain variable region comprising the amino acid sequence set forth as SEQ ID NO: 6, 7, or 8, wherein the isolated antibody or antigen binding fragment thereof specifically binds a lysyl oxidase- like 2(LOXL2) protein.

7. The method of any one of claims 1-5, wherein CDRl, CDR2, and CDR3 of the heavy chain variable region comprise the amino acid sequences set forth as SEQ ID NOs: 9, 10, and 11 respectively, and the CDRl, CDR2, and CDR3 of the light chain variable region comprise the amino acid sequences set forth as SEQ ID NOs: 12, 13, and 14, respectively.

8. The method of any one of claims 1-5, wherein the anti-LOXL2 antibody has a heavy chain variable region comprising the amino acid sequence set forth as SEQ ID NO: 2, 3, 4, or 5, and a light chain variable region comprising the amino acid sequence set forth as SEQ ID NO: 6, 7, or 8, wherein the isolated antibody or antigen binding fragment thereof specifically binds a lysyl oxidase- like 2 (LOXL2) protein.

9. The method of any one of claims 1-8, wherein the FXR agonist and the LOXL2 inhibitor are administered together.

10. The method of any one of claims 1-8, wherein the FXR agonist and the LOXL2 inhibitor are administered separately.

11. A pharmaceutical composition comprising a therapeutically effective amount of a FXR agonist inhibitor and a therapeutically effective amount of a LOXL2 inhibitor.

12. The pharmaceutical composition of claim 11, further comprising a pharmaceutically acceptable carrier.