Methods and compositions for treating pancreatic and liver diseases

Abstract

The present disclosure provides methods and compositions for treating pancreatic and hepatic diseases, such as pancreatic steatosis, pancreatic cancer, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, and hepatic cancer. The provided methods include administering to a subject suffering from or at risk of suffering from pancreatic and / or hepatic disease an effective amount of a HIF1-α pathway inhibitor or HIF1-α inhibitor, and an effective amount of a PFKFB3 inhibitor. The provided methods include administering to a subject suffering from or at risk of suffering from pancreatic and / or hepatic disease an effective amount of a HIF1-α pathway inhibitor or HIF1-α inhibitor, and an effective amount of a PFKFB3 inhibitor.

Description

[Background technology]

[0001] The present disclosure provides methods and compositions for treating pancreatic and hepatic diseases, such as pancreatic steatosis, pancreatic cancer, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, and hepatic cancer. The methods provided include administering to a subject suffering from or at risk of suffering from pancreatic and / or hepatic disease an effective amount of a HIF1-α pathway inhibitor or a HIF1-α inhibitor, and an effective amount of a PFKFB3 inhibitor.

[0002] Obesity and metabolic syndrome (including obesity, hyperglycemia, dyslipidemia, hypertension and insulin resistance) result in metabolic abnormalities that lead to lipid mishandling by adipocytes and fatty infiltration of the pancreas and liver. Imbalance of adipocytokines in the circulation and in the microenvironment of the pancreas and liver causes chronic low-grade inflammation, leading to pancreatic and hepatic dysfunction. Furthermore, these adipocytokines regulate cell proliferation, differentiation, and angiogenesis and lymphatic expansion.

[0003] Pancreatic steatosis refers to a disease ranging from fatty infiltration of the pancreas to pancreatic inflammation and the development of pancreatic fibrosis. The result of fatty cell infiltration of the pancreas is thought to cause pancreatic dysfunction and carcinogenesis, resulting in pancreatic intraepithelial neoplasia and pancreatic ductal adenocarcinoma (PDAC).

[0004] Nonalcoholic fatty liver disease (NAFLD) refers to the presence of hepatic accumulation of triglycerides in hepatocytes and is the most common cause of chronic liver disease in Western countries. Its clinical and histological phenotypes range from nonalcoholic fatty liver (NAFL) to nonalcoholic steatohepatitis (NASH), characterized by hepatic inflammation and progressive fibrosis, leading to cirrhosis and end-stage liver disease, as well as hepatocellular carcinoma. NASH-related cirrhosis is the leading indication for liver transplantation in the United States.

[0005] Type 2 diabetes mellitus (T2D) is a well-established risk factor for pancreatic adenocarcinoma (PDAC), with a 1.5- to 2-fold increased risk of malignancy, even after adjusting for correlating anthropometric factors such as BMI. Preneoplastic pancreatic intraepithelial lesions (PanINs) occur more frequently and pancreatic ductal gland (PDG) replication is increased twofold in pancreases from individuals with T2D when compared to BMI-matched and age-matched controls. In addition to acinar-to-ductal metaplasia, the PDG compartment has been implicated in PDAC as an initiating niche for tumor development and may respond to alterations in the pancreatic microenvironment (inflammation and metabolism) by increased proliferation in an attempt to regenerate ductal epithelium after injury during pancreatitis, for example. Chronic activation of PDGs may result in overall tumor promotion in PDGs, or in the progression of PanINs. Furthermore, islet-PanIN complexes were enriched in pancreata from brain-dead donors with T2D compared with non-diabetic patients, indicating that T2D diabetic islets are in anatomical proximity to PanINs and may exert significant paracrine effects on PanINs.

[0006] The available treatments for pancreatic and hepatic diseases, such as pancreatic cancer, hepatic cancer, pancreatitis and non-alcoholic fatty liver disease, are limited and have drawbacks.Given that the number of individuals suffering from pancreatic and hepatic diseases continues to increase and its impact on a global scale, there is an urgent need for treatments that can alleviate or ameliorate pancreatic and hepatic diseases. Summary of the Invention

[0007] The present disclosure provides methods and compositions for treating pancreatic and liver diseases, such as pancreatic steatosis, pancreatic cancer, such as pancreatic ductal adrenal carcinoma, nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, and liver cancer, such as hepatocellular carcinoma. The methods provided include administering to a subject suffering from or at risk of suffering from pancreatic and / or liver disease an effective amount of a HIF1-α pathway inhibitor or a HIF1-α inhibitor, and an effective amount of a PFKFB3 inhibitor.

[0008] In T2D, the exocrine pancreas undergoes various changes, including exocrine insufficiency, acinar fibrosis and inflammation, as well as activation of the HIF-1α / PFKFB3 signaling axis in diabetic β-cells, leading to β-cell dysfunction, inflammation, metabolic remodeling of islets, and lactate release—factors that can affect the microenvironment and provide clues for tumor promotion in the exocrine pancreas.

[0009] The present inventors have surprisingly found that the combination of HIF1-α inhibitor and PFKFB3 inhibitor of HIF1-α / PFKFB3 signaling axis can treat pancreatic cancer by reducing events related to the promotion or progression of pancreatic cancer and liver cancer.Without being bound by theory, it is believed that the disclosed method activates cell competition in pancreas and liver that promotes the survival of functional cells, thereby reducing factors such as cell dysfunction, inflammation, metabolic remodeling and lactate release that promote the survival and dysfunctional cells and lead to pancreatic and liver diseases and tumor growth and progression.

[0010] We also found that lactate secreted from diabetic islets in the exocrine pancreas activates GPR81 signaling in proximal acinar cells, pancreatic ductal glands (PDGs) and pre-existing PanINs. Chronic HIF1-α-PFKFB3-dependent release of lactate exerts tumor-promoting effects on the exocrine pancreas, thereby promoting tumor growth and progression. GPR81 receptors are overexpressed in 98% of all cell lines derived from PDAC patients. Activation of GPR81 is associated with inhibition of lipolysis, increased proliferation, transmigration, reactive oxygen species, and accumulation of intracellular lipids following detoxification of (ROS), multiple events leading to the growth of preneoplastic lesions in the pancreas.

[0011] In some embodiments, methods and compositions are provided to treat chronic pancreatic and / or liver disease, hi some embodiments, methods and compositions are provided to treat acute pancreatic and / or liver disease.

[0012] In some embodiments, the present disclosure provides: [1] A method for treating pancreatic steatosis in a subject in need thereof, comprising: (a) administering to a subject an effective amount of a HIF1-α pathway inhibitor and an effective amount of a PFKFB3 inhibitor; (b) administering to the subject an effective amount of a HIF1-α pathway inhibitor, wherein the subject has previously been administered a PFKFB3 inhibitor; or (c) administering to the subject an effective amount of a PFKFB3 inhibitor, wherein the subject has previously been administered a HIF1-α pathway inhibitor; The method, wherein the PFKFB3 inhibitor does not inhibit the PI3K / AKT / mTOR pathway or HIF1-α. [2] The method according to [1], wherein the subject is administered an effective amount of a HIF1-α pathway inhibitor and an effective amount of a PFKFB3 inhibitor. [3] The method of [1], wherein the subject is administered an effective amount of a HIF1-α pathway inhibitor, and the subject has previously been administered a PFKFB3 inhibitor. [4] The method of [1], wherein the subject is administered an effective amount of a PFKFB3 inhibitor, and the subject has previously been administered a HIF1-α pathway inhibitor. [5] The method according to any one of [1] to [4], wherein the method according to any one of 1(a) to 1(c) is administered as a preventive treatment of pancreatic steatosis (e.g., fatty infiltration of the pancreas, fatty infiltration of the pancreas accompanied by pancreatic inflammation, and fatty infiltration of the pancreas accompanied by the development of pancreatic fibrosis). [6] The method according to any one of [1] to [4], wherein the subject has or is at risk of having pancreatic steatosis (e.g., fatty infiltration of the pancreas, fatty infiltration of the pancreas with pancreatic inflammation, and fatty infiltration of the pancreas with the development of pancreatic fibrosis). [7] The method according to any one of [1] to [4], wherein the subject has or has been diagnosed as having pancreatic steatosis. [8] The method according to any one of [1] to [7], wherein pancreatic steatosis is associated with fatty infiltration of the pancreas, fatty infiltration of the pancreas accompanied by pancreatic inflammation, or fatty infiltration of the pancreas accompanied by the development of pancreatic fibrosis. [9] A method according to any one of [1] to [8], wherein pancreatic steatosis is accompanied by fatty infiltration of the pancreas.

[10] The method according to any one of [1] to [8], wherein pancreatic steatosis is associated with fatty infiltration of the pancreas accompanied by pancreatic inflammation.

[11] The method according to any one of [1] to [8], wherein pancreatic steatosis is associated with fatty infiltration of the pancreas with the development of pancreatic fibrosis.

[12] The method according to any one of [1] to [8], wherein pancreatic steatosis is accompanied by pancreatic inflammation and is associated with fatty infiltration of the pancreas with the development of pancreatic fibrosis.

[13] The method of any one of [1] to

[12] , wherein the administered HIF1-α pathway inhibitor is an antibody or antigen-binding antibody fragment (e.g., a single chain antibody, a single domain antibody (e.g., VHH), a Fab fragment, a F(ab')2 fragment, a Fd fragment, a Fv fragment, a scFv, a dAb fragment, or another engineered molecule, e.g., a diabody, a triabody, a tetrabody, a minibody, and a minimal recognition unit), a nucleic acid molecule (e.g., an aptamer, an antisense molecule, a ribozyme, a dicer substrate, MiRNA, dsRNA, ssRNA, and shRNA), a peptibody, a nanobody, a HIF1-α pathway binding polypeptide, or a small molecule HIF1-α pathway inhibitor.

[14] The method according to any one of [1] to

[13] , wherein the administered HIF1-α pathway inhibitor is silibinin, PX-478 or YC-1, or a salt thereof.

[15] The method according to any one of [1] to

[14] , wherein the administered HIF1-α pathway inhibitor is ganetespib (ST-9090), phenethyl isothocyanate or BAY-87-2243, or a salt thereof.

[16] The method according to any one of [1] to

[15] , wherein the administered HIF1-α pathway inhibitor is a HIF1-α inhibitor.

[17] The method of

[16] , wherein the HIF1-α inhibitor is an antibody or antigen-binding antibody fragment (e.g., a single chain antibody, a single domain antibody (e.g., VHH), a Fab fragment, a F(ab')2 fragment, a Fd fragment, a Fv fragment, a scFv, a dAb fragment, or another engineered molecule, e.g., a diabody, a triabody, a tetrabody, a minibody, and a minimal recognition unit), a nucleic acid molecule (e.g., an aptamer, an antisense molecule, a ribozyme, MiRNA, dsRNA, ssRNA, and shRNA), a peptibody, a nanobody, a HIF1-α binding polypeptide, or a small molecule HIF1-α inhibitor.

[18] The method according to

[16] or

[17] , wherein the administered HIF1-α inhibitor is the antisense oligonucleotide EZN-2968, or the nanobody AG-1, AG-2, AG-3, AG-4, AG-5, VHH212 or AHPC.

[19] The method of any one of [1] to

[18] , wherein the administered PFKFB3 inhibitor is an antibody or antigen-binding antibody fragment (e.g., a single chain antibody, a single domain antibody, a Fab fragment, a F(ab')2 fragment, a Fd fragment, a Fv fragment, a scFv, a dAb fragment, or another engineered molecule, e.g., a diabody, a triabody, a tetrabody, a minibody, and a minimal recognition unit), a nucleic acid molecule (e.g., an aptamer, an antisense molecule, a ribozyme, MiRNA, dsRNA, ssRNA, and shRNA), a peptibody, a nanobody, a PFKFB3-binding polypeptide, or a small molecule PFKFB3 inhibitor.

[20] The method according to any one of [1] to

[19] , wherein the administered PFKFB3 inhibitor is BrAcNHEtOP (N-bromoacetylethanolamine phosphate), PFK15 (1-(4-pyridinyl)-3-(2-quinolinyl)-2-propen-1-one), or PFK-158 ((E)-1-(4-pyridinyl)-3-[7-(trifluoromethyl)-2-quinolinyl]-2-propen-1-one), or a salt thereof.

[21] The method according to any one of [1] to

[20] , wherein the administered PFKFB3 inhibitor is (a) KAN0436151 or KAN0436067, or a salt thereof; (b) Formula 1 to Formula 53 or Formula 54, PQP, N4A, YN1, PK15, PFK-158, YZ29, Compound 26, KAN0436151, KAN0436067, or BrAcNHErOP, or a salt thereof, as shown in Figure 1A to Figure 1C or Figure 1D; (c) Formula AZ44 to Formula AZ70 or Formula AZ71, or a salt thereof, as shown in Figure 1E; or (d) AZ67 or a salt thereof.

[22] The method according to any one of [1] to

[21] , wherein the HIF1-α pathway inhibitor and the PFKFB3 inhibitor are co-administered to the subject.

[23] The method according to any one of [1] to

[22] , wherein the administration of the HIF1-α pathway inhibitor and / or the PFKFB3 inhibitor is oral, parenteral, orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal, intranasal, intratumoral, or intravenous.

[24] The method according to any one of [1] to

[23] , wherein the HIF1-α pathway inhibitor and the PFKFB3 inhibitor are administered before the onset of one or more symptoms of pancreatic steatosis.

[25] The method according to any one of [1] to

[24] , wherein treating pancreatic steatosis includes delaying the onset of pancreatic steatosis.

[26] The method according to any one of [1] to

[23] , wherein the HIF1-α pathway inhibitor and the PFKFB3 inhibitor are administered after the onset of one or more symptoms of pancreatic steatosis.

[27] The method according to

[26] , wherein the method results in a reduction in one or more symptoms of pancreatic steatosis in a subject administered a HIF1-α pathway inhibitor and a PFKFB3 inhibitor, compared to the subject before treatment.

[28] The method of

[27] , wherein the reduction in one or more symptoms of pancreatic steatosis is indicated by a reduction in the fat content of the pancreas to less than 25%, reduction in abdominal pain, reduction in nausea, increased appetite, weight gain, reduction in jaundice, reduction in edema; and reduction in fatigue, confusion or weakness; reduction in pancreatic inflammation, reduction in pancreatitis, reduction in pancreatic cellular fibrosis, and reduction in levels of pancreatic steatosis biomarkers or pro-inflammatory cytokines (e.g., TNFα, IL-1β, IL6, MCP1, IL8, PAF).

[29] The method of

[27] or

[28] , wherein one or more of the alleviated symptoms is a reduction of at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% in fat content in the subject compared to the subject prior to treatment with the HIF1-α pathway inhibitor and the PFKFB3 inhibitor.

[30] The method according to any one of

[27] to

[29] , wherein serum levels of at least one, two, three, four or five pancreatic steatosis biomarkers are reduced by at least 20% compared to a subject prior to treatment with a HIF1-α pathway inhibitor and a PFKFB3 inhibitor.

[31] The method of any one of

[27] to

[30] , wherein serum levels of at least one, two, three, four or five of the biomarkers: TNFα, IL-1β, IL6, MCP1, IL8 and / or PAF are reduced by at least 20% compared to the subject prior to treatment with a HIF1-α pathway inhibitor and a PFKFB3 inhibitor.

[32] The method according to any one of [1] to

[31] , further comprising administering an additional therapeutic agent to the subject.

[33] A method for treating pancreatic cancer in a subject in need thereof, comprising: (a) administering to a subject an effective amount of a HIF1-α pathway inhibitor and an effective amount of a PFKFB3 inhibitor; (b) administering to the subject an effective amount of a HIF1-α pathway inhibitor, wherein the subject has previously been administered a PFKFB3 inhibitor; or (c) administering to the subject an effective amount of a PFKFB3 inhibitor, wherein the subject has previously been administered a HIF1-α pathway inhibitor; The method, wherein the PFKFB3 inhibitor does not inhibit the PI3K / AKT / mTOR pathway or HIF1-α.

[34] The method of

[33] , wherein the subject is administered an effective amount of a HIF1-α pathway inhibitor and an effective amount of a PFKFB3 inhibitor.

[35] The method of

[33] , wherein the subject is administered an effective amount of a HIF1-α pathway inhibitor, and the subject has previously been administered a PFKFB3 inhibitor.

[36] The method of

[33] , wherein the subject is administered an effective amount of a PFKFB3 inhibitor, and the subject has previously been administered a HIF1-α pathway inhibitor.

[37] The method according to any one of

[33] to

[36] , wherein the method according to any one of 1(a) to 1(c) is administered as a prophylactic treatment for pancreatic cancer.

[38] The method according to any one of

[33] to

[36] , wherein the subject has or is at risk of having pancreatic cancer.

[39] The method according to any one of

[33] to

[36] , wherein the subject is suffering from or has been diagnosed with pancreatic cancer.

[40] The method according to any one of

[33] to

[39] , wherein the pancreatic cancer is exocrine pancreatic cancer or neuroendocrine pancreatic cancer.

[41] The method of

[40] , wherein the pancreatic cancer is exocrine pancreatic cancer (e.g., adenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma, or colloid carcinoma).

[42] The method according to

[40] , wherein the pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC).

[43] The method according to

[40] , wherein the pancreatic cancer is squamous cell carcinoma, adenosquamous carcinoma, or colloid carcinoma.

[44] The method according to

[40] , wherein the pancreatic cancer is neuroendocrine pancreatic cancer.

[45] The method of any one of

[33] -

[44] , wherein the administered HIF1-α pathway inhibitor is an antibody or antigen-binding antibody fragment (e.g., a single chain antibody, a single domain antibody (e.g., VHH), a Fab fragment, a F(ab')2 fragment, a Fd fragment, a Fv fragment, a scFv, a dAb fragment, or another engineered molecule, e.g., a diabody, a triabody, a tetrabody, a minibody, and a minimal recognition unit), a nucleic acid molecule (e.g., an aptamer, an antisense molecule, a ribozyme, a dicer substrate, dsRNA, ssRNA, and shRNA), a peptibody, a nanobody, a HIF1-α pathway binding polypeptide, or a small molecule HIF1-α pathway inhibitor.

[46] The method according to any one of

[33] to

[45] , wherein the administered HIF1-α pathway inhibitor is silibinin, PX-478 or YC-1, or a salt thereof.

[47] The method according to any one of

[33] to

[45] , wherein the administered HIF1-α pathway inhibitor is ganetespib (ST-9090), phenethyl isothocyanate or BAY-87-2243, or a salt thereof.

[48] ​​The method according to any one of

[33] to

[47] , wherein the administered HIF1-α pathway inhibitor is a HIF1-α inhibitor.

[49] The method of

[48] , wherein the HIF1-α inhibitor is an antibody or antigen-binding antibody fragment (e.g., a single chain antibody, a single domain antibody (e.g., VHH), a Fab fragment, a F(ab')2 fragment, a Fd fragment, a Fv fragment, a scFv, a dAb fragment, or another engineered molecule, e.g., a diabody, a triabody, a tetrabody, a minibody, and a minimal recognition unit), a nucleic acid molecule (e.g., an aptamer, an antisense molecule, a ribozyme, miRNA, dsRNA, ssRNA, and shRNA), a peptibody, a nanobody, a HIF1-α binding polypeptide, or a small molecule HIF1-α inhibitor.

[50] The method according to

[48] or

[49] , wherein the administered HIF1-α inhibitor is the antisense oligonucleotide EZN-2968, or the nanobody AG-1, AG-2, AG-3, AG-4, AG-5, VHH212 or AHPC.

[51] The method of any one of

[33] to

[50] , wherein the administered PFKFB3 inhibitor is an antibody or antigen-binding antibody fragment (e.g., a single chain antibody, a single domain antibody, a Fab fragment, a F(ab')2 fragment, a Fd fragment, a Fv fragment, a scFv, a dAb fragment, or another engineered molecule, e.g., a diabody, a triabody, a tetrabody, a minibody, and a minimal recognition unit), a nucleic acid molecule (e.g., an aptamer, an antisense molecule, a ribozyme, MiRNA, dsRNA, ssRNA, and shRNA), a peptibody, a nanobody, a PFKFB3-binding polypeptide, or a small molecule PFKFB3 inhibitor.

[52] The method according to any one of

[33] to

[51] , wherein the administered PFKFB3 inhibitor is BrAcNHEtOP (N-bromoacetylethanolamine phosphate), PFK15 (1-(4-pyridinyl)-3-(2-quinolinyl)-2-propen-1-one), or PFK-158 ((E)-1-(4-pyridinyl)-3-[7-(trifluoromethyl)-2-quinolinyl]-2-propen-1-one), or a salt thereof.

[53] The method according to any one of

[33] to

[51] , wherein the administered PFKFB3 inhibitor is (a) KAN0436151 or KAN0436067, or a salt thereof; (b) has the structure of formula 1 to formula 53 or formula 54, PQP, N4A, YN1, PK15, PFK-158, YZ29, compound 26, KAN0436151, KAN0436067, or BrAcNHErOP, or a salt thereof, as shown in Figure 1A to Figure 1C or Figure 1D; (c) has the structure of formula AZ44 to formula AZ70 or formula AZ71, or a salt thereof, as shown in Figure 1E; or (d) is AZ67 or a salt thereof.

[54] The method according to any one of

[33] to

[53] , wherein the HIF1-α pathway inhibitor and the PFKFB3 inhibitor are co-administered to the subject.

[55] The method according to any one of

[33] to

[54] , wherein the administration of the HIF1-α pathway inhibitor and / or the PFKFB3 inhibitor is oral, parenteral, orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal, intranasal, intratumoral, or intravenous.

[56] The method according to any one of

[33] to

[55] , wherein the HIF1-α pathway inhibitor and the PFKFB3 inhibitor are administered before the onset of one or more symptoms of pancreatic cancer.

[57] The method according to any one of

[33] to

[56] , wherein treating pancreatic cancer includes delaying the onset of pancreatic cancer.

[58] The method according to any one of

[33] to

[57] , wherein the HIF1-α pathway inhibitor and the PFKFB3 inhibitor are administered after the onset of one or more symptoms of pancreatic cancer.

[59] The method according to any one of

[33] to

[58] , wherein the method results in alleviation of one or more symptoms of pancreatic cancer in the subject compared to before administration of the HIF1-α pathway inhibitor and the PFKFB3 inhibitor.

[60] The method of

[59] , wherein alleviation of one or more symptoms of pancreatic cancer is indicated by a decrease in tumor size, inhibition or slowing of tumor growth, no formation of new tumors, a decrease in new tumor formation, an increase in survival or progression-free survival, no metastasis, an increase in treatment options, a delay in time from surgery to recurrence, a decrease in jaundice, an inhibition of liver infiltration, a decrease in pain, an improvement in appetite, an improvement in digestion, a decrease in size of the gallbladder, and a decrease in the incidence of blood clots.

[61] The method of

[59] or

[60] , wherein one or more symptoms of pancreatic cancer are alleviated by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% compared to the subject prior to treatment with the HIF1-α pathway inhibitor and the PFKFB3 inhibitor.

[62] The method according to any one of

[59] to

[61] , wherein at least one of the biomarkers creatine kinase (CK-MB), troponin, N-terminal pro-B-type natriuretic peptide, alpha-1 antitrypsin, C-reactive protein, apolipoprotein A1, apolipoprotein B, creatinine, alkaline phosphatase, and transferrin is decreased compared to the subject before treatment with a HIF1-α pathway inhibitor and a PFKFB3 inhibitor.

[63] (a) at least one, two, three, four, five or more of the following biomarkers are improved in the subject: creatine kinase (CK-MB), troponin, N-terminal pro-B-type natriuretic peptide, alpha-1 antitrypsin, C-reactive protein, apolipoprotein A1, apolipoprotein B, creatinine, alkaline phosphatase, and transferrin by at least 20%, at least 30%, at least 40%, or at least 50%, compared to the subject prior to treatment with a HIF1-α pathway inhibitor and a PFKFB3 inhibitor; or (b) the subject's tumor size is reduced by at least 20%, at least 30%, at least 40%, or at least 50% compared to the subject prior to treatment with the HIF1-α pathway inhibitor and the PFKFB3 inhibitor; The method according to any one of

[59] to

[62] .

[64] The method according to any one of

[33] to

[63] , further comprising administering an additional therapeutic agent to the subject.

[65] A method of treating non-alcoholic fatty liver disease (NAFLD) in a subject in need thereof, comprising: (a) administering to a subject an effective amount of a HIF1-α pathway inhibitor and an effective amount of a PFKFB3 inhibitor; (b) administering to the subject an effective amount of a HIF1-α pathway inhibitor, wherein the subject has previously been administered a PFKFB3 inhibitor; or (c) administering to the subject an effective amount of a PFKFB3 inhibitor, wherein the subject has previously been administered a HIF1-α pathway inhibitor; The method, wherein the PFKFB3 inhibitor does not inhibit the PI3K / AKT / mTOR pathway or HIF1-α.

[66] The method of

[65] , wherein the subject is administered an effective amount of a HIF1-α pathway inhibitor and an effective amount of a PFKFB3 inhibitor.

[67] The method of

[65] , wherein the subject is administered an effective amount of a HIF1-α pathway inhibitor, and the subject has previously been administered a PFKFB3 inhibitor.

[68] The method of

[65] , wherein the subject is administered an effective amount of a PFKFB3 inhibitor, and the subject has previously been administered a HIF1-α pathway inhibitor.

[69] The method according to any one of 1(a) to 1(c), wherein the method according to any one of

[65] to

[68] is administered as a preventative treatment for non-alcoholic fatty disease (e.g., non-alcoholic fatty liver (NAFL), non-alcoholic steatohepatitis (NASH), or NAFLD-associated liver fibrosis).

[70] The method of any one of

[65] -

[68] , wherein the subject has or is at risk of having non-alcoholic fatty liver disease (e.g., non-alcoholic fatty liver (NAFL), non-alcoholic steatohepatitis (NASH), or NAFLD-associated liver fibrosis).

[71] The method of any one of

[65] -

[68] , wherein the subject has or has been diagnosed with non-alcoholic fatty liver disease (e.g., non-alcoholic fatty liver (NAFL), non-alcoholic steatohepatitis (NASH), or NAFLD-associated liver fibrosis).

[72] The method of any one of

[65] -

[71] , wherein the administered HIF1-α pathway inhibitor is an antibody or antigen-binding antibody fragment (e.g., a single chain antibody, a single domain antibody (e.g., VHH), a Fab fragment, a F(ab')2 fragment, a Fd fragment, a Fv fragment, a scFv, a dAb fragment, or another engineered molecule, e.g., a diabody, a triabody, a tetrabody, a minibody, and a minimal recognition unit), a nucleic acid molecule (e.g., an aptamer, an antisense molecule, a ribozyme, a dicer substrate, dsRNA, ssRNA, and shRNA), a peptibody, a nanobody, a HIF1-α pathway binding polypeptide, or a small molecule HIF1-α pathway inhibitor.

[73] The method according to any one of

[65] to

[72] , wherein the administered HIF1-α pathway inhibitor is silibinin, PX-478 or YC-1, or a salt thereof.

[74] The method according to any one of

[65] to

[72] , wherein the administered HIF1-α pathway inhibitor is ganetespib (ST-9090), phenethyl isothocyanate or BAY-87-2243, or a salt thereof.

[75] The method according to any one of

[65] to

[72] , wherein the administered HIF1-α pathway inhibitor is a HIF1-α inhibitor.

[76] The method of

[75] , wherein the HIF1-α inhibitor is an antibody or antigen-binding antibody fragment (e.g., a single chain antibody, a single domain antibody (e.g., VHH), a Fab fragment, a F(ab')2 fragment, a Fd fragment, a Fv fragment, a scFv, a dAb fragment, or another engineered molecule such as a diabody, a triabody, a tetrabody, a minibody, and a minimal recognition unit), a nucleic acid molecule (e.g., an aptamer, an antisense molecule, a ribozyme, miRNA, dsRNA, ssRNA, and shRNA), a peptibody, a nanobody, a HIF1-α binding polypeptide, or a small molecule HIF1-α inhibitor.

[77] The method of

[75] or

[76] , wherein the administered HIF1-α inhibitor is the antisense oligonucleotide EZN-2968, or the nanobody AG-1, AG-2, AG-3, AG-4, AG-5, VHH212 or AHPC.

[78] The method of any one of

[65] -

[77] , wherein the administered PFKFB3 inhibitor is an antibody or antigen-binding antibody fragment (e.g., a single chain antibody, a single domain antibody, a Fab fragment, a F(ab')2 fragment, a Fd fragment, a Fv fragment, a scFv, a dAb fragment, or another engineered molecule, e.g., a diabody, a triabody, a tetrabody, a minibody, and a minimal recognition unit), a nucleic acid molecule (e.g., an aptamer, an antisense molecule, a ribozyme, MiRNA, dsRNA, ssRNA, and shRNA), a peptibody, a nanobody, a PFKFB3-binding polypeptide, or a small molecule PFKFB3 inhibitor.

[79] The method according to any one of

[65] to

[78] , wherein the administered PFKFB3 inhibitor is BrAcNHEtOP (N-bromoacetylethanolamine phosphate), PFK15 (1-(4-pyridinyl)-3-(2-quinolinyl)-2-propen-1-one), or PFK-158 ((E)-1-(4-pyridinyl)-3-[7-(trifluoromethyl)-2-quinolinyl]-2-propen-1-one), or a salt thereof.

[80] The method of any one of

[65] to

[78] , wherein the administered PFKFB3 inhibitor is (a) KAN0436151 or KAN0436067, or a salt thereof; (b) has the structure of formula 1 to formula 53 or formula 54, PQP, N4A, YN1, PK15, PFK-158, YZ29, compound 26, KAN0436151, KAN0436067, or BrAcNHErOP, or a salt thereof, as shown in Figure 1A to Figure 1C or Figure 1D; (c) has the structure of formula AZ44 to formula AZ70 or formula AZ71, or a salt thereof, as shown in Figure 1E; or (d) AZ67 or a salt thereof.

[81] The method according to any one of

[65] to

[80] , wherein the HIF1-α pathway inhibitor and the PFKFB3 inhibitor are co-administered to the subject.

[82] The method according to any one of

[65] to

[81] , wherein the administration of the HIF1-α pathway inhibitor and / or the PFKFB3 inhibitor is oral, parenteral, orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal, intranasal, intratumoral, or intravenous.

[83] The method of any one of

[65] to

[82] , wherein the HIF1-α pathway inhibitor and the PFKFB3 inhibitor are administered prior to the onset of one or more symptoms of non-alcoholic fatty liver disease (e.g., non-alcoholic fatty liver (NAFL), non-alcoholic steatohepatitis (NASH), or NAFLD-associated liver fibrosis).

[84] Treating non-alcoholic fatty liver disease (NAFLD) includes delaying the onset of non-alcoholic fatty liver disease (e.g., non-alcoholic fatty liver (NAFL), non-alcoholic steatohepatitis (NASH), or NAFLD-associated liver fibrosis), according to any one of

[65] -

[83] .

[85] The method according to any one of

[65] to

[84] , wherein the HIF1-α pathway inhibitor and the PFKFB3 inhibitor are administered after onset of one or more symptoms of non-alcoholic fatty liver disease (e.g., non-alcoholic fatty liver (NAFL), non-alcoholic steatohepatitis (NASH), or NAFLD-associated liver fibrosis).

[86] The method according to any one of

[65] to

[85] , wherein the method results in alleviation of one or more symptoms of non-alcoholic fatty liver disease in the subject compared to before administration of the HIF1-α pathway inhibitor and the PFKFB3 inhibitor.

[87] Reduction in one or more symptoms of nonalcoholic fatty liver disease may be (a) reduction of fatigue, pain or discomfort in the right upper abdomen, reduction of splenomegaly, reduction of jaundice, reduction of edema, increase and decrease of appetite, (b) Mitigation of NAS; or (c) decreased levels of NAFLD biomarkers (e.g., TNFα, IL-6, CRP, IL-1RA, PAI1, CXCL10, CK18, FGF21, oxLDL, hyaluronic acid, laminin, procollagen II, and TIMP1); The method according to

[86] , as shown by:

[88] The method of

[86] or

[87] , wherein one or more symptoms of nonalcoholic fatty liver disease are reduced by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% compared to the subject prior to treatment with the HIF1-α pathway inhibitor and the PFKFB3 inhibitor.

[89] (a) at least one plasma NAFLD biomarker (e.g., TNFα, IL-6, CRP, IL-1RA, PAI1, CXCL10, CK18, FGF21, oxLDL, ALT, hyaluronic acid, laminin, procollagen II, and TIMP1) is improved compared to a subject prior to treatment with a HIF1-α pathway inhibitor and a PFKFB3 inhibitor; or (b) the subject's NAS score is reduced by 1, 2, 3, or more compared to the subject prior to treatment with the HIF1-α pathway inhibitor and the PFKFB3 inhibitor; or (c) treated subjects have a NAS of less than 3 points; The method according to any one of

[86] to

[88] .

[90] The method of any one of

[86] to

[89] , wherein at least one, two, three, four or five biomarkers selected from TNFα, IL-6, CRP, IL-1RA, PAI1, CXCL10, CK18, FGF21, oxLDL, hyaluronic acid, laminin, procollagen II, and TIMP1 are reduced by at least 20%, at least 30%, at least 40%, or at least 50% compared to the subject prior to treatment with a HIF1-α pathway inhibitor and a PFKFB3 inhibitor.

[91] The method according to any one of

[65] to

[90] , further comprising administering an additional therapeutic agent to the subject.

[92] A method of treating hepatocellular carcinoma in a subject in need thereof, comprising: (a) administering to a subject an effective amount of a HIF1-α pathway inhibitor and an effective amount of a PFKFB3 inhibitor; (b) administering to the subject an effective amount of a HIF1-α pathway inhibitor, wherein the subject has previously been administered a PFKFB3 inhibitor; or (c) administering to the subject an effective amount of a PFKFB3 inhibitor, wherein the subject has previously been administered a HIF1-α pathway inhibitor; The method, wherein the PFKFB3 inhibitor does not inhibit the PI3K / AKT / mTOR pathway or HIF1-α.

[93] The method of

[92] , wherein the subject is administered an effective amount of a HIF1-α pathway inhibitor and an effective amount of a PFKFB3 inhibitor.

[94] The method of

[92] , wherein the subject is administered an effective amount of a HIF1-α pathway inhibitor, and the subject has previously been administered a PFKFB3 inhibitor.

[95] The method of

[92] , wherein the subject is administered an effective amount of a PFKFB3 inhibitor, and the subject has previously been administered a HIF1-α pathway inhibitor. The method according to any one of

[92] to

[95] , wherein the method according to any one of

[96] [92(a) to (c)] is administered as a prophylactic treatment for hepatocellular carcinoma.

[97] The method according to any one of

[92] to

[95] , wherein the subject is suffering from or at risk of suffering from hepatocellular carcinoma.

[98] The method according to any one of

[92] to

[95] , wherein the subject has hepatocellular carcinoma or has been diagnosed with hepatocellular carcinoma.

[99] The method of any one of

[92] -

[98] , wherein the administered HIF1-α pathway inhibitor is an antibody or antigen-binding antibody fragment (e.g., a single chain antibody, a single domain antibody (e.g., VHH), a Fab fragment, a F(ab')2 fragment, a Fd fragment, a Fv fragment, a scFv, a dAb fragment, or another engineered molecule, e.g., a diabody, a triabody, a tetrabody, a minibody, and a minimal recognition unit), a nucleic acid molecule (e.g., an aptamer, an antisense molecule, a ribozyme, a dicer substrate, dsRNA, ssRNA, and shRNA), a peptibody, a nanobody, a HIF1-α pathway binding polypeptide, or a small molecule HIF1-α pathway inhibitor.

[0100] The method according to any one of

[92] to

[99] , wherein the administered HIF1-α pathway inhibitor is silibinin, PX-478 or YC-1, or a salt thereof.

[0101] The method according to any one of

[92] to

[99] , wherein the administered HIF1-α pathway inhibitor is ganetespib (ST-9090), phenethyl isothocyanate or BAY-87-2243, or a salt thereof.

[0102] The method according to any one of

[92] to

[99] , wherein the administered HIF1-α pathway inhibitor is a HIF1-α inhibitor.

[0103] The method of any one of claims 1 to 5, wherein the HIF1-α inhibitor is an antibody or antigen-binding antibody fragment (e.g., a single chain antibody, a single domain antibody (e.g., VHH), a Fab fragment, a F(ab')2 fragment, a Fd fragment, a Fv fragment, a scFv, a dAb fragment, or another engineered molecule, e.g., a diabody, a triabody, a tetrabody, a minibody, and a minimal recognition unit), a nucleic acid molecule (e.g., an aptamer, an antisense molecule, a ribozyme, miRNA, dsRNA, ssRNA, and shRNA), a peptibody, a nanobody, a HIF1-α binding polypeptide, or a small molecule HIF1-α inhibitor.

[0104] The method of

[0102] or

[0103] , wherein the administered HIF1-α inhibitor is the antisense oligonucleotide EZN-2968, or the nanobody AG-1, AG-2, AG-3, AG-4, AG-5, VHH212 or AHPC.

[0105] The method according to any one of

[92] to

[0104] , wherein the administered PFKFB3 inhibitor is an antibody or antigen-binding antibody fragment (e.g., a single chain antibody, a single domain antibody, a Fab fragment, a F(ab')2 fragment, a Fd fragment, a Fv fragment, a scFv, a dAb fragment, or another engineered molecule, e.g., a diabody, a triabody, a tetrabody, a minibody, and a minimal recognition unit), a nucleic acid molecule (e.g., an aptamer, an antisense molecule, a ribozyme, MiRNA, dsRNA, ssRNA, and shRNA), a peptibody, a nanobody, a PFKFB3-binding polypeptide, or a small molecule PFKFB3 inhibitor.

[0106] The method according to any one of

[92] to

[0105] , wherein the administered PFKFB3 inhibitor is BrAcNHEtOP (N-bromoacetylethanolamine phosphate), PFK15 (1-(4-pyridinyl)-3-(2-quinolinyl)-2-propen-1-one), or PFK-158 ((E)-1-(4-pyridinyl)-3-[7-(trifluoromethyl)-2-quinolinyl]-2-propen-1-one), or a salt thereof.

[0107] The method according to any one of

[92] to

[0105] , wherein the PFKFB3 inhibitor administered is (a) KAN0436151 or KAN0436067, or a salt thereof; (b) having the structure of formula 1 to formula 53 or formula 54, PQP, N4A, YN1, PK15, PFK-158, YZ29, compound 26, KAN0436151, KAN0436067, or BrAcNHErOP, or a salt thereof, as shown in Figure 1A to Figure 1C or Figure 1D; (c) having the structure of formula AZ44 to formula AZ70 or formula AZ71, or a salt thereof, as shown in Figure 1E; or (d) AZ67 or a salt thereof.

[0108] The method according to any one of

[92] to

[0107] , wherein the HIF1-α pathway inhibitor and the PFKFB3 inhibitor are co-administered to the subject.

[0109] The method according to any one of

[92] to

[0108] , wherein the administration of the HIF1-α pathway inhibitor and / or the PFKFB3 inhibitor is oral, parenteral, orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal, intranasal, intratumoral, or intravenous.

[0110] The method according to any one of

[92] to

[0109] , wherein the HIF1-α pathway inhibitor and the PFKFB3 inhibitor are administered before the onset of one or more symptoms of hepatocellular carcinoma.

[0111] The method according to any one of

[92] to

[0110] , wherein treating hepatocellular carcinoma includes delaying the onset of hepatocellular carcinoma.

[0112] The method according to any one of

[92] to

[0111] , wherein the HIF1-α pathway inhibitor and the PFKFB3 inhibitor are administered after the onset of one or more symptoms of hepatocellular carcinoma.

[0113] A method according to any one of

[92] to

[0112] , wherein the method results in the alleviation of one or more symptoms of hepatocellular carcinoma in a subject administered a HIF1-α pathway inhibitor and a PFKFB3 inhibitor, compared to the subject before treatment.

[0114] One or more alleviated symptoms of hepatocellular carcinoma (a) weight gain, increased appetite, decreased fever, decreased nausea, decreased fatigue, decreased upper abdominal pain, decreased swelling of the abdomen and lower extremities, decreased yellowing of the skin (jaundice), or decreased subcutaneous bleeding, (b) a decrease in at least one biomarker that is typically elevated in a subject with HCC (e.g., EpCam, VEGF, EGFR, FLT1, theophylline, HCC-22-5, KRT23, AHSG, FTL, C16Cer, C16DHC, C18DHC, S1P, C24DHC, C24:1DHC, C18Cer, C20Cer, C24Cer, C24:1Cer, sphingosine, and SA1P, CTSD, HYOU1, PSAP, and LAMP-2); or (c) a reduction in tumor size, an inhibition or slowing of tumor growth, no formation of new tumors, a reduction in the formation of new tumors, an increase in survival or progression-free survival, no metastasis, an increase in treatment options, or a delay in the time from surgery to recurrence. The method according to

[0113] , wherein

[0115] The method according to

[0113] or

[0114] , wherein at least one of the following symptoms: weight loss, loss of appetite, fever, nausea, fatigue, upper abdominal pain, swelling of the abdomen and lower limbs, yellowing of the skin (jaundice), subcutaneous bleeding or tendency to bleed, and liver failure is improved in the subject compared to before treatment with the HIF1-α pathway inhibitor and the PFKFB3 inhibitor.

[0116] The method of any one of

[0113] to

[0115] , wherein at least one, two, three, four or five biomarkers selected from EpCam, VEGF, EGFR, FLT1, theophylline, HCC-22-5, KRT23, AHSG, FTL, C16Cer, C16DHC, C18DHC, S1P, C24DHC, C24:1DHC, C18Cer, C20Cer, C24Cer, C24:1Cer, sphingosine, and SA1P, CTSD, HYOU1, PSAP, and LAMP-2 are reduced by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% compared to the subject prior to treatment with the HIF1-α pathway inhibitor and PFKFB3.

[0117] The method of any one of

[0113] to

[0116] , wherein the subject's tumor size is reduced by at least 20%, at least 30%, at least 40%, or at least 50% compared to the subject before treatment with a HIF1-α pathway inhibitor and a PFKFB3 inhibitor.

[0118] The method according to any one of

[92] to

[0117] , further comprising administering an additional therapeutic agent to the subject.

[0119] The method according to any one of [1] to

[0118] , further comprising administering a GPR81 inhibitor to a subject. [Brief description of the drawings]

[0013] [Figure 1A] 1 shows exemplary PFKFB3 small molecule inhibitors. [Figure 1B] 1 shows exemplary PFKFB3 small molecule inhibitors. [Figure 1C] 1 shows exemplary PFKFB3 small molecule inhibitors. [Figure 1D] 1 shows exemplary PFKFB3 small molecule inhibitors. [Figure 1E] 1 shows exemplary PFKFB3 small molecule inhibitors. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs.Methods and materials similar or equivalent to those described herein can be used in the practice or testing of the compositions provided, but suitable methods and materials are described below.Each publication, patent application, patent, and other references mentioned herein are incorporated herein by reference in their entirety.In case of conflict, the present specification, including definitions, will take precedence.In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.

[0015] Other features and advantages of the disclosed methods and compositions will become apparent from the following disclosure, the drawings, and the claims.

[0016] Whenever embodiments are described herein using the word "comprising," it should be understood that other similar embodiments described in terms of "containing," "consisting of," and / or "consisting essentially of" are also provided. However, when used as transitional phrases within the claims, each must be interpreted separately and within the appropriate legal and factual context (e.g., within the claims, the transitional phrase "comprising" is considered more open-ended, "consisting of" is more exclusive, and "consisting essentially of" is intermediate).

[0017] As used herein, the singular forms "a", "an" and "the" include the plural unless expressly stated or clearly evident from the context that such is not the intention. The singular forms "a", "an" and "the" also include the statistical average composition, characteristics or size of particles within a particle population (e.g., average polyethylene glycol molecular weight, average liposome diameter, average liposome zeta potential). The average particle size and zeta potential of liposomes in a pharmaceutical composition can be routinely measured using methods known in the art, such as dynamic light scattering. The average amount of therapeutic agent in a nanoparticle composition can be routinely measured, for example, using absorption spectroscopy (e.g., ultraviolet-visible spectroscopy).

[0018] As used herein, the term "approximately" or "about" as applied to one or more values ​​of interest refers to a value similar to the stated reference value. In certain embodiments, the term "approximately" or "about" refers to a range of values ​​that are included within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% in either direction (above or below) of the stated reference value, unless otherwise stated or otherwise clear from the context (except where such number may exceed 100% of the possible values). For example, when used in the context of the amount of a given compound in the lipid component of a nanoparticle composition, "about" can mean + / - 10% of the stated value. For example, a nanoparticle composition that includes a lipid component having about 40% of a given compound can contain 30-50% of that compound.

[0019] The term "and / or," when used herein in phrases such as "A and / or B," is intended to include both A and B, A or B, A (alone), and B (alone). Similarly, the term "and / or," when used in phrases such as "A, B, and / or C," is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0020] The recitation of ranges of values ​​herein, unless otherwise indicated herein, is intended to serve merely as a shorthand method of referring individually to each separate value falling within the range, and each separate value is incorporated herein as if it were individually recited herein.

[0021] When embodiments of the present disclosure are described in terms of a Markush group or other alternative grouping, the compositions or methods of the present disclosure not only encompass the entire group recited as a whole, but also encompass each member of the group individually, and also encompass all possible subgroups of the main group, and also encompass the main group in the absence of one or more of the group members. The methods and compositions of the present disclosure also contemplate the explicit exclusion of any one or more of the group members in the compositions or methods of the present disclosure.

[0022] As used herein, terms such as "antibody" and "antigen-binding antibody fragment" include any protein- or peptide-containing molecule that contains at least a portion of an immunoglobulin molecule, such as, but not limited to, at least one complementarity determining region (CDR) of a heavy or light chain, or an antigen-binding portion thereof.

[0023] The term "antibody" also includes fragments, specified portions and variants thereof, including antibody mimetics or portions of antibodies that mimic the structure and / or function of antibodies or specified fragments or portions thereof, including single chain antibodies, single binding domain antibodies and antigen-binding antibody fragments.

[0024] The term "antibody fragment" refers to a portion of an intact antibody, typically the antigen-binding or variable region of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, single chain (scFv) and Fv fragments, diabodies; linear antibodies; single chain antibody molecules; single Fab arm "one-arm" antibodies, and multispecific antibodies formed from antibody fragments. Antibody fragments include any protein or peptide-containing molecule that includes at least a portion of an immunoglobulin molecule, such as, but not limited to, at least one complementarity determining region (CDR) or ligand-binding portion thereof of a heavy or light chain, a heavy or light chain variable region, a heavy or light chain constant region, a framework region or any portion thereof, or at least a portion of an antigen or antigen receptor or binding protein, which can be incorporated into the antibodies provided herein.

[0025] Antibody fragments can be produced by enzymatic cleavage, synthetically, or recombinantly, as known in the art. Antibodies can also be produced in a variety of truncated forms using antibody genes in which one or more stop codons have been introduced upstream of the natural stop site. For example, a combined gene encoding a F(ab')2 heavy chain portion can be designed to include DNA sequences encoding the CH1 domain and / or hinge region of the heavy chain. The various portions of the antibody can be joined together chemically by conventional techniques, or prepared as a contiguous protein using genetic engineering techniques.

[0026] The terms "nucleic acid" or "oligonucleotide" are used interchangeably herein and refer to at least two nucleotides covalently linked together. In some embodiments, the HIF1-α pathway inhibitor and / or PFKFB3 inhibitor administered according to the provided methods is a therapeutic nucleic acid. In some embodiments, the nucleic acid administered is ENMD-1198, shRNA, dicer substrate (e.g., dsRNA), miRNA, anti-miRNA, antisense molecule, decoy, or aptamer, or a plasmid expressing ENMD-1198, shRNA, dicer substrate, miRNA, anti-miRNA, antisense molecule, decoy, or aptamer.

[0027] Nucleic acids administered according to the provided methods are preferably single-stranded or double-stranded and generally contain phosphodiester bonds, although in some cases nucleic acid / oligonucleotide analogs include those with alternative backbones, including, for example, phosphoramide, phosphorothioate, phosphorodithioate, O-methyl phosphoramidite bonds, as well as peptide nucleic acid backbones and bonds. Other analog nucleic acids / oligonucleotides include those with cationic backbones, non-ionic backbones, and non-ribose backbones. Nucleic acids / oligonucleotides containing one or more carbocyclic sugars are also included in the definition of nucleic acids and oligonucleotides. These modifications of the ribose-phosphate backbone can be made, for example, to facilitate the addition of additional moieties, such as labels, or to increase the stability and half-life of such molecules in physiological environments. The nucleic acid / oligonucleotide backbones of the oligonucleotides used according to the provided methods can range from about 5 nucleotides to about 750 nucleotides. Preferred nucleic acid / oligonucleotide backbones range in length from about 5 nucleotides to about 500 nucleotides, preferably from about 10 nucleotides to about 100 nucleotides.

[0028] The oligonucleotides administered according to the provided methods are polymeric structures of nucleoside and / or nucleotide monomers that can specifically hybridize to at least some region of a nucleic acid target. As described above, the "nucleic acids" and "oligonucleotides" used according to the provided methods include, but are not limited to, compounds that contain naturally occurring bases, sugars and intersugar (backbone) linkages, non-naturally occurring modified monomers that function similarly to their naturally occurring counterparts, or portions thereof (e.g., oligonucleotide analogs or mimetics), and combinations of these naturally occurring and non-naturally occurring monomers. As used herein, the term "modified" or "modification" includes any substitution and / or any change from a starting or natural oligomeric compound such as a nucleic acid. Modifications to nucleic acids include substitutions or changes to internucleoside linkages, sugar moieties, or base moieties, such as those described herein and otherwise known in the art.

[0029] As used herein, "small molecule" refers to an organic compound that is synthesized by conventional organic chemistry methods (e.g., in a laboratory) or found in nature. Typically, small molecules are characterized by containing several carbon-carbon bonds and having a molecular weight of less than about 1500 grams / mole. In certain embodiments, small molecules are less than about 1000 grams / mole. In certain embodiments, small molecules are less than about 550 grams / mole. In certain embodiments, small molecules are between about 200 and about 550 grams / mole. In certain embodiments, small molecules do not include peptides (e.g., compounds that include two or more amino acids joined by peptidyl bonds). In certain embodiments, small molecules do not include nucleic acids.

[0030] The terms "condition" and "disease" are used interchangeably herein and refer to any condition or disorder that damages, disrupts, or dysregulates the normal function of a cell, tissue, or organ.

[0031] "Fibrosis" is a pathological condition in which fibrous connective tissue invades any organ (eg, the pancreas and liver), usually as a result of inflammation or other injury.

[0032] As used herein, the term "pancreatic cancer" refers to a disease recognized in the art, and includes cancer originating from tissues including pancreas. In various embodiments, pancreatic cancer is exocrine pancreatic cancer, pancreatic cystic tumor, or pancreatic endocrine tumor. A subject who is "afflicted with pancreatic cancer" or "diagnosed with pancreatic cancer" is a subject who has been clinically diagnosed with such cancer by a qualified clinician, or who shows one or more signs or symptoms of such cancer (e.g., decreased levels of pancreatic cancer biomarkers in gastrointestinal lavage or fecal matter) and is then clinically diagnosed with cancer by a qualified physician.

[0033] As used herein, the term "hepatocellular carcinoma" refers to a disease recognized in the art, including cancer originating from tissues including the liver. A subject "suffering from hepatocellular carcinoma" or "diagnosed with hepatocellular carcinoma" is a subject who has been clinically diagnosed with such cancer by a qualified clinician, or who shows one or more signs or symptoms of such cancer (e.g., a decreased level of hepatocellular carcinoma biomarkers) and is subsequently clinically diagnosed with cancer by a qualified physician.

[0034] As used herein, the term pancreatic or hepatic cancer "biomarker" refers to a protein or non-proteinaceous substance that is differentially present in biological samples (e.g., gastrointestinal lavage fluid or fecal material) of subjects with pancreatic or hepatic cancer compared to subjects without pancreatic or hepatic cancer.In certain embodiments, pancreatic or hepatic cancer biomarkers are proteins that originate from the pancreas or liver, respectively.In other embodiments, pancreatic or hepatic cancer biomarkers are proteins that originate from non-pancreatic or non-hepatic sources (e.g., gastrointestinal tract, e.g., intestinal tract), respectively.

[0035] As used herein, the "level" of a pancreatic or hepatic cancer biomarker refers to the level of the pancreatic or hepatic cancer biomarker in a biological sample, such as serum, digestive tract washings, or fecal material, determined using a method for measuring the level of a protein or non-proteinaceous substance. Such methods include, for example, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), superdiffusion chromatography, fluid or gel precipitation reaction, absorption spectroscopy, colorimetric assay, spectrophotometric assay, flow cytometry, immunodiffusion (single or double), liquid phase assay, immunoelectrophoresis, Western blotting, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay, and electrochemiluminescence immunoassay. In a preferred embodiment, the level is determined using an ELISA-based assay.

[0036] As used herein, the term "sample" refers to similar fluids, cells or tissues isolated from a subject, as well as collections of fluids, cells or tissues present within a subject. In a preferred embodiment, the sample is a biological fluid containing a pancreatic or liver cancer biomarker. Examples of biological fluids include gastrointestinal washings, fecal material, blood, serum and serous fluid, plasma, semen, pancreatic juice, bile, lymphatic fluid, urine, cerebrospinal fluid, saliva, ocular fluid, cyst fluid, tears, sputum, mucosal secretions of secretory tissues and organs, vaginal secretions, gynecological fluids, ascites associated with non-solid tumors; fluids of the pleura, pericardium, peritoneum, abdominal cavity and other body cavities; fluids collected by bronchial lavage, etc. In a particular embodiment, the biological sample is serum, gastrointestinal washings or fecal material.

[0037] As used herein, the term "pancreatic disease progression" or "liver disease progression" refers to the gradual worsening of the disease over time, whereby symptoms and neurochemical deficits become increasingly debilitating and / or severe.

[0038] As used herein, the terms "inhibiting the progression of pancreatic disease" or "inhibiting the progression of liver disease" refer to slowing and / or halting the progression of symptoms of pancreatic disease or liver disease, respectively.

[0039] As used herein, "delaying the onset" of a pancreatic disease, such as pancreatic steatosis or pancreatic cancer, or a liver disease, such as non-alcoholic fatty liver (NAFL), non-alcoholic steatohepatitis (NASH) or liver cancer, means delaying, preventing, slowing, inhibiting, stabilizing, and / or postponing the onset of one or more symptoms of the disease, including slowing the rate at which the patient's disease progresses (e.g., to transition the patient from a rapidly progressing disease to a more slowly progressing disease). This delay can be of different lengths of time depending on the medical and / or medical history of the individual being treated. As will be apparent to those skilled in the art, a sufficient or significant delay can, in effect, encompass prevention in that the individual does not develop detectable disease. A method that "delays" the onset of a disease is one that reduces the extent of the disease within a given time frame compared to when the method is not used. Such comparisons are typically based on clinical studies using a statistically significant number of subjects, although this knowledge can be based on anecdotal evidence. "Delayed onset" can mean that the severity of clinical symptoms and / or undesirable clinical symptoms are reduced and / or the time course of progression is slowed or prolonged compared to when the agent is not administered. Thus, the term also includes, but is not limited to, alleviation of symptoms, whether detectable or undetectable, reduction in the extent of disease, stabilization of disease pathology (i.e., not worsening), delay or slowing of disease progression, and remission (whether partial or complete).

[0040] As used herein, an "effective amount" refers to a dose of an agent sufficient to provide a medically desirable prophylactic and / or therapeutic effect against a pancreatic disease (e.g., pancreatic cancer, such as pancreatic steatosis and pancreatic ductal adrenal carcinoma (PDAC)) or liver disease (e.g., liver cancer, such as NAFL, NASH, and hepatocellular carcinoma (HCC)). The effective amount will vary depending on the desired outcome, the particular pancreatic or liver disease being treated (or prevented), the age and physical condition of the subject being treated, the severity of the condition, the duration of treatment, the nature of concurrent or combined treatment (if any), the particular route of administration, and similar factors within the knowledge and skill of the medical practitioner. An "effective amount" can be determined empirically and in a routine manner in relation to the stated purpose. Prophylactic and / or therapeutic effects associated with cancer include, but are not limited to, a reduction in tumor size, inhibition or reduction in tumor growth, no formation of new tumors, a reduction in new tumor formation, an increase in survival or progression-free survival, no metastasis, an increase in treatment options, a reduction in biomarkers typically elevated in the cancer, and a delay in the time from surgery to recurrence.

[0041] The terms "subject," "patient," "individual," and "animal" are used interchangeably and refer to mammals, such as human patients and non-human primates, as well as laboratory animals, such as rabbits, rats, and mice, and other laboratory animals. Animals include all vertebrates, e.g., mammals and non-mammals, such as chickens, amphibians, and reptiles. As used herein, "mammal" refers to any member of the class Mammalia, including, but not limited to, humans and non-human primates, such as chimpanzees and other ape and monkey species; farm animals, such as cows, sheep, pigs, goats, and horses; domesticated mammals, such as dogs and cats; laboratory animals, including rodents, such as mice, rats, and guinea pigs; and other members of the class Mammalia known in the art. In certain embodiments, the patient is a human.

[0042] The terms "treating" or "treatment," "treat," or "therapy" refer to both (a) therapeutic measures that cure, slow, attenuate, alleviate the symptoms of, and / or halt the progression of, a pathological condition, and (b) prophylactic or preventative measures that prevent and / or delay the onset of the targeted condition and / or its associated symptoms.

[0043] Thus, subjects in need of treatment include those already suffering from pancreatic and / or liver disease, those at risk of suffering from pancreatic and / or liver disease, and those for whom it is desired to prevent pancreatic and / or liver disease. A subject may be routinely identified as "having or at risk of having" pancreatic and / or liver disease, or another condition referred to herein, using medical and diagnostic techniques known in the art. In certain embodiments, a subject is successfully "treated" according to the methods provided when the subject exhibits, for example, a total, partial, or transient remission or disappearance of at least one symptom associated with the condition.

[0044] In other embodiments, the terms "treat" or "treatment", "treating" or "therapy" refer to inhibiting the progression of pancreatic and / or liver disease either physically (e.g., stabilization of a discernible symptom), physiologically (e.g., stabilization of a physical parameter), or both. In other embodiments, the terms "treat" or "treatment", "treating" or "therapy" refer to reducing symptom relief, reducing inflammation, and restoring cellular function. Treatment can be performed using the HIF1-α pathway inhibitor and PFKFB3 inhibitor compositions disclosed herein or in further combination with one or more additional therapeutic agents.

[0045] The term "pharmaceutical acceptable carrier" refers to an ingredient in a pharmaceutical preparation other than active ingredient that is non-toxic to subjects. Pharmaceutically acceptable carriers include, but are not limited to, buffers, carriers, excipients, stabilizers, diluents or preservatives. Pharmaceutically acceptable carriers can include, for example, one or more compatible solid or liquid fillers, diluents or encapsulating materials suitable for administration to humans or other subjects.

[0046] The "therapeutic agent(s)" used in accordance with the disclosed methods and compositions can further include any agent for treating a condition of a subject. "Therapeutic agent" also refers to the salt, acid, and free base forms of the aforementioned agents.

[0047] PFKFB3 inhibitors PFKFB3 (6-phosphofructo-2-kinase-fructose-2,6-bisphosphatase 3) is a bifunctional protein involved in both the synthesis and degradation of fructose-2,6-bisphosphate, a regulatory molecule that controls eukaryotic glycolysis and is required for cell cycle progression and prevention of apoptosis.

[0048] In some embodiments, the present disclosure provides a method of treating a pancreatic or hepatic disease in a subject in need thereof, comprising: (a) administering to a subject an effective amount of a HIF1-α pathway inhibitor or a HIF1-α inhibitor and an effective amount of a PFKFB3 inhibitor; (b) administering to the subject an effective amount of a HIF1-α pathway inhibitor or a HIF1-α inhibitor, wherein the subject has previously been administered a PFKFB3 inhibitor; or (c) administering to the subject an effective amount of a PFKFB3 inhibitor, wherein the subject has previously been administered a HIF1-α pathway inhibitor or a HIF1-α inhibitor; The method provides said wherein the PFKFB3 inhibitor does not inhibit the PI3K / AKT / mTOR pathway or HIF1-α.

[0049] The PFKFB3 inhibitor that can be used according to the provided method is not particularly limited.In some embodiments, the PFKFB3 inhibitor administered is an antibody or PFKFB3 binding antibody (e.g., single chain antibody, single domain antibody, Fab fragment, F(ab')2 fragment, Fd fragment, Fv fragment, scFv, dAb fragment, or another engineered molecule, such as diabody, triabody, tetrabody, minibody, and minimal recognition unit), a nucleic acid molecule (e.g., aptamer, antisense molecule, ribozyme, dicer substrate, miRNA, dsRNA, ssRNA, and shRNA), a peptibody, a nanobody, a PFKFB3 inhibitory binding polypeptide, or a small molecule PFKFB3 inhibitor.

[0050] In some embodiments, the PFKFB3 inhibitor administered according to the provided methods has a concentration of PFKFB3 activity with an IC50 of 100 μM or less for PFKFB3 activity / function. In some embodiments, the PFKFB3 inhibitor has an IC50 of at least or up to about 200, 100, 80, 50, 40, 20, 10, 5 or 1 μM, or at least or up to about 100, 10 or 1 nM or less (or any range or value derivable therein). In some embodiments, the PFKFB3 inhibitor inhibits the expression of PFKFB3. Assays for determining the ability of a compound to inhibit PFKFB3 activity are known in the art. In some embodiments, the inhibition of PFKFB3 activity or expression is a decrease compared to a control level or sample. In some embodiments, a functional assay such as an MTT assay, a cell proliferation assay, a BRDU or Ki67 immunofluorescence assay, an apoptosis assay or a glycolysis assay is used to assay the ability of the composition to inhibit PFKFB3 activity.

[0051] In some embodiments, the PFKFB3 inhibitor administered according to the provided methods is an antibody or a PFKFB3-binding antibody fragment (e.g., a single chain antibody, a single domain antibody, a Fab fragment, a F(ab')2 fragment, a Fd fragment, a Fv fragment, a scFv, a dAb fragment, or another engineered molecule such as a diabody, a triabody, a tetrabody, a minibody, and a minimal recognition unit). In certain embodiments, the PFKFB3 inhibitor administered is a nanobody (e.g., a VHH).

[0052] In some embodiments, the HIF1-A inhibitor administered according to the provided methods is a therapeutic nucleic acid. In some embodiments, the therapeutic nucleic acid is an aptamer, an antisense molecule, a ribozyme, a dicer substrate, miRNA, dsRNA, ssRNA, and shRNA. In certain embodiments, the HIF1-α inhibitor administered according to the provided methods is an siRNA or an antisense oligonucleotide. In one embodiment, the PFKFB3 inhibitor administered is EZN-4178.

[0053] Representative examples of human PFKFB3 coding sequences are provided in GenBank Accession Nos. NM_004566.3, NM_001145443.2, NP_001138915.1, NM_001282630.2, NM_001314063.1, NM_001323016.1, NM_001323017.1, and NM_001363545.2. The sequences associated with each of these Genbank Accession Nos. are incorporated herein by reference in their entirety for all purposes. Therapeutic nucleic acids that inhibit PFKFB3 activity can be routinely designed and prepared based on each of the above human PFKFB3 transcript sequences using methods known in the art.

[0054] Certain embodiments of the provided methods contemplate administration of a PFKFB3 inhibitory nucleic acid or any method of inhibiting gene expression of PFKFB3 known in the art. Examples of inhibitory nucleic acids include, but are not limited to, antisense nucleic acids, such as small interfering RNA (SiRNA), small hairpin RNA (shRNA), double-stranded RNA, and any other antisense oligonucleotides. Also included are ribozymes or nucleic acids encoding any of the inhibitors described herein. The inhibitory nucleic acid may inhibit transcription of PFKFB3 in cells or prevent translation of PFKFB3 gene transcripts. In some embodiments, the PFKFB3 inhibitory nucleic acid administered according to the provided methods is between 16 and 1000 nucleotides in length. In certain embodiments, the PFKFB3 inhibitory nucleic acid administered is between 18 and 100 nucleotides in length. In certain embodiments, the PFKFB3 inhibitory nucleic acid administered is at least or up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 40, 50, 60, 70, 80, 90 nucleotides, or any range derivable therein.

[0055] In some embodiments, the PFKFB3 inhibitory nucleic acid administered according to the provided methods can reduce expression of PFKFB3 by at least 10%, 20%, 30% or 40%, more particularly at least 50%, 60%, or 70%, most particularly at least 75%, 80%, 90%, 95% or more, or any range or value therebetween.

[0056] In some embodiments, the PFKFB3 inhibitory nucleic acid administered in accordance with the provided methods is 17-25 nucleotides in length and comprises a 5' to 3' sequence that is at least 90% complementary to the 5' to 3' sequence of mature PFKFB3 mRNA (e.g., a sequence disclosed in any one or more of GenBank Accession Nos. NM_004566.3, NM_001145443.2, NM_001282630.2, NM_001314063.1, NM_001323016.1, NM_001323017.1, and NM_001363545.2). In some embodiments, the PFKFB3 inhibitory nucleic acid administered is 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, or any range derivable therein. In some embodiments, the PFKFB3 inhibitory nucleic acid administered has a sequence (5' to 3') that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9 or 100% complementary to the corresponding 5' to 3' sequence of mature PFKFB3 mRNA (e.g., a sequence disclosed in any one or more of GenBank Accession Nos. NM_004566.3, NM_001145443.2, NM_001282630.2, NM_001314063.1, NM_001323016.1, NM_001323017.1, and NM_001363545.2), or any range derivable therein. One skilled in the art could use the portion of the probe sequence that is complementary to the sequence of the mature mRNA as the sequence of the mRNA inhibitor, and furthermore, that portion of the probe sequence could be altered so that it is still 90% complementary to the sequence of the mature mRNA.

[0057] In some embodiments, the PFKFB3 inhibitory nucleic acid administered according to the provided methods is a miRNA. In further embodiments, the administered miRNA is hsa-mir-26b-5p (MIRT028775), hsa-mir-330-3p (MIRT043840), hsa-mir-6779-5p (MIRT454747), hsa-mir-6780a-5p (MIRT454748), hsa-mir-3689c (MIRT454749), hsa-mir-3689b-3p (MIRT454749), hsa-mir-3689c-3p ...c-3p (MIRT454749), hsa-mir-3689b-3p (MIRT454749), hsa-mir-3689c-3p (MIRT454749), hsa-mir-3689c-3p (MIRT454749), hsa-mir-3689c-3p (MIRT454749), hsa-mir-3689c-3p (MIRT454749), hsa-mir-3689c-3p (MIRT454749), hsa-mir-3689c-3p (MIRT454749), hsa p(MIRT454750), hsa-mir-3689a-3p(MIRT454751), hsa-mir-30b-3p(MIRT454752), hsa-mir-1273h-5p( MIRT454753), hsa-mir-6778-5p(MIRT454754), hsa-mir-1233-5p(MIRT454755), hsa-mir-6799-5p(MIRT 454756), hsa-mir-7106-5p(MIRT454757), hsa-mir-6775-3p(MIRT454758), hsa-mir-1291(MIRT454759 ), hsa-mir-765(MIRT454760), hsa-mir-423-5p(MIRT454761), hsa-mir-3184-5p(MIRT454762), hsa-mi A member selected from r-6856-5p (MIRT454763), hsa-mir-6758-5p (MIRT454764), hsa-mir-3185 (MIRT527973), hsa-mir-6892-3p (MIRT527974), hsa-mir-6840-5p (MIRT527975), and hsa-mir-6865-3p (MIRT527976).

[0058] In some embodiments, the PFKFB3 inhibitor administered according to the provided method is a small molecule.The small molecule PFKFB3 inhibitor administered can be any small molecule that is determined to inhibit the function or activity of PFKFB3.Such small molecules can be determined based on in vitro or in vivo functional assays.

[0059] In some embodiments, the PFKFB3 inhibitor small molecule administered according to the provided methods is a small molecule PFKFB3 inhibitor molecule disclosed in U.S. Patent Publication Nos. 20130059879, 20120177749, 20100267815, 20100267815, and 20090074884, the disclosures of each of which are incorporated by reference in their entireties herein.

[0060] In some embodiments, the PFKFB3 inhibitor administered in accordance with the provided methods is (1H-benzo[g]indol-2-yl)-phenyl-methanone, (3H-benzo[e]indol-2-yl)-phenyl-methanone, (3H-benzo[e]indol-2-yl)-(4-methoxy-phenyl)-methanone, (3H-benzo[e]indol-2-yl)-pyridin-4-yl-methanone, the HCl salt of (3H-benzo[e]indol-2-yl)-pyridin-4-yl-methanone, (3H-benzo[e]indol-2-yl)-pyridin-4-yl-methanone, (3H-benzo[e]indol-2-yl) -(3-Methoxy-phenyl)-methanone, (3H-benzo[e]indol-2-yl)-pyridin-3-yl-methanone, (3H-benzo[e]indol-2-yl)-(2-methoxy-phenyl)-methanone, (3H-benzo[e]indol-2-yl)-(2-hydroxy-phenyl)-methanone, (3H-benzo[e]indol-2-yl)-(4-hydroxy-phenyl)-methanone, (5-methyl-3H-benzo[e]indol-2-yl)-phenyl-methanone, phenyl-(7H-pyrrolo[2,3-h]quinoline-8 -yl)-methanone, (3H-benzo[e]indol-2-yl)-(3-hydroxy-phenyl)-methanone, (3H-benzo[e]indol-2-yl)-(2-chloro-pyridin-4-yl)-methanone, (3H-benzo[e]indol-2-yl)-(1-oxy-pyridin-4-yl)-methanone, phenyl-(6,7,8,9-tetrahydro-3H-benzo[e]indol-2-yl)-methanone, (3H-benzo[e]indol-2-yl)-(4-hydroxy-3-methoxyrutheny)-methanone, (3H-benzo[e]indol-2-yl)-(4-hydroxy-3-methoxyrutheny)-methanone, e]indol-2-yl)-(4-benzyloxy-3-methoxy-phenyl)-methanone, 4-(3H-benzo[e]indole-2-carbonyl)-benzoic acid methyl ester, 4-(3H-benzo[e]indole-2-carbonyl)-benzoic acid, (4-amino-phenyl)-(3H-benzo[e]indol-2-yl)-methanone, 5-(3H-benzo[e]indole-2-carbonyl)-2-benzyloxy-benzoic acid methyl ester, 5-(3H-benzo[e]indole-2-carbonyl)-2-benzyloxy-benzoic acid methanone,(3H-benzo[e]indol-2-yl)-(2-methoxy-pyridin-4-yl)-methanone, (5-fluoro-3H-benzo[e]indol-2-yl)-(3-methoxy-phenyl)-methanone, (5-fluoro-3H-benzo[e]indol-2-yl)-pyridin-4-yl-methanone, (4-benzyloxy-3-methoxy-phenyl)-(5-fluoro-3H-benzo[e]indol-2-yl)-methanone, (5-fluoro-3H-benzo[e]indol-2-yl)-(4-hydroxy-3-methoxy- phenyl)-methanone, (3H-benzo[e]indol-2-yl)-(3-hydroxymethyl-phenyl)-methanone, cyclohexyl-(5-fluoro-3H-benzo[e]indol-2-yl)-methanone, (5-fluoro-3H-benzo[e]indol-2-yl)-(3-fluoro-4-hydroxy-phenyl)-methanone, (3H-benzo[e]indol-2-yl)-p-tolyl-methanone, (3H-benzo[e]indol-2-yl)-(3-methoxy-phenyl-methanol, (3H-benzo[e]indol (4-amino-3-methoxy-phenyl)-(3H-benzo[e]indol-2-yl)-pyridin-4-yl-methanol, 3H-benzo[e]indole-2-carboxylic acid phenylamide, 3H-benzo[e]indole-2-carboxylic acid (3-methoxy-phenyl)-amide, (3H-benzo[e]indol-2-yl)-(4-dimethylamino-phenyl)-methanone, (4-amino-3-methoxy-phenyl)-(3H-benzo[e]indol-2-yl)-methanone, (4-amino-3-methoxy-phenyl)-(5-hydroxy-3H-benzo[e]indol-2-yl)-methanone, (4-amino-3-methoxy-phenyl)-(5-methoxy-3H-benzo[e]indol-2-yl)-methanone, N-[4-(3H-benzo[e]indole-2-carbonyl)-phenyl]-methanesulfonamide, 3H-benzo[e]indole-2-carboxylic acid (4-amino-phenyl)-amide, (4-amino-phenyl)-(5-methoxy-3H-benzo[e]indol-2-yl)-methanone, (4-amino-2-fluoro-phenyl)-(5-methoxy-3H-benzo[e]indol-2-yl)-methanone,(4-amino-3-fluoro-phenyl)-(5-methoxy-3H-benzo[e]indol-2-yl)-methanone, (4-amino-2-methoxy-phenyl)-(5-methoxy-3H-benzo[e]indol-2-yl)-methanone, (4-amino-phenyl)-(9-methoxy-3H-benzo[e]indol-2-yl)-methanone, (4-amino-3-methoxy-phenyl)-(9-methoxy-3H-benzo[e]indol-2-yl)-methanone Methanone, (4-amino-2-methoxy-phenyl)-(9-methoxy-3H-benzo[e]indol-2-yl)-methanone, (4-amino-3-fluoro-phenyl)-(9-methoxy-3H-benzo[e]indol-2-yl)-methanone, (4-amino-2-fluoro-phenyl)-(9-methoxy-3H-benzo[e]indol-2-yl)-methanone, (4-amino-3-fluoro-phenyl)-(3H-benzo[e]indol-2-yl )-methanone, (4-amino-2-fluoro-phenyl)-(3H-benzo[e]indol-2-yl)-methanone, (4-amino-phenyl)-(7-methoxy-3H-benzo[e]indol-2-yl)-methanone, (4-amino-phenyl)-(5-hydroxy-3-methyl-3H-benzo[e]indol-2-yl)-methanone, (7-amino-5-fluoro-9-hydroxy-3H-benzo[e]indol-2-yl)-(3-methyl- pyridin-4-yl)-methanone, (5-amino-3H-pyrrolo[3,2-f]isoquinolin-2-yl)-(3-methoxy-pyridin-4-yl)-methanone, (4-amino-2-methyl-phenyl)-(9-hydroxy-3H-pyrrolo[2,3-c]quinolin-2-yl)-methanone, and (4-amino-phenyl)-(7-methanesulfonyl-3H-benzo[e]indol-2-yl)-methanone, or at least one of their salts.

[0061] In some embodiments, the PFKFB3 inhibitor administered in accordance with the provided methods is 1-pyridin-4-yl-3-quinolin-4-yl-propenone, 1-pyridin-4-yl-3-quinolin-3-yl-propenone, 1-pyridin-3-yl-3-quinolin-2-yl-propenone, 1-pyridin-3-yl-3-quinolin-4 ... 3-yl-propenone, 1-naphthalen-2-yl-3-quinolin-2-yl-propenone, 1-naphthalen-2-yl-3-quinolin-3-yl-propenone, 1-pyridin-4-yl-3-quinolin-3-yl-propenone, 3-(4-hydroxy-quinolin-2-yl)-1-pyridin-4-yl-propenone, 3-(8-hydroxy-quinolin-2-yl)-1-pyridin-3-yl-propenone 3-quinolin-2-yl-1-p-tolyl-propenone, 3-(8-hydroxy-quinolin-2-yl)-1-pyridin-4-yl-propenone, 3-(8-hydroxy-quinolin-2-yl)-1-p-tolyl-propenone, 3-(4-hydroxy-quinolin-2-yl)-1-p-tolyl-propenone, 1-phenyl-3-quinolin-2-yl-propenone, 1-pyridin-2-yl In one embodiment, the quinolin-2-yl-propenone is at least one of 1-(2-hydroxy-phenyl)-3-quinolin-2-yl-propenone, 1-(4-hydroxy-phenyl)-3-quinolin-2-yl-propenone, 1-(2-amino-phenyl)-3-quinolin-2-yl-propenone, 1-(4-amino-phenyl)-3-quinolin-2-yl-propenone, or a salt thereof.

[0062] In some embodiments, the PFKFB3 inhibitor administered in accordance with the provided methods is 4-(3-quinolin-2-yl-acryloyl)-benzamide, 4-(3-quinolin-2-yl-acryloyl)-benzoic acid, 3-(8-methyl-quinolin-2-yl)-1-pyridin-4-yl-propenone, 1-(2-fluoro-pyridin-4-yl)-3-quinolin-2-yl-propenone, 3-(8-fluoro-quinolin-2-yl)-1-pyridinyl and at least one of 1-methyl-4-[3-(8-methyl-quinolin-2-yl)-acryloyl]-pyridinium, or a salt thereof.

[0063] In some embodiments, the PFKFB3 inhibitor administered according to the provided methods is at least one of PFK15 (1-(4-pyridinyl)-3-(2-quinolinyl)-2-propen-1-one), (2S)-N-[4-[[3-cyano-1-(2-methyl-propyl)-1H-indol-5-yl]oxy]phenyl]-2-pyrrolidine-carboxamide 3PO (3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one), (2S)-N-[4-[[3-cyano-1-[(3,5-dimethyl-4-isoxazolyl)methyl]-1H-indol-5-yl]oxy]phenyl]-2-pyrrolidine-carboxamide, and ethyl 7-hydroxy-2-oxo-2H-1-benzopyran-3-carboxylate, or a salt thereof.

[0064] In certain embodiments, the PFKFB3 inhibitor administered in accordance with the methods provided is PFK15 or a salt thereof.

[0065] In certain embodiments, the PFKFB3 inhibitor administered in accordance with the methods provided is PFK158 ((E)-1-(4-pyridinyl)-3-[7-(trifluoromethyl)-2-quinolinyl]-2-propen-1-one), or a salt thereof.

[0066] In certain embodiments, the PFKFB3 inhibitor administered in accordance with the methods provided is BrAcNHEtOP (N-bromoacetylethanolamine phosphate), or a salt thereof.

[0067] In certain embodiments, the PFKFB3 inhibitor administered in accordance with the methods provided is AZ67, or a salt thereof.

[0068] In some embodiments, the PFKFB3 inhibitor administered according to the provided methods is at least one PFKFB3 inhibitor having the structure of Formula 1-53 or Formula 54, PQP, N4A, YN1, PK15, PFK-158, YZ29, Compound 26, KAN0436151, KAN0436067, or BrAcNHErOP, or a salt thereof, as shown in Figure 1A-1D. In some embodiments, the PFKFB3 inhibitor administered according to the provided methods is at least one PFKFB3 inhibitor having the structure of Formula AZ44-AZ70 or AZ71, or a salt thereof, as shown in Figure 1E.

[0069] In certain embodiments, the PFKFB3 inhibitor administered in accordance with the methods provided is KAN0436151, or a salt thereof.

[0070] In certain embodiments, the PFKFB3 inhibitor administered in accordance with the methods provided is KAN0436067, or a salt thereof.

[0071] HIF1-α pathway inhibitors Hypoxia-inducible factor 1-α (HIF-1-α) is a subunit of the heterodimeric transcription factor hypoxia-inducible factor 1 (HIF-1), which is thought to be the master transcriptional regulator of the cellular and developmental response to hypoxia.

[0072] In some embodiments, the present disclosure provides a method of treating a pancreatic or hepatic disease in a subject in need thereof, comprising: (a) administering to a subject an effective amount of a HIF1-α pathway inhibitor or a HIF1-α inhibitor and an effective amount of a PFKFB3 inhibitor; (b) administering to the subject an effective amount of a HIF1-α pathway inhibitor or a HIF1-α inhibitor, wherein the subject has previously been administered a PFKFB3 inhibitor; or (c) administering to the subject an effective amount of a PFKFB3 inhibitor, wherein the subject has previously been administered a HIF1-α pathway inhibitor or a HIF1-α inhibitor; The method provides said wherein the PFKFB3 inhibitor does not inhibit the PI3K / AKT / mTOR pathway or HIF1-α.

[0073] As used herein, the term "HIF1-α pathway-α inhibitor" refers to a composition that inhibits or reduces HIF1-α directly or indirectly through inhibiting one or more activities of the PI3K / AKT / mTOR pathway upstream of the HIF1-α pathway. The term "HIF1-α inhibitor" is used herein to refer to a composition that directly inhibits or reduces HIF1-α. Thus, for example, mTOR pathway inhibitors such as temsirolimus, everolimus, and sirolimus are considered "HIF1-α pathway-α inhibitors" and not "HIF1-α inhibitors" herein.

[0074] The "HIF1-α pathway-α inhibitor" that can be administered according to the provided method is not particularly limited. In some embodiments, the administered HIF1-α pathway inhibitor is an antibody or HIF1-α binding antibody fragment (e.g., single chain antibody, single domain antibody (e.g., VHH), Fab fragment, F(ab')2 fragment, Fd fragment, Fv fragment, scFv, dAb fragment, or another engineered molecule, e.g., diabody, triabody, tetrabody, minibody, and minimal recognition unit), a nucleic acid molecule (e.g., aptamer, antisense molecule, ribozyme, dicer substrate, ENMD-1198, miRNA, dsRNA, ssRNA, and shRNA), a peptibody, a nanobody, a HIF1-α pathway binding polypeptide, or a small molecule HIF1-α pathway inhibitor.

[0075] In some embodiments, the administered HIF1-α pathway inhibitor administered according to the provided method is at a concentration of HIF1-α activity / function IC50 of 100 μM or less. In some embodiments, the HIF1-α pathway inhibitor has an IC50 of at least or up to about 200, 100, 80, 50, 40, 20, 10, 5 or 1 μM, or at least or up to about 100, 10 or 1 nM or less (or any range or value derivable therein). In some embodiments, the HIF1-α pathway inhibitor inhibits the expression of HIF1-α. Assays for determining the ability of a compound to inhibit HIF1-α activity are known in the art. In some embodiments, the inhibition of HIF1-α activity or expression is a decrease compared to a control level or sample. In some embodiments, a functional assay such as an MTT assay, a cell proliferation assay, a BRDU or Ki67 immunofluorescence assay, an apoptosis assay or a glycolysis assay is used to assay the ability of the composition to inhibit HIF1-α activity.

[0076] The HIF1-α inhibitor that can be administered according to the provided method is not particularly limited. In some embodiments, the HIF1-α inhibitor modulates one or more of HIF-1α mRNA expression, HIF-1α protein translation or degradation, HIF-1α / HIF-1β dimerization, HIF-1α-DNA binding (e.g., HIF-1α / HRE), and / or HIF-1α transcriptional activity (e.g., CH-1 of p300 / C-TAD of HIF-1α).

[0077] In some embodiments, the HIF1-α inhibitor administered according to the provided method is a small molecule.In some embodiments, the HIF1-α inhibitor administered according to the provided method is a protein or polypeptide (e.g., anti-HIF1 antibody or antibody fragment that binds to HIF1).In some embodiments, the HIF1-α inhibitor administered according to the provided method is a therapeutic nucleic acid (e.g., aptamer, antisense molecule, ribozyme, dicer substrate, siRNA, miRNA, dsRNA, ssRNA, or shRNA).

[0078] In some embodiments, the HIF1-α pathway inhibitor administered in accordance with the provided methods is a HIF1-α pathway inhibitor (e.g., a PI3K pathway inhibitor, a MAPK pathway inhibitor, an Akt pathway inhibitor, and / or an mTOR inhibitor); a HIF translation inhibitor (e.g., a topoisomerase inhibitor, a microtubule targeting drug, a cardiac glycoside, or an antisense HIF-1a mRNA); an inhibitor of HIF stability, nuclear localization or dimerization (e.g., acriflavine or an HDAC inhibitor); an inhibitor of HIF transactivation (e.g., a HIF1 coactivator recruitment inhibitor or a HIF1 DNA binding inhibitor).

[0079] In some embodiments, the HIF1-α inhibitor administered according to the provided methods is a HIF1-α pathway inhibitor (e.g., a PI3K pathway inhibitor, a MAPK pathway inhibitor, an Akt pathway inhibitor, and / or an mTOR inhibitor). In some embodiments, the HIF1-α inhibitor administered according to the provided methods is a PI3K pathway inhibitor. In one embodiment, the HIF1-α pathway inhibitor administered is P3155, LY29, LY294002, wortmannin, or GDC-0941. In one embodiment, the HIF1-α pathway inhibitor administered is resveratrol. In another embodiment, the HIF1-α pathway inhibitor administered is glyceollin. In some embodiments, the HIF1-α pathway inhibitor administered according to the provided methods is an mTOR inhibitor. In one embodiment, the HIF1-α pathway inhibitor administered is rapamycin, temsirolimus (CC1-779), everolimus, sirolimus, or PP242.

[0080] In certain embodiments, the HIF1-α inhibitor administered is silibinin.

[0081] In some embodiments, the HIF1-α inhibitor administered according to the provided method is a HIF translation inhibitor.In one embodiment, the HIF1-α inhibitor administered is PX-478 (S-2-amino-3-[4'-N,N-bis(chloroethyl)[amino]phenylpropionic acid N-oxide dihydrochloride), NSC-64421, camptothecin (CPT), SN38, irinotecan, topotecan, NSC-644221, cycloheximide, or apigenin, or their salts. In one embodiment, the HIF1-α inhibitor administered is aminoflavone, KC7F2 (N,N'-(disulfanediylbis(ethane-2,1-diyl))bis(2,5-dichlorobenzenesulfonamide), 2-methoxyestradiol (2ME2), or an analog or salt thereof. In one embodiment, the HIF1-α inhibitor administered is ENMD-1198, ENMD-1200, or ENMD-1237, or a salt thereof. In one embodiment, the HIF1-α inhibitor administered is EZN-2208, or a salt thereof. In one embodiment, the HIF1-α inhibitor administered is EZN-2968, or a salt thereof.

[0082] In a particular embodiment, the HIF1-α inhibitor administered is PX-478 or a salt thereof.

[0083] In some embodiments, the HIF1-α inhibitor administered according to the provided method is a cardiac glycoside. In one embodiment, the cardiac glycoside administered is digoxin or a salt thereof. In another embodiment, the cardiac glycoside administered is ouabain or proscillaridin A or a salt thereof.

[0084] In some embodiments, the HIF1-α pathway inhibitor administered according to the provided method is a topoisomerase inhibitor.In one embodiment, the topoisomerase inhibitor administered is camptothecin (CPT), SN38, irinotecan, or topotecan (e.g., PEG-SN38), or salts thereof.

[0085] In some embodiments, the HIF1-α pathway inhibitor administered according to the provided method is a microtubule targeting drug.In one embodiment, the microtubule targeting drug administered is 2 methoxyestradiol (2ME2), ENMD-1198, ENMD-1200, ENMD-1237, or taxotere, or their salts.

[0086] In some embodiments, the HIF1-α inhibitor administered according to the provided methods is a therapeutic nucleic acid. In some embodiments, the therapeutic nucleic acid is an aptamer, an antisense molecule, a ribozyme, a dicer substrate, an siRNA, an miRNA, a dsRNA, a ssRNA, and an shRNA. In some embodiments, the therapeutic nucleic acid is an antisense oligonucleotide.

[0087] In certain embodiments, the HIF1-α inhibitor administered according to the provided methods is an siRNA or an antisense oligonucleotide.In one embodiment, the HIF1-α inhibitor administered is EZN-2968.In one embodiment, the HIF1-α inhibitor administered is RX-0047.

[0088] Representative examples of human HIF1-A coding sequences are provided in GenBank Accession Nos. NM_004566.3, NM_001145443.2, NP_001138915.1, NM_001282630.2, NM_001314063.1, NM_001323016.1, NM_001323017.1, and NM_001363545.2. The sequences associated with each of these Genbank Accession Nos. are incorporated herein by reference in their entirety for all purposes. Therapeutic nucleic acids that inhibit HIF1-A activity can be routinely designed and prepared based on each of the above human HIF1-A transcript sequences using methods known in the art.

[0089] Certain embodiments of the provided methods contemplate administration of HIF1-A inhibitory nucleic acid or any method of inhibiting gene expression of HIF1-A known in the art. Examples of inhibitory (therapeutic) nucleic acids include, but are not limited to, antisense nucleic acids such as small interfering RNA (siRNA), small hairpin RNA (shRNA), double-stranded RNA, and any other antisense oligonucleotides. Also included are ribozymes or nucleic acids encoding any of the inhibitors described herein. The inhibitory nucleic acid may inhibit transcription of HIF1-A in cells or prevent translation of HIF1-A gene transcripts. In some embodiments, the HIF1-A inhibitory nucleic acid administered according to the provided methods is between 16 and 1000 nucleotides in length. In certain embodiments, the HIF1-A inhibitory nucleic acid administered is between 18 and 100 nucleotides in length. In certain embodiments, the HIF1-A inhibitory nucleic acid administered is at least or up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 40, 50, 60, 70, 80, 90 nucleotides, or any range derivable therein.

[0090] In some embodiments, the HIF1-A inhibitory nucleic acid administered in accordance with the provided methods can reduce expression of HIF1-A by at least 10%, 20%, 30% or 40%, more particularly at least 50%, 60%, or 70%, and most particularly at least 75%, 80%, 90%, 95% or more, or any range or value therebetween.

[0091] In some embodiments, the HIF1-A inhibitory nucleic acid administered in accordance with the provided methods is 17-25 nucleotides in length and comprises a 5' to 3' sequence that is at least 90% complementary to the 5' to 3' sequence of mature HIF1-A mRNA (e.g., as disclosed in any one or more of GenBank Accession Nos. NM_001530.4, NM_181054.3, and NM_001243084.2). In some embodiments, the HIF1-A inhibitory nucleic acid administered is 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, or any range derivable therein. In some embodiments, the administered HIF1-A inhibitory nucleic acid has a sequence (5' to 3') that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9 or 100% complementary to the corresponding 5' to 3' sequence of the mature HIF1-A mRNA (e.g., as disclosed in any one or more of GenBank Accession Nos. NM_001530.4, NM_181054.3, and NM_001243084.2), or any range derivable therein. One of skill in the art would be able to use the portion of the probe sequence that is complementary to the sequence of the mature mRNA as the sequence of the mRNA inhibitor. Furthermore, that portion of the probe sequence can be modified so that it is still 90% complementary to the sequence of the mature mRNA.

[0092] In some embodiments, the HIF1-α inhibitory nucleic acid administered according to the provided methods is an miRNA mimic. In some embodiments, the HIF1-α inhibitor administered is an miR-483 mimic.

[0093] In some embodiments, the HIF1-α inhibitor administered according to the provided methods is an inhibitor of HIF stability, nuclear localization or dimerization. In one embodiment, the inhibitor administered according to the provided methods destabilizes HIF. In one embodiment, the inhibitor administered according to the provided methods is a histone deacetylase inhibitor (HDACI). In further embodiments, the HDACI administered is LW6 / CAY10585, vorinostat, romidepsin (FK228), panobinostat, belinostat, trichostatin A (TSA), LAQ824, or phenethyl isothiocyanate, or a salt thereof. In one embodiment, the inhibitor administered according to the provided methods is PX-12 / pleurotin, HIF-1α inhibitor (CAS number 934593-90-5), cryptotanshinone, or BAY87-2243 (1-cyclopropyl-4-[4-[[5-methyl-3-[3-[4-(trifluoromethoxy)phenyl]-1,2,4-oxadiazol-5-yl]-1H-pyrazol-1-yl]methyl]-2-pyridinyl]-piperazine), or a salt thereof. In one embodiment, the inhibitor administered according to the provided methods is IDF-11774, bisphenol A / dimethylbisphenol A, or a salt thereof. Chrysin (5,7-dihydroxy-flavone), or SCH66336, or a salt thereof. In one embodiment, the inhibitor administered according to the provided methods is geldanamycin or an analog thereof, 17-AAG (Tanespimycin: allylamino-17-demethoxygeldanamycin), 17-DMAG (alvespimycin), 17AG, radicicol, KF58333, ENMD-1198, ENMD-1237, or ganetespib, or a salt thereof. In one embodiment, the inhibitor administered according to the provided methods prevents HIF dimerization. In one embodiment, the inhibitor administered according to the provided methods is acriflavine or a salt thereof. In one embodiment, the inhibitor administered according to the provided methods is TC-S7009, PT2385, or TAT-cyclo-CLLFVY, or a salt thereof.

[0094] In certain embodiments, the inhibitor administered in accordance with the methods provided is ganetespib or a salt thereof.

[0095] In certain embodiments, the inhibitor administered in accordance with the methods provided is BAY87-2243.

[0096] In some embodiments, the HIF1-α pathway inhibitor administered according to the provided method is a histone deacetylase inhibitor (HDACI). In one embodiment, the HDACI administered is LW6 / CAY10585 (methyl 3-(2-(4-(adamantan-1-yl)phenoxy)acetamido)-4-hydroxy-benzoate), vorinostat, romidepsin (FK228), panobinostat, belinostat, trichostatin A (TSA), LAQ824, or phenethyl isothiocyanate, or a salt thereof.

[0097] In some embodiments, the HIF1-α pathway inhibitor administered according to the provided methods is a heat shock protein inhibitor. In one embodiment, the HIF1-α pathway inhibitor administered is an HSP90 inhibitor. In one embodiment, the HSP90 inhibitor administered is geldanamycin or an analog thereof, 17-AAG (Tanespimycin: Allylamino-17-demethoxygeldanamycin), 17-DMAG (Alvespimycin), 17AG, radicicol, KF58333, ENMD-1198, ENMD-1237, or ganetespib, or a salt thereof. In certain embodiments, the heat shock protein inhibitor administered is ganetespib or a salt thereof. In one embodiment, the HIF1-α pathway inhibitor administered is an HSP70 inhibitor. In one embodiment, the HSP70 inhibitor administered is triptolide or a salt thereof.

[0098] In some embodiments, the HIF1-α inhibitor administered according to the provided methods is a HIF transactivation inhibitor. In one embodiment, the HIF1-α inhibitor administered according to the provided methods inhibits the recruitment of HIF coactivators. In one embodiment, the HIF1-α inhibitor administered is ketomin, YC-1 or KCN-1 (3,4-dimethoxy-N-[(2,2-dimethyl-2H-chromen-6-yl)methyl]-N-phenylbenzenesulfonamide), or a salt thereof. In another particular embodiment, the HIF1-α inhibitor administered is NSC607097 or a salt thereof. In one embodiment, the HIF1-α inhibitor administered is a proteasome inhibitor. In a further embodiment, the inhibitor administered is bortezomib or carfilzomib, or a salt thereof. In one embodiment, the HIF1-α inhibitor administered is indenopyrazole 21, FM19G11, flavopiridol, amphotericin B, actinomycin, AJM290, or AW464, or a salt thereof.In one embodiment, the HIF1-α inhibitor administered is triptolide or a salt thereof.

[0099] In certain embodiments, the HIF1-α inhibitor administered in accordance with the methods provided is YC-1, or a salt thereof.

[0100] In some embodiments, the HIF1-α inhibitor administered according to the provided methods is an antibody or HIF1-α binding antibody fragment that binds to HIF1-α (e.g., a single chain antibody, a single domain antibody (e.g., AG1-5 VHH), a Fab fragment, a F(ab')2 fragment, a Fd fragment, a Fv fragment, a scFv, a dAb fragment, or another engineered molecule such as a diabody, a triabody, a tetrabody, a minibody, and a minimal recognition unit). In certain embodiments, the HIF1-α inhibitor administered is a VHH or a nanobody. In one embodiment, the antibody administered is AGI-5. In one embodiment, the antibody administered is AHPC.

[0101] In some embodiments, the HIF1-α inhibitor administered according to the provided methods is a HIF1 DNA binding inhibitor. In one embodiment, the HIF1-α inhibitor administered is echinomycin (NSC-13502) or the compound DJ12.162. In one embodiment, the HIF1-α inhibitor administered is an anthracycline. In a further embodiment, the inhibitor administered is doxorubicin or daunorubicin. In one embodiment, the HIF1-α inhibitor administered is a polyamide. In some embodiments, the HIF1-α inhibitor is an antibody that binds to HIF1-α, or a HIF1-α binding antibody fragment, such as a VHH or a nanobody.

[0102] In some embodiments, the HIF1-A inhibitor administered according to the provided methods is a therapeutic nucleic acid. In some embodiments, the therapeutic nucleic acid is an aptamer, an antisense molecule, a ribozyme, a dicer substrate, ENMD-1198, miRNA, dsRNA, ssRNA, and shRNA. In some embodiments, the therapeutic nucleic acid is ENMD-1198 or an antisense oligonucleotide.

[0103] In some embodiments, the HIF1-A inhibitor administered according to the provided methods is an siRNA or an antisense oligonucleotide. In some embodiments, the HIF1-A inhibitor administered is RX-0047. In some embodiments, the HIF1-A inhibitor administered is EZN-2968.

[0104] Representative examples of human HIF1-A coding sequences are provided in GenBank Accession Nos. NM_004566.3, NM_001145443.2, NP_001138915.1, NM_001282630.2, NM_001314063.1, NM_001323016.1, NM_001323017.1, and NM_001363545.2. The sequences associated with each of these Genbank Accession Nos. are incorporated herein by reference in their entirety for all purposes. Therapeutic nucleic acids that inhibit HIF1-A activity can be routinely designed and prepared based on each of the above human HIF1-A transcript sequences using methods known in the art.

[0105] Certain embodiments of the provided methods contemplate administration of a HIF1-A inhibitory nucleic acid or any method of inhibiting gene expression of HIF1-A known in the art. Examples of inhibitory nucleic acids include, but are not limited to, antisense nucleic acids, such as small interfering RNA (siRNA), small hairpin RNA (shRNA), double-stranded RNA, and any other antisense oligonucleotides. Also included are ribozymes or nucleic acids encoding any of the inhibitors described herein. The inhibitory nucleic acid may inhibit transcription of HIF1-A in cells or prevent translation of HIF1-A gene transcripts. In some embodiments, the HIF1-A inhibitory nucleic acid administered according to the provided methods is between 16 and 1000 nucleotides in length. In certain embodiments, the HIF1-A inhibitory nucleic acid administered is between 18 and 100 nucleotides in length. In certain embodiments, the HIF1-A inhibitory nucleic acid administered is at least or up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 40, 50, 60, 70, 80, 90 nucleotides, or any range derivable therein.

[0106] In some embodiments, the HIF1-A inhibitory nucleic acid administered in accordance with the provided methods can reduce expression of HIF1-A by at least 10%, 20%, 30% or 40%, more particularly at least 50%, 60%, or 70%, and most particularly at least 75%, 80%, 90%, 95% or more, or any range or value therebetween.

[0107] In some embodiments, the HIF1-A inhibitory nucleic acid administered in accordance with the provided methods is 17-25 nucleotides in length and comprises a 5' to 3' sequence that is at least 90% complementary to the 5' to 3' sequence of mature HIF1-A mRNA (e.g., as disclosed in any one or more of GenBank Accession Nos. NM_001530.4, NM_181054.3, and NM_001243084.2). In some embodiments, the HIF1-A inhibitory nucleic acid administered is 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, or any range derivable therein. In some embodiments, the administered HIF1-A inhibitory nucleic acid has a sequence (5' to 3') that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9 or 100% complementary to the corresponding 5' to 3' sequence of the mature HIF1-A mRNA (e.g., as disclosed in any one or more of GenBank Accession Nos. NM_001530.4, NM_181054.3, and NM_001243084.2), or any range derivable therein. One of skill in the art would be able to use the portion of the probe sequence that is complementary to the sequence of the mature mRNA as the sequence of the mRNA inhibitor. Furthermore, that portion of the probe sequence can be modified so that it is still 90% complementary to the sequence of the mature mRNA.

[0108] In some embodiments, the HIF1-α inhibitory nucleic acid administered according to the provided methods is an miRNA mimic. In some embodiments, the HIF1-α inhibitor administered is an miR-483 mimic.

[0109] In some embodiments, the HIF1-α inhibitor administered according to the provided methods is a therapeutic nucleic acid. In some embodiments, the therapeutic nucleic acid is an ENMD-1198 molecule, or an antisense oligonucleotide.

[0110] GPR81 Inhibitors Lactate receptor (also known as G protein-coupled receptor 81, GPR81, hydroxy-carboxylic acid receptor 1 and HCA1) is a G protein-coupled receptor that has been reported to play a central role in promoting abnormal tissue remodeling, as well as cancer-induced angiogenesis, immune evasion and chemotherapy resistance. In the context of the present disclosure, chronic HIF1α-PFKFB3-dependent release of lactate from abnormal cells in the pancreas promotes the abnormal function of cells in the pancreas and plays an autocrine role in spreading the growth and proliferation of pancreatic cancer.

[0111] In some embodiments, the present disclosure provides a method of treating pancreatic and / or hepatic disease in a subject in need thereof, comprising: (a) administering to a subject an effective amount of a HIF1-α pathway inhibitor or a HIF1-α inhibitor and an effective amount of a GPR81 inhibitor; (b) administering to the subject an effective amount of a PFKFB3 pathway inhibitor and an effective amount of a GPR81 inhibitor; (c) administering to the subject an effective amount of a HIF1-α pathway inhibitor or a HIF1-α inhibitor, an effective amount of a PFKFB3 inhibitor, and an effective amount of a GPR81 inhibitor; (d) administering to the subject an effective amount of a GPR81 inhibitor, wherein the subject has previously been administered a PFKFB3 inhibitor; (e) administering to a subject an effective amount of a GPR81 inhibitor, wherein the subject has previously been administered a HIF1-α pathway inhibitor or a HIF1-α inhibitor; or (f) administering to the subject an effective amount of a GPR81 inhibitor, wherein the subject has previously been administered a HIF1-α pathway inhibitor or a HIF1-α inhibitor, and a PFKFB3 inhibitor; The method provides said wherein the PFKFB3 inhibitor does not inhibit the PI3K / AKT / mTOR pathway or HIF1-α.

[0112] As used herein, the term "GPR81 inhibitor" refers to a composition that inhibits or reduces GPR81 directly, or indirectly by inhibiting one or more activities of the GPR81 signaling cascade.

[0113] The "GPR81 inhibitor" that can be administered according to the provided method is not particularly limited. In some embodiments, the GPR81 inhibitor administered is an antibody or GPR81-binding antibody fragment (e.g., a single chain antibody, a single domain antibody (e.g., VHH), a Fab fragment, a F(ab')2 fragment, a Fd fragment, a Fv fragment, a scFv, a dAb fragment, or another engineered molecule, such as a diabody, a triabody, a tetrabody, a minibody, and a minimal recognition unit), a nucleic acid molecule (e.g., an aptamer, an antisense molecule, a ribozyme, a dicer substrate, ENMD-1198, miRNA, dsRNA, ssRNA, and shRNA), a peptibody, a nanobody, or a small molecule.

[0114] In some embodiments, the GPR81 inhibitor administered according to the provided method has a concentration of GPR81 activity with an IC50 of GPR81 activity / function of 100 μM or less. In some embodiments, the GPR81 inhibitor has an IC50 of at least or up to about 200, 100, 80, 50, 40, 20, 10, 5 or 1 μM, or at least or up to about 100, 10 or 1 nM or less (or any range or value derivable therein). In some embodiments, the GPR81 inhibitor inhibits the expression of GPR81. Assays for determining the ability of a compound to inhibit GPR81 activity are known in the art. In some embodiments, the inhibition of GPR81 activity or expression is a decrease compared to a control level or sample. The GPR81 inhibitor that can be administered according to the provided method is not particularly limited. In some embodiments, the GPR81 inhibitor regulates one or more of GPR81 binding to lactate, GPR81 signal transduction, GPR81 mRNA expression, translation or degradation of GPR81 protein.

[0115] In some embodiments, the GPR81 inhibitor administered according to the provided methods is a small molecule.In some embodiments, the GPR81 inhibitor administered according to the provided methods is a protein or polypeptide (e.g., anti-GPR81 antibody or antibody fragment that binds to GPR81).In some embodiments, the GPR81 inhibitor administered according to the provided methods is a therapeutic nucleic acid (e.g., an aptamer, an antisense molecule, a ribozyme, a dicer substrate, an siRNA, an miRNA, a dsRNA, a ssRNA, or an shRNA).

[0116] A representative example of a human GPR81 coding sequence is provided in GenBank Accession No. NM_032554. The sequence associated with each of these Genbank Accession Nos. is incorporated herein by reference in its entirety for all purposes. Therapeutic nucleic acids that inhibit GPR81 activity can be routinely designed and prepared based on each of the above-mentioned human GPR81 transcript sequences using methods known in the art.

[0117] Kit for administration of active agents In another embodiment, the disclosure provides a kit comprising a HIF1-α pathway inhibitor and a PFKFB3 inhibitor, and / or other therapeutic and delivery agents. In some embodiments, a kit for preparing and / or administering the therapeutic methods described herein may be provided. The kit may comprise one or more sealed vials comprising any of the pharmaceutical compositions, therapeutic agents, and / or other therapeutic and delivery agents. In some embodiments, the kit comprises a lipid delivery system. In some embodiments, the lipid is in one vial and the therapeutic agent is in another vial. The kit may comprise, for example, at least one inhibitor of PFKFB3 expression / activity, at least one inhibitor of HIF1-α expression / activity, and one or more reagents for preparing, formulating and / or administering the components described herein or for carrying out one or more steps of the method. In some embodiments, the kit comprises at least one inhibitor of PFKFB3 expression / activity, at least one inhibitor of HIF1-α expression / activity, and at least one inhibitor of GPR81 expression / activity. In some embodiments, the kits may also include a suitable container means, which is a container that will not react with the kit components, such as an Eppendorf tube, an assay plate, a syringe, a bottle, or a tube, etc. The container may be made from a sterilizable material such as plastic or glass.

[0118] The kit may further include instructions outlining the procedural steps of the methods described herein, following substantially the same procedures as described herein or known to one of skill in the art. The instructional information may be in a computer readable medium that includes machine readable instructions that, when executed using a computer, display an actual or virtual procedure for delivering a pharma- ceutical effective amount of a therapeutic agent.

[0119] In some embodiments, kits may be provided for evaluating the expression of PFKFB3 and / or HIF-α, or related molecules. Such kits may be prepared from readily available materials and reagents. For example, such kits may include any one or more of the following substances: enzymes, reaction tubes, buffers, detergents, primers and probes, nucleic acid amplification, and / or hybridization agents. In certain embodiments, these kits allow a physician to obtain samples of blood, tears, semen, saliva, urine, tissue, serum, stool, colon, rectum, sputum, cerebrospinal fluid, and supernatants from cell lysates. In another embodiment, these kits include the equipment necessary to perform RNA extraction, RT-PCR, and gel electrophoresis. Instructions for carrying out the assay may also be included in the kit.

[0120] The kits may include components that may be individually packaged or placed in containers such as tubes, bottles, vials, syringes, or other suitable container means. The components may include probes, primers, antibodies, arrays, negative and / or positive controls. Individual components may also be provided in the kit in concentrated amounts. In some embodiments, components are provided individually at the same concentration that they would be in solution with other components. Concentrations of components may be provided as 1x, 2x, 5x, 10x, or 20x or greater.

[0121] The kit may further comprise a reagent for labeling PFKFB3 and / or HIF-1α in the sample. The kit may also comprise a labeling reagent, comprising at least one of an amine-modified nucleotide, a poly(A) polymerase, and a poly(A) polymerase buffer. The labeling reagent may comprise an amine-reactive dye or any dye known in the art.

[0122] The components of the kit may be packaged in either aqueous media or lyophilized form. The container means of the kit will generally include at least one vial, test tube, flask, bottle, syringe or other container means into which the components may be placed and preferably appropriately aliquoted. Where there are multiple components in the kit (labeling reagents and labels may be packaged together), the kit will generally also include a second, third or other additional container into which the additional components may be placed separately. However, various combinations of components may be included within a vial. The kit may also include means for containing the nucleic acid, antibody or other reagent containers in seal for commercial sale. Such containers may include injection or blow molded plastic containers into which the desired vials are retained.

[0123] When the components of the kit are provided in one and / or more solutions, the solution is an aqueous solution, with a sterile aqueous solution being particularly preferred. Alternatively, the components of the kit may be provided as dry powder(s). When the reagents and / or components are provided as dry powders, the powder may be reconstituted by adding a suitable solvent. It is envisioned that the solvent may be provided in another container means. The container means will generally include at least one vial, test tube, flask, bottle, syringe and / or other container means into which the nucleic acid formulation may be placed, and preferably appropriately aliquoted. The kit may also include a second container means for containing a sterile pharma- ceutically acceptable buffer and / or other diluent.

[0124] The kits may also include a means for sealingly containing the vials for commercial sale, such as, for example, injection and / or blow molded plastic containers into which the desired vials are retained. The kits may also include instructions for using the components of the kit, as well as instructions for the use of other reagents not included in the kit. The instructions may include operable variations.

[0125] Method of administration The dosing regimen (e.g., dosage combined with dosing frequency) according to the methods provided herein generally includes administration in an amount and frequency that provides a desired effect, e.g., administration of an amount effective to provide improvement of one or more symptoms of pancreatic disease and / or liver disease in a subject, such as one or more symptoms associated with pancreatic disease and / or liver disease. The combined administration of each drug may be by any suitable means that can combine with the other component to alleviate the patient's condition or increase the concentration of the drug to effectively treat the disease or disorder. Possible compositions include those suitable for oral, rectal, topical (including transdermal, oral and sublingual), or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration.

[0126] In some embodiments of the invention, the composition is administered to a patient alone or in combination with other therapies, medicines, supplements and / or specific diets, or as a pharmaceutical composition mixed with excipient(s) or other pharma- ceutical carriers. Depending on the goal of administration (e.g., severity of condition, duration of treatment, etc.), the composition (e.g., containing a compound of formula I, such as DMB) can be formulated and administered systemically or locally. Techniques for formulation and administration can be found in the latest edition of "Remington's Pharmaceutical Sciences" (Mack Publishing Co, Easton Pa.). Suitable routes can include, for example, oral or mucosal administration, and parenteral delivery, including intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, or intranasal administration. In some embodiments, the compound of formula I (e.g., DMB) can be administered in the form of a solid, semi-solid, or liquid formulation appropriately formulated to provide the desired therapeutic profile, such as tablets, capsules, tablets, powders, suppositories, solutions, elixirs, syrups, suspensions, creams, lozenges, pastes, and sprays. As one of ordinary skill in the art would recognize, the form of the composition will be selected depending on the route of administration selected.

[0127] As used herein, the phrases "parenteral administration" and "parenterally administered" refer to modes of administration other than enteral and topical administration, e.g., by injection, including, but not limited to, intravenous, intramuscular, intrapleural, intravascular, intrapericardial, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion.

[0128] Pharmaceutical compositions may be formulated according to conventional pharmaceutical practice (see, e.g., Remington: The Science and Practice of Pharmacy (20th ed.), Ed. A. R. Gennaro, Lippincott Williams & Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology, eds J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York).

[0129] Formulations suitable for oral administration may be presented as discrete units, such as capsules, cachets, lozenges or tablets, each containing a predetermined amount of the active ingredient; as powders or granules; as a solution or suspension in an aqueous or non-aqueous liquid; or as an oil-in-water or water-in-oil emulsion. Such formulations can be prepared by any suitable method of pharmacy, including the step of combining the active compound with a suitable carrier, which may contain one or more accessory ingredients as described above. In general, the formulations of the present invention are prepared by uniformly and thoroughly mixing the active compound with a liquid carrier or finely divided solid carrier or both, and then, if necessary, shaping the resulting mixture. For example, a tablet can be prepared by compressing or molding a powder or granules containing the active agent, optionally with one or more accessory ingredients. A compressed tablet can be prepared by compressing the compound in a free-flowing form as a powder or granules, optionally mixed with a binder, lubricant, inert diluent and / or surface active / dispersing agent(s), in a suitable machine. Molded tablets may be made by molding in a suitable machine the powdered compound moistened with an inert liquid binder.

[0130] Formulations suitable for buccal (sublingual) administration include lozenges, which usually contain the active agent in a flavored base such as sucrose and acacia or tragacanth; and pastilles, which contain the active agent in an inert base such as gelatin and glycerin, or sucrose and acacia.

[0131] The preparation for parenteral administration is preferably a sterile aqueous preparation of active agent, and this preparation is preferably isotonic with the blood of intended recipient.These preparations can be administered by subcutaneous, intravenous, intramuscular or intradermal injection.This preparation can be conveniently prepared by mixing compound with water or glycine buffer, and making the resulting solution sterile and isotonic with blood.

[0132] Formulations suitable for topical application (e.g., in the mouth, nasopharynx, or oropharynx) take the form of an ointment, cream, lotion, paste, gel, spray, aerosol, or oil. Carriers that can be used include petrolatum, lanolin, polyethylene glycols, alcohols, transdermal enhancers, and combinations of two or more of these.

[0133] In some embodiments, the present disclosure provides a method of treatment in which the compositions provided herein are administered in combination with one or more additional therapeutic agents. The combination of the compositions provided and the therapeutic agent(s) can be administered in any conventional dosage form or co-administered (e.g., sequentially, simultaneously, at different times). Co-administration herein refers to administering multiple therapeutic agents to a subject in the course of coordinated treatment to achieve improved clinical outcomes. Such co-administration can be co-extensive, i.e., during overlapping periods. For example, a first therapeutic agent can be administered to a patient before, simultaneously with, before, after, or after administration of a second active agent. In some embodiments, the therapeutic agents are combined / formulated in a single composition and thus administered to a subject simultaneously.

[0134] Treatment and Methods of Use Pancreatic steatosis Obesity and metabolic syndrome (including obesity, hyperglycemia, dyslipidemia, hypertension and insulin resistance) result in metabolic abnormalities that lead to lipid mishandling by adipocytes and fatty infiltration of the pancreas. Imbalance of adipocytokines in the circulation and in the pancreatic microenvironment causes chronic low-grade inflammation, resulting in apoptosis of beta cells and acinar cells, leading to endocrine and exocrine pancreatic insufficiency. Furthermore, these adipocytokines regulate cell proliferation, differentiation, and angiogenesis and lymphatic expansion.

[0135] Pancreatic steatosis refers to a disease ranging from fatty infiltration of the pancreas to inflammation of the pancreas and the development of pancreatic fibrosis. The consequences of adipose cell infiltration of the pancreas are thought to be carcinogenic, leading to pancreatic intraepithelial neoplasia and pancreatic ductal adenocarcinoma.

[0136] In some embodiments, the present disclosure provides methods and compositions for treating pancreatic steatosis (PS).

[0137] In some embodiments, the present disclosure provides methods and compositions for treating pancreatic steatosis in a subject in need thereof, comprising: (a) administering to a subject an effective amount of a HIF1-α pathway inhibitor and an effective amount of a PFKFB3 inhibitor; (b) administering to the subject an effective amount of a HIF1-α pathway inhibitor, wherein the subject has previously been administered a PFKFB3 inhibitor; or (c) administering to the subject an effective amount of a PFKFB3 inhibitor, wherein the subject has previously been administered a HIF1-α pathway inhibitor; The methods and compositions provide wherein the PFKFB3 inhibitor does not inhibit the PI3K / AKT / mTOR pathway or HIF1-α.

[0138] In one embodiment, the subject is administered an effective amount of a HIF1-α pathway inhibitor and an effective amount of a PFKFB3 inhibitor. In one embodiment, the subject is administered an effective amount of a HIF1-α pathway inhibitor and the subject has previously been administered a PFKFB3 inhibitor. In one embodiment, the subject is administered an effective amount of a PFKFB3 inhibitor and the subject has previously been administered a HIF1-α pathway inhibitor.

[0139] The terms "pancreatic steatosis" and fatty pancreas are used interchangeably to refer to the accumulation of fat in the pancreas.

[0140] In some embodiments, the PS treated according to the provided method is non-alcoholic fatty pancreatic disease.The non-alcoholic fatty pancreatic disease treated includes but is not limited to non-alcoholic fatty pancreatitis, pancreatic lipomatosis, pancreatic lipomatous pseudohypertrophy, pancreatic fatty replacement, and pancreatic fatty infiltration.

[0141] In certain embodiments, the PS treated according to the provided methods is non-alcoholic fatty pancreatic disease (NASP). In further embodiments, the PS treated is NAFPD non-alcoholic fatty pancreatic disease, including obesity and metabolic syndrome. Metabolic syndrome is a group of five syndromes that can lead to heart disease, diabetes, stroke, and other health problems. Pathologies of metabolic syndrome include diabetes with insulin resistance, arterial hypertension, obesity, and dyslipidemia (hypertriglyceridemia, reduced HDL cholesterol).

[0142] In some embodiments, the PS treated according to the provided methods is alcoholic fatty pancreatic disease. Treated alcoholic fatty pancreatic disease includes, but is not limited to, alcoholic fatty pancreatitis and alcoholic pancreatitis.

[0143] In some embodiments, the subject is at risk of developing PS. In some embodiments, the methods provided herein (e.g., any of (a)-(c) above) are performed as a prophylactic treatment for PS.

[0144] In some embodiments, the methods and compositions provided prevent PS in subjects at risk of developing PS, e.g., subjects with one or more risk factors associated with the development of PS. In some embodiments, the subject has one or more risk factors selected from the following: obesity, metabolic syndrome, arterial hypertension, hypertriglyceridemia, altered HDL cholesterol, diabetes (e.g., MD-T2), hepatic steatosis, NAFLD, malnutrition; taking certain medications, e.g., rosiglitazone, corticosteroids, octreotide and gemcitabine; hemochromatosis, infectious diseases (viral infections caused by reovirus), chronic hepatitis B infection; personal or family history of congenital diseases (Shwachman-Diamond syndrome, Johanson-Blizzard syndrome, cystic fibrosis, heterozygous carboxylester lipase mutations); alcohol abuse (excessive alcohol consumption), history of necrotizing pancreatitis, history of recurrent acute pancreatitis, family history of chronic pancreatitis. In certain embodiments, the risk factor is obesity, or obesity as part of the metabolic syndrome.

[0145] In some embodiments, the disclosure provides methods and compositions for preventing, inhibiting, or delaying the onset of PS by administering a provided composition to a subject prior to the onset of PS, e.g., prior to the onset of one or more symptoms of PS.

[0146] In some embodiments, the HIF1-α pathway inhibitor and the PFKFB3 inhibitor are administered before the onset of one or more symptoms of PS. In some embodiments, the provided methods prevent PS. In some embodiments, the provided methods delay the onset of PS. In some embodiments, the provided methods are administered to a subject at risk of developing PS. In such a subject, the prevention of PS can be monitored by the absence of typical characteristics of PS. For example, a subject to which an effective amount of a HIF1-α inhibitor and a PFKFB3 inhibitor is administered prophylactically may not experience or may experience a reduced incidence of one or more of the following symptoms: pancreatic fat content greater than 25%, abdominal pain (or right upper abdominal bloating), nausea, loss of appetite or weight loss, jaundice (yellowish skin and / or whites of the eyes), edema (swelling of the abdomen and / or lower extremities), extreme fatigue or confusion, and weakness.

[0147] In some embodiments, the subject has been diagnosed with PS. A variety of suitable methods for routinely diagnosing and monitoring PS are commonly known in the art and include MRI proton density fat fraction, computed tomography, endoscopic ultrasound, ultrasound elastography, and transperitoneal ultrasound comparing pancreatic parenchymal echogenicity with renal or hepatic echogenicity.

[0148] In some embodiments, the HIF1-α pathway inhibitor and the PFKFB3 inhibitor are administered after the onset of one or more symptoms of PS.In some embodiments, the subject shows one or more of the following: pancreatic fat content of 25% or more, abdominal pain (or right upper abdominal bloating), nausea, loss of appetite or weight loss, jaundice (yellowish skin and / or whites of eyes), edema (abdominal and / or lower limb swelling), extreme fatigue or confusion, and weakness.In some embodiments, the provided methods and compositions can reduce the incidence, severity, or level of one or more of the above symptoms.

[0149] In some embodiments, the HIF1-α pathway inhibitor administered in accordance with the methods provided herein is an antibody or antigen-binding fragment thereof (e.g., a single chain antibody, a single domain antibody (e.g., VHH), a Fab fragment, a F(ab')2 fragment, an Fd fragment, an Fv fragment, a scFv, a dAb fragment, or another engineered molecule such as a diabody, a triabody, a tetrabody, a minibody, and a minimal recognition unit), a nucleic acid molecule (e.g., an aptamer, an antisense molecule, a ribozyme, miRNA, dsRNA, ssRNA, and shRNA), a peptibody, a nanobody, a HIF1-α pathway binding polypeptide, or a small molecule HIF1-α pathway inhibitor.

[0150] In some embodiments, the HIF1-α pathway inhibitor administered is silibinin, PX-478 or YC-1, or a salt thereof.

[0151] In some embodiments, the administered HIF1-α pathway inhibitor is ganetespib (ST-9090), phenethyl isothocyanate, or BAY-87-2243, or a salt thereof.

[0152] In some embodiments, the HIF1-α pathway inhibitor administered according to the methods provided herein is a HIF1-α inhibitor. In some embodiments, the HIF1-α inhibitor does not inhibit the PI3K / AKT / mTOR pathway. In some embodiments, the HIF1-α inhibitor is an antibody or an antigen-binding fragment thereof (e.g., a single chain antibody, a single domain antibody (e.g., VHH), a Fab fragment, a F(ab')2 fragment, a Fd fragment, a Fv fragment, a scFv, a dAb fragment, or another engineered molecule, e.g., a diabody, a triabody, a tetrabody, a minibody, and a minimal recognition unit), a nucleic acid molecule (e.g., an aptamer, an antisense molecule, a ribozyme, a miRNA, a dsRNA, a ssRNA, and a shRNA), a peptibody, a nanobody, a HIF1-α binding polypeptide, or a small molecule HIF1-α inhibitor.

[0153] In some embodiments, the HIF1-α inhibitor administered is the antisense oligonucleotide EZN-2968, or nanobody AG-1, AG-2, AG-3, AG-4, AG-5, VHH212 or AHPC.

[0154] In some embodiments, the PFKFB3 inhibitor administered according to the methods provided herein is an antibody or antigen-binding antibody fragment (e.g., a single chain antibody, a single domain antibody, a Fab fragment, a F(ab')2 fragment, an Fd fragment, an Fv fragment, a scFv, a dAb fragment, or another engineered molecule such as a diabody, a triabody, a tetrabody, a minibody, and a minimal recognition unit), a nucleic acid molecule (e.g., an aptamer, an antisense molecule, a ribozyme, miRNA, dsRNA, ssRNA, and shRNA), a peptibody, a nanobody, a PFKFB3 binding polypeptide, or a small molecule PFKFB3 inhibitor.

[0155] In some embodiments, the administered PFKFB3 inhibitor is BrAcNHEtOP (N-bromoacetylethanolamine phosphate), PFK15 (1-(4-pyridinyl)-3-(2-quinolinyl)-2-propen-1-one), or PFK-158 ((E)-1-(4-pyridinyl)-3-[7-(trifluoromethyl)-2-quinolinyl]-2-propen-1-one), or a salt thereof.

[0156] In some embodiments, the PFKFB3 inhibitor administered is KAN0436151 or KAN0436067, or a salt thereof.

[0157] In some embodiments, the PFKFB3 inhibitor administered is AZ67, or a salt thereof.

[0158] In some embodiments, the administered PFKFB3 inhibitor has the structure of Formula 1-53 or Formula 54, PQP, N4A, YN1, PK15, PFK-158, YZ29, Compound 26, KAN0436151, KAN0436067, or BrAcNHErOP, or a salt thereof, as shown in Figure 1A-1C or Figure 1D. In some embodiments, the administered PFKFB3 inhibitor has the structure of Formula AZ44-AZ70 or AZ71, or a salt thereof, as shown in Figure 1E.

[0159] In some embodiments, the methods provided herein for treating PS are carried out by co-administering to a subject a HIF1-α pathway inhibitor and a PFKFB3 inhibitor.

[0160] In some embodiments, the HIF1-α pathway inhibitor and / or PFKFB3 inhibitor is administered orally. In some embodiments, the HIF1-α pathway inhibitor and / or PFKFB3 inhibitor is administered transmucosally, as a syrup, topically, parenterally, by injection, subcutaneously, rectally, bucally, or transdermally.

[0161] In some embodiments, treating PS in accordance with the methods provided herein comprises alleviating one or more symptoms of PS in the subject compared to a control subject or the subject prior to treatment with a HIF1-α pathway inhibitor and a PFKFB3 inhibitor.

[0162] In some embodiments, the provided methods result in a reduction in pancreatic fat (e.g., steatosis), such as a reduction in fibrosis in adipose tissue and / or pancreas, improved function of adipocytes and / or pancreas, a reduction in hypoxia in adipose tissue and / or pancreas, a reduction in inflammation in adipose tissue and / or pancreas, a reduction in pancreatitis, and / or a reduction in pancreatic pathology caused by fatty pancreas. In some embodiments, the provided methods result in a reduction in PS biomarkers (e.g., associated with angiotensin II (AngII) type 1 receptor (AT1) signaling and / or pro-inflammatory cytokines, e.g., IL-1β). In some embodiments, one or more symptoms of PS are reduced by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% compared to a control subject or compared to a subject prior to treatment with a HIF1-α pathway inhibitor and a PFKFB3 inhibitor.

[0163] In some embodiments, the subject's pancreatic fat is reduced by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% compared to the subject prior to treatment with a HIF1-α pathway inhibitor and a PFKFB3 inhibitor.

[0164] In some embodiments, adipose tissue inflammation and / or pancreatic inflammation is reduced by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% compared to a subject prior to treatment with a HIF1-α pathway inhibitor and a PFKFB3 inhibitor.

[0165] In some embodiments, the pancreatitis is alleviated by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% compared to the subject prior to treatment with the HIF1-α pathway inhibitor and the PFKFB3 inhibitor.

[0166] In additional embodiments, the methods provided include further administering an additional therapeutic agent to the subject.

[0167] Pancreatic cancer In some embodiments, the present disclosure provides methods and compositions for treating pancreatic cancer in a subject. Pancreatic cancer is a malignant growth of the pancreas that occurs primarily in the cells of the pancreatic duct. The disease is the ninth most common form of cancer, but is the fourth and fifth leading cause of cancer death in men and women, respectively. Pancreatic cancer is almost always fatal, with a five-year survival rate of less than 3%.

[0168] In some embodiments, the present disclosure provides methods and compositions for treating pancreatic cancer in a subject in need thereof, comprising: (a) administering to a subject an effective amount of a HIF1-α pathway inhibitor and an effective amount of a PFKFB3 inhibitor; (b) administering to the subject an effective amount of a HIF1-α pathway inhibitor, wherein the subject has previously been administered a PFKFB3 inhibitor; or (c) administering to the subject an effective amount of a PFKFB3 inhibitor, wherein the subject has previously been administered a HIF1-α pathway inhibitor; The methods and compositions provide wherein the PFKFB3 inhibitor does not inhibit the PI3K / AKT / mTOR pathway or HIF1-α.

[0169] In one embodiment, the subject is administered an effective amount of a HIF1-α pathway inhibitor and an effective amount of a PFKFB3 inhibitor. In one embodiment, the subject is administered an effective amount of a HIF1-α pathway inhibitor and the subject has previously been administered a PFKFB3 inhibitor. In one embodiment, the subject is administered an effective amount of a PFKFB3 inhibitor and the subject has previously been administered a HIF1-α pathway inhibitor.

[0170] In some embodiments, the pancreatic cancer treated in accordance with the methods provided is an exocrine pancreatic cancer or a neuroendocrine pancreatic cancer.

[0171] In some embodiments, the pancreatic cancer is an exocrine pancreatic cancer. In some embodiments, the pancreatic cancer is an exocrine pancreatic cancer selected from adenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma, and collagenous carcinoma. In some embodiments, the pancreatic cancer is squamous cell carcinoma, adenosquamous carcinoma, or collagenous carcinoma. In certain embodiments, the pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC).

[0172] In some embodiments, the pancreatic cancer is a neuroendocrine pancreatic cancer.

[0173] In some embodiments, the subject is at risk of developing pancreatic cancer (e.g., PDAC). In some embodiments, the methods provided herein (e.g., any of (a)-(c) above) are performed as a preventative treatment for pancreatic cancer.

[0174] In some embodiments, the methods and compositions provided prevent pancreatic cancer (e.g., PDAC) in a subject at risk of developing pancreatic cancer, e.g., a subject having one or more risk factors associated with the development of pancreatic cancer. In some embodiments, the subject has one or more risk factors selected from: over 40 years of age, smoking, excessive alcohol consumption, history of diabetes (e.g., type 2 diabetes), onset of diabetes at an older age, obesity, history of chronic pancreatitis, history of fatty pancreas, nonalcoholic fatty liver disease (NAFLD), male sex; family history of chronic pancreatitis, pancreatic cancer, or BRCA-associated cancer (e.g., breast cancer); African-American or Ashkenazi Jewish race, and exposure to chemicals used by dry cleaners and metalworkers.

[0175] In some embodiments, the disclosure provides methods and compositions for preventing, inhibiting, or delaying the onset of pancreatic cancer (e.g., PDAC) by administering a provided composition to a subject prior to the onset of pancreatic cancer, e.g., prior to the onset of one or more symptoms of pancreatic cancer.

[0176] In some embodiments, the HIF1-α pathway inhibitor and the PFKFB3 inhibitor are administered prior to the onset of one or more symptoms of pancreatic cancer (e.g., PDAC). In some embodiments, the methods provided prevent pancreatic cancer. In some embodiments, the methods provided delay the onset of pancreatic cancer (e.g., PDAC).

[0177] In some embodiments, the provided method is administered to a subject at risk of developing pancreatic cancer.In such a subject, the prevention of pancreatic cancer can be monitored by the absence of typical characteristics of pancreatic cancer.For example, the subject who is administered an effective amount of HIF1-α inhibitor and PFKFB3 inhibitor prophylactically may not experience or may experience a reduced incidence of one or more of the following symptoms: pancreatitis, jaundice or yellowing of eyes and skin, loss of appetite, abdominal pain, change in stool, enlargement of gallbladder, sudden and unexplained weight loss; itchy skin, palms; diabetes, and change in taste.

[0178] In some embodiments, the biological sample from the subject does not have a level of a pancreatic cancer biomarker (e.g., CA19-9) consistent with pancreatic cancer. The level of the pancreatic cancer biomarker in the serum of the subject is not elevated in one or more of the pancreatic cancer biomarkers (e.g., creatine kinase (CK-MB), troponin, N-terminal pro-B-type natriuretic peptide, alpha-1 antitrypsin, C-reactive protein, apolipoprotein A1, apolipoprotein B, creatinine, alkaline phosphatase, and transferrin).

[0179] In some embodiments, the subject is diagnosed as suffering from pancreatic cancer. Current methods for diagnosing and monitoring pancreatic cancer generally include clinical symptoms, electrocardiography (ECG), and measurement of peripheral circulating biomarkers. Angiography is also used for severe chest pain, usually associated with unstable angina and acute fatty pancreas (APS). Pancreatic cancer patients often experience tight chest pain that often radiates to the neck, jaw, shoulder, or inner left or both arms, and may be accompanied by symptoms of dyspnea, sweating, palpitations, confusion, and nausea. Myocardial ischemia may cause changes in diagnostic ECG, such as Q waves and ST segment changes. Elevated plasma concentrations of myocardial enzymes in subjects reflect the degree of cardiac tissue necrosis associated with severe unstable angina and fatty pancreas.

[0180] In some embodiments, the HIF1-α pathway inhibitor and PFKFB3 inhibitor are administered after the onset of one or more symptoms of pancreatic cancer.In some embodiments, the subject shows at least one of the following: pancreatitis, jaundice or yellowing of eyes and skin, loss of appetite, abdominal pain, change in stool, enlarged gallbladder, sudden and unexplained weight loss; itchy skin, palms; diabetes, and change in taste.In some embodiments, the provided methods and compositions can reduce the incidence, severity, or level of one or more of the above symptoms.

[0181] In one embodiment, the pancreatic cancer is stage I, II, III or IV.

[0182] In certain embodiments, the pancreatic cancer is an exocrine pancreatic cancer selected from the group consisting of pancreatic ductal adenocarcinoma (PDAC), adenosquamous carcinoma, squamous cell carcinoma, giant cell carcinoma, acinar cell carcinoma, and small cell carcinoma.

[0183] In one particular embodiment, the pancreatic cancer is a ductal adenocarcinoma, e.g., a resectable pancreatic ductal adenocarcinoma (PDAC) arising within the exocrine component of the pancreas. As used herein, "adenocarcinoma" refers to a cancer tumor, as opposed to "adenoma," which refers to a benign (non-cancerous) tumor composed of cells that form glands (a collection of cells surrounding a cavity). As used herein, "pancreatic ductal adenocarcinoma cells" refer to cancer cells that have the ability to form or originate from the pancreas' luminal lining. Pancreatic ductal adenocarcinoma cells may be found within the pancreas forming glands, or may be found within any organ as metastatic cells, or may be found within the lymphatic system's bloodstream. As used herein, "ductal cells" in relation to the pancreas refer to any cell that forms, has the ability to form, or originates from, the luminal lining of the ducts that exit and enter the pancreas.

[0184] In another embodiment, the pancreatic cancer is a pancreatic endocrine tumor, also known as islet cell tumor, pancreatic endocrine tumor arising from pancreatic islet cells (PET), and pancreatic neuroendocrine tumor (PNET). In a particular embodiment, the pancreatic cancer is an endocrine pancreatic cancer selected from the group consisting of insulinoma (i.e., arising from insulin-producing cells), glucagonoma (i.e., arising from glucagon-producing cells), somatostatinoma (i.e., arising from somatostatin-producing cells), gastrinoma (i.e., arising from gastrin-producing cells), VIPomas (arising from vasoactive intestinal peptide-producing cells), and non-secretory islet tumors of the pancreas.

[0185] In one embodiment, the subject is afflicted with one or more of hepatocellular carcinoma, unresectable pancreatic cancer, locally advanced pancreatic cancer, borderline resectable pancreatic cancer, locally advanced pancreatic ductal adenocarcinoma, borderline resectable pancreatic ductal adenocarcinoma, metastatic pancreatic cancer, chemotherapy-resistant pancreatic ductal carcinoma, pancreatic ductal adenocarcinoma, squamous pancreatic carcinoma, pancreatic progenitor cell, immunogenic pancreatic carcinoma, abnormally differentiated endocrine-exocrine (ADEX) tumor, exocrine pancreatic carcinoma, pancreatic intraepithelial neoplasia, intraductal papillary mucinous neoplasm, mucinous cystic neoplasm, mucinous pancreatic carcinoma, adenosquamous carcinoma, signet ring cell carcinoma, hepatoid carcinoma, colloid carcinoma, undifferentiated carcinoma, undifferentiated carcinoma with osteoclast-like giant cells, pancreatic cystic tumor, pancreatic islet cell tumor, pancreatic endocrine tumor, or pancreatic neuroendocrine tumor.

[0186] In some embodiments, the HIF1-α pathway inhibitor administered in accordance with the methods provided herein is an antibody or antigen-binding fragment thereof (e.g., a single chain antibody, a single domain antibody (e.g., VHH), a Fab fragment, a F(ab')2 fragment, an Fd fragment, an Fv fragment, a scFv, a dAb fragment, or another engineered molecule such as a diabody, a triabody, a tetrabody, a minibody, and a minimal recognition unit), a nucleic acid molecule (e.g., an aptamer, an antisense molecule, a ribozyme, miRNA, dsRNA, ssRNA, and shRNA), a peptibody, a nanobody, a HIF1-α pathway binding polypeptide, or a small molecule HIF1-α pathway inhibitor.

[0187] In some embodiments, the HIF1-α pathway inhibitor administered is silibinin, PX-478 or YC-1, or a salt thereof.

[0188] In some embodiments, the administered HIF1-α pathway inhibitor is ganetespib (ST-9090), phenethyl isothocyanate, or BAY-87-2243, or a salt thereof.

[0189] In some embodiments, the HIF1-α pathway inhibitor administered according to the methods provided herein is a HIF1-α inhibitor. In some embodiments, the HIF1-α inhibitor does not inhibit the PI3K / AKT / mTOR pathway. In some embodiments, the HIF1-α inhibitor is an antibody or an antigen-binding fragment thereof (e.g., a single chain antibody, a single domain antibody (e.g., VHH), a Fab fragment, a F(ab')2 fragment, a Fd fragment, a Fv fragment, a scFv, a dAb fragment, or another engineered molecule, e.g., a diabody, a triabody, a tetrabody, a minibody, and a minimal recognition unit), a nucleic acid molecule (e.g., an aptamer, an antisense molecule, a ribozyme, a miRNA, a dsRNA, a ssRNA, and a shRNA), a peptibody, a nanobody, a HIF1-α binding polypeptide, or a small molecule HIF1-α inhibitor.

[0190] In some embodiments, the HIF1-α inhibitor administered is the antisense oligonucleotide EZN-2968, or nanobody AG-1, AG-2, AG-3, AG-4, AG-5, VHH212 or AHPC.

[0191] In some embodiments, the PFKFB3 inhibitor administered according to the methods provided herein is an antibody or antigen-binding antibody fragment (e.g., a single chain antibody, a single domain antibody, a Fab fragment, a F(ab')2 fragment, an Fd fragment, an Fv fragment, a scFv, a dAb fragment, or another engineered molecule such as a diabody, a triabody, a tetrabody, a minibody, and a minimal recognition unit), a nucleic acid molecule (e.g., an aptamer, an antisense molecule, a ribozyme, miRNA, dsRNA, ssRNA, and shRNA), a peptibody, a nanobody, a PFKFB3 binding polypeptide, or a small molecule PFKFB3 inhibitor.

[0192] In some embodiments, the administered PFKFB3 inhibitor is BrAcNHEtOP (N-bromoacetylethanolamine phosphate), PFK15 (1-(4-pyridinyl)-3-(2-quinolinyl)-2-propen-1-one), or PFK-158 ((E)-1-(4-pyridinyl)-3-[7-(trifluoromethyl)-2-quinolinyl]-2-propen-1-one), or a salt thereof.

[0193] In some embodiments, the PFKFB3 inhibitor administered is KAN0436151 or KAN0436067, or a salt thereof.

[0194] In some embodiments, the PFKFB3 inhibitor administered is AZ67, or a salt thereof.

[0195] In some embodiments, the administered PFKFB3 inhibitor has the structure of Formula 1-53 or Formula 54, PQP, N4A, YN1, PK15, PFK-158, YZ29, Compound 26, KAN0436151, KAN0436067, or BrAcNHErOP, or a salt thereof, as shown in Figure 1A-1C or Figure 1D. In some embodiments, the administered PFKFB3 inhibitor has the structure of Formula AZ44-AZ70 or AZ71, or a salt thereof, as shown in Figure 1E.

[0196] In some embodiments, the methods provided herein for treating pancreatic cancer are carried out by co-administering to a subject a HIF1-α pathway inhibitor and a PFKFB3 inhibitor.

[0197] In some embodiments, the HIF1-α pathway inhibitor and / or PFKFB3 inhibitor is administered orally. In some embodiments, the HIF1-α pathway inhibitor and / or PFKFB3 inhibitor is administered transmucosally, as a syrup, topically, parenterally, by injection, subcutaneously, rectally, bucally, or transdermally.

[0198] In some embodiments, treating pancreatic cancer according to the methods provided herein includes alleviating one or more symptoms of pancreatic cancer in a subject compared to a control subject or a subject prior to treatment with a HIF1-α pathway inhibitor and a PFKFB3 inhibitor. In some embodiments, the methods provided result in one or more of a reduction in tumor size, inhibition or reduction in tumor growth, no new tumor formation, reduced new tumor formation, increased survival or progression-free survival, no metastasis, increased treatment options, delayed time from surgery to recurrence, reduced jaundice, reduced liver infiltration, reduced pain, improved appetite, improved digestion, reduced gallbladder size, and reduced incidence of blood clots.

[0199] In some embodiments, the provided methods result in normalization of the ECG (e.g., Q waves and ST segment changes returning to normal) or reduced levels of plasma concentrations of cardiac enzymes or other biomarkers (e.g., creatine kinase (CK-MB), troponin, N-terminal pro-B-type natriuretic peptide, alpha-1 antitrypsin, C-reactive protein, apolipoprotein A1, apolipoprotein B, creatinine, alkaline phosphatase, and transferrin). In some embodiments, one or more symptoms of pancreatic cancer are alleviated by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% compared to a control subject or compared to a subject prior to treatment with a HIF1-α pathway inhibitor and a PFKFB3 inhibitor.

[0200] In some embodiments, at least one, two, three, four or five or more pancreatic cancer biomarkers (e.g., creatine kinase (CK-MB), troponin, N-terminal pro-B-type natriuretic peptide, alpha-1 antitrypsin, C-reactive protein, apolipoprotein A1, apolipoprotein B, creatinine, alkaline phosphatase, and transferrin) in a subject's biological sample is reduced by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% compared to a control subject or compared to a subject prior to treatment with a HIF1-α pathway inhibitor and a PFKFB3 inhibitor.

[0201] In one embodiment, the subject achieves a complete response. In one embodiment, the subject achieves a partial response. In one embodiment, the subject achieves a stable state of disease. In one embodiment, the subject achieves a slowly progressive state of disease.

[0202] In additional embodiments, the methods provided include further administering an additional therapeutic agent to the subject.

[0203] Nonalcoholic fatty liver disease (NAFLD) Fatty liver disease is a chronic condition characterized by excessive deposition of triglycerides in the liver. It can be attributed to multiple causes, and the two main forms are related to excessive alcohol consumption or metabolic dysregulation in the absence of excessive alcohol intake. The latter is called nonalcoholic fatty liver disease (NAFLD).

[0204] NAFLD is characterized by hepatocyte steatosis accompanied by intralobular inflammation and fibrosis and is commonly associated with the metabolic syndrome and its individual components: obesity, type 2 diabetes, dyslipidemia and hypertension.

[0205] The spectrum of NAFLD ranges from isolated fatty liver, also called nonalcoholic fatty liver (NAFL), to nonalcoholic steatohepatitis (NASH). NASH is a common and often "asymptomatic" liver disease. Three main features characterize NASH and distinguish it from other liver diseases of metabolic origin: abnormal fat accumulation or deposition in the liver (hepatic steatosis), liver inflammation, and liver damage or liver tissue injury (fibrosis). NASH is a potentially serious disease with a significant risk of progression to end-stage liver disease, cirrhosis, and hepatocellular carcinoma. Some patients who develop cirrhosis are at risk for liver failure and may ultimately require a liver transplant.

[0206] In some embodiments, the disclosure provides methods and compositions for treating non-alcoholic fatty liver disease (NAFLD) in a subject.

[0207] In some embodiments, the present disclosure provides methods and compositions for treating NAFLD in a subject in need thereof, comprising: (a) administering to a subject an effective amount of a HIF1-α pathway inhibitor and an effective amount of a PFKFB3 inhibitor; (b) administering to the subject an effective amount of a HIF1-α pathway inhibitor, wherein the subject has previously been administered a PFKFB3 inhibitor; or (c) administering to the subject an effective amount of a PFKFB3 inhibitor, wherein the subject has previously been administered a HIF1-α pathway inhibitor; The methods and compositions provide wherein the PFKFB3 inhibitor does not inhibit the PI3K / AKT / mTOR pathway or HIF1-α.

[0208] In one embodiment, the subject is administered an effective amount of a HIF1-α pathway inhibitor and an effective amount of a PFKFB3 inhibitor. In one embodiment, the subject is administered an effective amount of a HIF1-α pathway inhibitor and the subject has previously been administered a PFKFB3 inhibitor. In one embodiment, the subject is administered an effective amount of a PFKFB3 inhibitor and the subject has previously been administered a HIF1-α pathway inhibitor.

[0209] In some embodiments, the NAFLD treated according to the provided method is non-alcoholic fatty liver disease (NAFL).In some embodiments, the NAFLD treated according to the provided method is non-alcoholic steatohepatitis (NASH).In some embodiments, the NAFLD treated according to the provided method is NAFLD-associated liver fibrosis.

[0210] In some embodiments, the subject is at risk of suffering from NAFLD. In some embodiments, the method provided herein (e.g., any of (a)-(c) above) is performed as a preventative treatment for NAFLD. In some embodiments, the subject is at risk of suffering from NAFLD. In some embodiments, the method provided herein (e.g., any of (a)-(c) above) is performed as a preventative treatment for NASH. In some embodiments, the method provided herein (e.g., any of (a)-(c) above) is performed as a preventative treatment for NAFLD-associated liver fibrosis.

[0211] In some embodiments, the provided methods and compositions prevent NAFLD in subjects at risk of developing NAFLD, for example, subjects with one or more risk factors associated with the development of NAFLD.In some embodiments, the subject has one or more risk factors selected from obesity, metabolic syndrome, hypertriglyceridemia, type 2 diabetes, sleep apnea, hypothyroidism, hypopituitarism, and polycystic ovarian syndrome.NASH is more likely to occur in elderly people, diabetic patients, and subjects with body fat concentrated in the abdomen.

[0212] In some embodiments, the subject treated in accordance with the methods provided herein also suffers from type II diabetes, type I diabetes, pre-diabetes, insulin resistance, or obesity, where obesity is defined as a patient having a body mass index of 30 or greater.

[0213] In some embodiments, the present disclosure provides methods and compositions for preventing, inhibiting, or delaying the onset of NAFL, NASH, and / or NAFLD associated liver fibrosis by administering to a subject a provided composition prior to the onset of NAFL, NASH, and / or NAFLD associated liver fibrosis.

[0214] In some embodiments, the present disclosure provides methods and compositions for preventing, inhibiting, or delaying the onset of NAFL.

[0215] In some embodiments, the present disclosure provides methods and compositions for preventing, inhibiting, or delaying the onset of NASH. The preferred means in the art for distinguishing between NASH and steatosis with inflammation is currently histological evaluation of liver biopsy using the so-called "NAFLD activity score" or "NAS" (Kleiner et al., Hepatology 2005, vol. 41, pp. 1313-1321). Other scoring systems can also be used to diagnose the severity of NAFLD and its components. NAS. NAS is defined as the unweighted sum of subscores for (i) steatosis, (ii) lobular inflammation, and (iii) hepatocyte hypertrophy, and specifically includes features of active injury that may be reversible in the short term. Each score is semi-quantitatively graded as described in the following table. [Table 1]

[0216] A subject is diagnosed with or considered to have NAFLD according to the present disclosure if the fatty liver score ("Fatty liver score") using NRA is at least 1. NASH can be distinguished from NAFL or simple fatty liver by the presence of hepatocyte vacuolation ("Vacuolation score") with or without some inflammation ("Inflammation score"). A NAS of less than 3 corresponds to NAFLD, 3-4 corresponds to borderline NASH, and >5 corresponds to NASH. This definition was used in the diagnostic algorithm shown in Table 2. [Table 2]

[0217] While NAS determines the extent of NAFL and NASH (higher scores mean higher disease activity), Kleiner fibrosis score can be used to determine the degree of fibrosis progression. Table 3 shows the definition of Kleiner fibrosis score. [Table 3]

[0218] In some embodiments, the HIF1-α pathway inhibitor and the PFKFB3 inhibitor are administered before the onset of one or more symptoms of NAFLD (e.g., NASH). In some embodiments, the provided methods prevent NAFLD. In some embodiments, the provided methods delay the onset of NAFLD. In some embodiments, the provided methods are administered to subjects at risk of developing NAFLD (e.g., NASH). In such subjects, prevention of NAFLD can be monitored by the absence of typical characteristics of NAFLD (e.g., NASH). For example, subjects to whom an effective amount of a HIF1-α inhibitor and a PFKFB3 inhibitor is administered prophylactically may not experience, or may experience a reduced incidence of, one or more of the following symptoms (e.g., NASH):

[0219] In some embodiments, the HIF1-α pathway inhibitor and PFKFB3 inhibitor are administered after the onset of one or more symptoms of NAFLD.In some embodiments, the subject shows at least one of the following: fatigue, right upper abdominal pain or discomfort, splenomegaly, jaundice (yellowish skin and / or whites of the eyes), edema (abdominal swelling), bruising or bleeding easily, loss of appetite, and nausea.In some embodiments, the provided methods and compositions can reduce the incidence, severity, or level of one or more of the above symptoms.

[0220] In some embodiments, the HIF1-α pathway inhibitor administered in accordance with the methods provided herein is an antibody or antigen-binding fragment thereof (e.g., a single chain antibody, a single domain antibody (e.g., VHH), a Fab fragment, a F(ab')2 fragment, an Fd fragment, an Fv fragment, a scFv, a dAb fragment, or another engineered molecule such as a diabody, a triabody, a tetrabody, a minibody, and a minimal recognition unit), a nucleic acid molecule (e.g., an aptamer, an antisense molecule, a ribozyme, miRNA, dsRNA, ssRNA, and shRNA), a peptibody, a nanobody, a HIF1-α pathway binding polypeptide, or a small molecule HIF1-α pathway inhibitor.

[0221] In some embodiments, the HIF1-α pathway inhibitor administered is silibinin, PX-478 or YC-1, or a salt thereof.

[0222] In some embodiments, the administered HIF1-α pathway inhibitor is ganetespib (ST-9090), phenethyl isothocyanate, or BAY-87-2243, or a salt thereof.

[0223] In some embodiments, the HIF1-α pathway inhibitor administered according to the methods provided herein is a HIF1-α inhibitor. In some embodiments, the HIF1-α inhibitor does not inhibit the PI3K / AKT / mTOR pathway. In some embodiments, the HIF1-α inhibitor is an antibody or an antigen-binding fragment thereof (e.g., a single chain antibody, a single domain antibody (e.g., VHH), a Fab fragment, a F(ab')2 fragment, a Fd fragment, a Fv fragment, a scFv, a dAb fragment, or another engineered molecule, e.g., a diabody, a triabody, a tetrabody, a minibody, and a minimal recognition unit), a nucleic acid molecule (e.g., an aptamer, an antisense molecule, a ribozyme, a miRNA, a dsRNA, a ssRNA, and a shRNA), a peptibody, a nanobody, a HIF1-α binding polypeptide, or a small molecule HIF1-α inhibitor.

[0224] In some embodiments, the HIF1-α inhibitor administered is the antisense oligonucleotide EZN-2968, or nanobody AG-1, AG-2, AG-3, AG-4, AG-5, VHH212 or AHPC.

[0225] In some embodiments, the PFKFB3 inhibitor administered according to the methods provided herein is an antibody or antigen-binding antibody fragment (e.g., a single chain antibody, a single domain antibody, a Fab fragment, a F(ab')2 fragment, an Fd fragment, an Fv fragment, a scFv, a dAb fragment, or another engineered molecule such as a diabody, a triabody, a tetrabody, a minibody, and a minimal recognition unit), a nucleic acid molecule (e.g., an aptamer, an antisense molecule, a ribozyme, miRNA, dsRNA, ssRNA, and shRNA), a peptibody, a nanobody, a PFKFB3 binding polypeptide, or a small molecule PFKFB3 inhibitor.

[0226] In some embodiments, the administered PFKFB3 inhibitor is BrAcNHEtOP (N-bromoacetylethanolamine phosphate), PFK15 (1-(4-pyridinyl)-3-(2-quinolinyl)-2-propen-1-one), or PFK-158 ((E)-1-(4-pyridinyl)-3-[7-(trifluoromethyl)-2-quinolinyl]-2-propen-1-one), or a salt thereof.

[0227] In some embodiments, the PFKFB3 inhibitor administered is KAN0436151 or KAN0436067, or a salt thereof.

[0228] In some embodiments, the PFKFB3 inhibitor administered is AZ67, or a salt thereof.

[0229] In some embodiments, the administered PFKFB3 inhibitor has the structure of Formula 1-53 or Formula 54, PQP, N4A, YN1, PK15, PFK-158, YZ29, Compound 26, KAN0436151, KAN0436067, or BrAcNHErOP, or a salt thereof, as shown in Figure 1A-1C or Figure 1D. In some embodiments, the administered PFKFB3 inhibitor has the structure of Formula AZ44-AZ70 or AZ71, or a salt thereof, as shown in Figure 1E.

[0230] In some embodiments, the methods provided herein for treating NAFLD are carried out by co-administering to a subject a HIF1-α pathway inhibitor and a PFKFB3 inhibitor.

[0231] In some embodiments, the HIF1-α pathway inhibitor and / or PFKFB3 inhibitor is administered orally. In some embodiments, the HIF1-α pathway inhibitor and / or PFKFB3 inhibitor is administered transmucosally, as a syrup, topically, parenterally, by injection, subcutaneously, rectally, bucally, or transdermally.

[0232] In some embodiments, treating NAFLD in accordance with the methods provided herein includes alleviating one or more symptoms of NAFLD in a subject compared to a control subject or a subject prior to treatment with a HIF1-α pathway inhibitor and a PFKFB3 inhibitor.

[0233] In some embodiments, the provided methods result in a reduction in liver fat (e.g., steatosis), such as a reduction in fibrosis in adipose tissue and / or liver, improved adipocyte and / or liver function, a reduction in hypoxia in adipose tissue and / or liver, a reduction in inflammation in adipose tissue and / or liver, a reduction in hepatitis, a reduction in alanine transaminase (ALT) and insulin levels, and / or a reduction in liver pathology caused by fatty liver. In some embodiments, the provided methods result in a reduction in NAFLD biomarkers (e.g., NAFLD biomarkers-increased TNFα, IL-6, CRP, IL-1RA, PAI1, CXCL10, CK18, FGF21, and oxLDL, and decreased ADP and leptin; NASH biomarkers-increased TNFα and IL-6, and decreased ADP and leptin; fibrosis biomarkers-decreased ADP, and increased leptin, hyaluronic acid, laminin, procollagen II, and TIMP1). In some embodiments, one or more symptoms of NAFLD are alleviated by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% compared to a control subject or compared to a subject prior to treatment with a HIF1-α pathway inhibitor and a PFKFB3 inhibitor.

[0234] In some embodiments, the methods provided result in a reduction in (a) hepatic steatosis, triglyceride content, inflammation and / or apoptosis in liver tissue, (b) serum aminotransferase levels, (c) insulin resistance, and optionally, improved glucose tolerance in a subject.

[0235] In some embodiments, the methods provided result in a reduction in the NAS score of 1, 2, 3 or more points in the subject. In some embodiments, the methods provided result in the subject having an NAS of less than 3.

[0236] In some embodiments, the method provided treats a subject suffering from NASH. Such a subject may suffer from NASH, may be diagnosed with NASH, or may be genetically predisposed to developing NASH, or the subject may be predisposed to developing NASH because of suffering from metabolic syndrome, obesity, diabetes or prediabetes. In yet other embodiments, a patient suffering from NASH is a patient who has been tested and found to show clinical findings characteristic of NASH (abnormal accumulation of fat in the liver, liver inflammation and liver fibrosis) even if the patient does not yet show physical symptoms of NASH. In some cases, a patient suffering from NASH shows symptoms of NASH even if he / she has not yet been diagnosed.

[0237] In some embodiments, the provided method results in the subject's slowing or stopping the progression of NASH to cirrhosis.In some embodiments, the treatment results in the improvement of at least one measurable physical symptom of NASH, such as weight loss, weakness, or fatigue.In other embodiments, the treatment results in the amelioration of at least one clinical parameter or sign of NASH, such as abnormal hepatic fat accumulation, liver fibrosis determined by biopsy, liver inflammation, abnormal levels of liver enzymes (e.g., ALT), abnormal levels of inflammatory cytokines, or NAS score.In other embodiments, the treatment results in the alleviation, inhibition, or slowing down of the progression of NASH, either or both physically, such as by stabilizing a measurable symptom or set of symptoms (e.g., fatigue, weight loss, or weakness), or clinically / physiologically, such as by stabilizing a measurable parameter, such as abnormal hepatic fat accumulation, abnormal levels of liver enzymes, abnormal levels of hepatic inflammatory markers, abnormal findings in liver biopsy, NAS score. In another embodiment, the treatment also results in preventing the cause and / or effects or clinical symptoms of NASH, or one of the symptoms that develop as a result of NASH, before the disease or disorder fully manifests. In some embodiments, the treatment results in an increase in the survival rate or duration of a patient with NASH. In some embodiments, the treatment results in a decrease in the likelihood that a patient with NASH will require a liver transplant. In other embodiments, the treatment results in a patient with NASH not needing to undergo a liver transplant. In other embodiments, the treatment results in a decrease in the likelihood that a patient with NASH will develop cirrhosis. In other embodiments, the treatment results in the prevention of progression to cirrhosis, as determined by histology.

[0238] In some embodiments, the subject's liver fat is reduced by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% compared to the subject prior to treatment with a HIF1-α pathway inhibitor and a PFKFB3 inhibitor.

[0239] In some embodiments, adipose tissue inflammation and / or liver inflammation is reduced by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% compared to a subject prior to treatment with a HIF1-α pathway inhibitor and a PFKFB3 inhibitor.

[0240] In some embodiments, the pancreatitis is alleviated by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% compared to the subject prior to treatment with the HIF1-α pathway inhibitor and the PFKFB3 inhibitor.

[0241] In additional embodiments, the methods provided include further administering an additional therapeutic agent to the subject.

[0242] hepatocellular carcinoma In some embodiments, the present disclosure provides methods and compositions for treating hepatocellular carcinoma (HCC) in a subject, which arises from malignant transformation of liver cells following chronic hepatitis or cirrhosis.

[0243] In some embodiments, the present disclosure provides methods and compositions for treating hepatocellular carcinoma (HCC) in a subject in need thereof, comprising: (a) administering to a subject an effective amount of a HIF1-α pathway inhibitor and an effective amount of a PFKFB3 inhibitor; (b) administering to the subject an effective amount of a HIF1-α pathway inhibitor, wherein the subject has previously been administered a PFKFB3 inhibitor; or (c) administering to the subject an effective amount of a PFKFB3 inhibitor, wherein the subject has previously been administered a HIF1-α pathway inhibitor; The methods and compositions provide wherein the PFKFB3 inhibitor does not inhibit the PI3K / AKT / mTOR pathway or HIF1-α.

[0244] In one embodiment, the subject is administered an effective amount of a HIF1-α pathway inhibitor and an effective amount of a PFKFB3 inhibitor. In one embodiment, the subject is administered an effective amount of a HIF1-α pathway inhibitor and the subject has previously been administered a PFKFB3 inhibitor. In one embodiment, the subject is administered an effective amount of a PFKFB3 inhibitor and the subject has previously been administered a HIF1-α pathway inhibitor.

[0245] In some embodiments, the subject is at risk of developing hepatocellular carcinoma. In some embodiments, the method provided herein (e.g., any of (a) to (c) above) is performed as a preventative treatment for hepatocellular carcinoma.

[0246] In some embodiments, the methods and compositions provided prevent hepatocellular carcinoma in subjects at risk of developing hepatocellular carcinoma, for example, subjects with one or more risk factors associated with the development of hepatocellular carcinoma. In some embodiments, the subject has one or more risk factors selected from male, over 55 years old, viral chronic hepatitis (e.g., hepatitis B, hepatitis C), autoimmune hepatitis, NAFLD, NASH, fatty pancreas, smoking, excessive alcohol consumption, diabetes (e.g., type 2 diabetes), obesity, history of chronic pancreatitis, cirrhosis, primary biliary cirrhosis, family history of HCC, inherited metabolic disease, hereditary hemochromatosis, alpha 1-antitrypsin deficiency, Wilson's disease, and exposure to certain environmental factors such as aflatoxin.

[0247] In some embodiments, the present disclosure provides methods and compositions for preventing, inhibiting, or delaying the onset of hepatocellular carcinoma by administering a provided composition to a subject prior to the onset of hepatocellular carcinoma, e.g., prior to the onset of one or more symptoms of hepatocellular carcinoma.

[0248] Symptoms of HCC often do not appear until the cancer is in its early stages, after which symptoms may include weight loss, loss of appetite, fever, nausea, fatigue, upper abdominal pain, swelling of the abdomen and legs, yellowing of the skin (jaundice), easy bruising or bleeding, and liver failure.

[0249] In some embodiments, the HIF1-α pathway inhibitor and the PFKFB3 inhibitor are administered prior to the onset of one or more symptoms of hepatocellular carcinoma. In some embodiments, the provided methods prevent hepatocellular carcinoma. In some embodiments, the provided methods delay the onset of hepatocellular carcinoma.

[0250] In some embodiments, the provided method is administered to a subject at risk of developing hepatocellular carcinoma.In such subjects, the prevention of hepatocellular carcinoma can be monitored by the absence of typical characteristics of hepatocellular carcinoma.For example, the subject who is administered an effective amount of HIF1-α inhibitor and PFKFB3 inhibitor prophylactically may not experience or may experience a reduced incidence of one or more of the following symptoms: weight loss, loss of appetite, fever, nausea, fatigue, upper abdominal pain, abdominal and lower limb swelling, yellowing of the skin (jaundice), prone to subcutaneous bleeding or bleeding, and liver failure.

[0251] In some embodiments, the biological sample from the subject does not have a level of hepatocellular carcinoma biomarkers (e.g., EpCam, VEGF, EGFR, FLT1, theophylline, HCC-22-5, KRT23, AHSG, FTL, C16Cer, C16DHC, C18DHC, S1P, C24DHC, C24:1DHC, C18Cer, C20Cer, C24Cer, C24:1Cer, sphingosine, and SA1P, CTSD, HYOU1, PSAP and LAMP-2) consistent with hepatocellular carcinoma. The biological sample for analysis is usually blood, plasma, serum, mucosa, tissue biopsy, tumor, ascites or cerebrospinal fluid from the patient. The sample can be analyzed for signs of tumor.

[0252] In some embodiments, the subject is diagnosed as suffering from hepatocellular carcinoma.Current methods for diagnosing and monitoring hepatocellular carcinoma generally include clinical symptoms, computed tomography (CAT scan), magnetic resonance imaging (MRI), abdominal ultrasound, coagulation test, arteriogram, or biopsy.Patients suffering from hepatocellular carcinoma often suffer from weight loss, loss of appetite, fever, nausea, fatigue, upper abdominal pain, abdominal and lower limb swelling, yellowing of the skin (jaundice), prone to subcutaneous bleeding or bleeding, and liver failure.

[0253] In some embodiments, the HIF1-α pathway inhibitor and PFKFB3 inhibitor are administered after the onset of one or more symptoms of hepatocellular carcinoma.In some embodiments, the subject shows at least one of the following: weight loss, loss of appetite, fever, nausea, fatigue, upper abdominal pain, abdominal and lower limb swelling, yellowing of the skin (jaundice), easy bleeding or bleeding under the skin, and liver failure.In some embodiments, the provided methods and compositions can reduce the incidence, severity, or level of one or more of the above symptoms.

[0254] In some embodiments, the subject is susceptible to developing HCC due to a genetic predisposition or for other reasons (eg, having a risk factor).

[0255] In one embodiment, the hepatocellular carcinoma is early stage HCC. In one embodiment, the hepatocellular carcinoma is non-metastatic HCC or primary HCC. In one embodiment, the hepatocellular carcinoma is advanced HCC, locally advanced HCC or metastatic HCC. In one embodiment, the hepatocellular carcinoma is HCC in remission or recurrent HCC. In some embodiments, the HCC is resectable localized, unresectable localized, or unresectable (i.e., the tumor involves an entire lobe of the liver and / or spreads to other organs (e.g., lung, lymph nodes, bone)). In some embodiments, the HCC is a stage I tumor (single tumor without vascular invasion), stage II tumor (single tumor with vascular invasion or multiple tumors, <5 cm), stage III tumor (multiple tumors, >5 cm, or tumors involving major branches of the portal or hepatic veins), stage IV tumor (tumor with direct invasion of adjacent organs other than the gallbladder or perforation of the visceral peritoneum), N1 tumor (regional lymph node metastasis), or M1 tumor (distant metastasis) according to the TNM classification. In some embodiments, the HCC is a stage T1, T2, T3, or T4 HCC according to the AJCC (American Joint Commission on Cancer) staging criteria.

[0256] In one embodiment, the subject is afflicted with hepatocellular carcinoma, the fibrolamellar variant of HCC, or mixed hepatocellular-cholangiocarcinoma.

[0257] In some embodiments, the HIF1-α pathway inhibitor administered in accordance with the methods provided herein is an antibody or antigen-binding fragment thereof (e.g., a single chain antibody, a single domain antibody (e.g., VHH), a Fab fragment, a F(ab')2 fragment, an Fd fragment, an Fv fragment, a scFv, a dAb fragment, or another engineered molecule such as a diabody, a triabody, a tetrabody, a minibody, and a minimal recognition unit), a nucleic acid molecule (e.g., an aptamer, an antisense molecule, a ribozyme, miRNA, dsRNA, ssRNA, and shRNA), a peptibody, a nanobody, a HIF1-α pathway binding polypeptide, or a small molecule HIF1-α pathway inhibitor.

[0258] In some embodiments, the HIF1-α pathway inhibitor administered is silibinin, PX-478 or YC-1, or a salt thereof.

[0259] In some embodiments, the administered HIF1-α pathway inhibitor is ganetespib (ST-9090), phenethyl isothocyanate, or BAY-87-2243, or a salt thereof.

[0260] In some embodiments, the HIF1-α pathway inhibitor administered according to the methods provided herein is a HIF1-α inhibitor. In some embodiments, the HIF1-α inhibitor does not inhibit the PI3K / AKT / mTOR pathway. In some embodiments, the HIF1-α inhibitor is an antibody or an antigen-binding fragment thereof (e.g., a single chain antibody, a single domain antibody (e.g., VHH), a Fab fragment, a F(ab')2 fragment, a Fd fragment, a Fv fragment, a scFv, a dAb fragment, or another engineered molecule, e.g., a diabody, a triabody, a tetrabody, a minibody, and a minimal recognition unit), a nucleic acid molecule (e.g., an aptamer, an antisense molecule, a ribozyme, a miRNA, a dsRNA, a ssRNA, and a shRNA), a peptibody, a nanobody, a HIF1-α binding polypeptide, or a small molecule HIF1-α inhibitor.

[0261] In some embodiments, the HIF1-α inhibitor administered is the antisense oligonucleotide EZN-2968, or nanobody AG-1, AG-2, AG-3, AG-4, AG-5, VHH212 or AHPC.

[0262] In some embodiments, the PFKFB3 inhibitor administered according to the methods provided herein is an antibody or antigen-binding antibody fragment (e.g., a single chain antibody, a single domain antibody, a Fab fragment, a F(ab')2 fragment, an Fd fragment, an Fv fragment, a scFv, a dAb fragment, or another engineered molecule such as a diabody, a triabody, a tetrabody, a minibody, and a minimal recognition unit), a nucleic acid molecule (e.g., an aptamer, an antisense molecule, a ribozyme, miRNA, dsRNA, ssRNA, and shRNA), a peptibody, a nanobody, a PFKFB3 binding polypeptide, or a small molecule PFKFB3 inhibitor.

[0263] In some embodiments, the administered PFKFB3 inhibitor is BrAcNHEtOP (N-bromoacetylethanolamine phosphate), PFK15 (1-(4-pyridinyl)-3-(2-quinolinyl)-2-propen-1-one), or PFK-158 ((E)-1-(4-pyridinyl)-3-[7-(trifluoromethyl)-2-quinolinyl]-2-propen-1-one), or a salt thereof.

[0264] In some embodiments, the PFKFB3 inhibitor administered is KAN0436151 or KAN0436067, or a salt thereof.

[0265] In some embodiments, the PFKFB3 inhibitor administered is AZ67, or a salt thereof.

[0266] In some embodiments, the administered PFKFB3 inhibitor has the structure of Formula 1-53 or Formula 54, PQP, N4A, YN1, PK15, PFK-158, YZ29, Compound 26, KAN0436151, KAN0436067, or BrAcNHErOP, or a salt thereof, as shown in Figure 1A-1C or Figure 1D. In some embodiments, the administered PFKFB3 inhibitor has the structure of Formula AZ44-AZ70 or AZ71, or a salt thereof, as shown in Figure 1E.

[0267] In some embodiments, the methods provided herein for treating hepatocellular carcinoma are carried out by co-administering to a subject a HIF1-α pathway inhibitor and a PFKFB3 inhibitor.

[0268] In some embodiments, the HIF1-α pathway inhibitor and / or PFKFB3 inhibitor is administered orally. In some embodiments, the HIF1-α pathway inhibitor and / or PFKFB3 inhibitor is administered transmucosally, as a syrup, topically, parenterally, by injection, subcutaneously, rectally, bucally, or transdermally.

[0269] In some embodiments, treating hepatocellular carcinoma according to the methods provided herein includes alleviating one or more symptoms of hepatocellular carcinoma in a subject, compared to a control subject or a subject prior to treatment with a HIF1-α pathway inhibitor and a PFKFB3 inhibitor. In some embodiments, the methods provided result in a reduction in HCC cell count, a reduction in tumor size, inhibition or reduction in tumor growth, no new tumor formation, reduction in new tumor formation; inhibition, delay, slowing to some extent, and preferably stopping the invasion of HCC cancer cells into peripheral organs; no metastasis, inhibition of tumor growth; prevention or delay of tumor development and / or recurrence; alleviation to some extent of one or more symptoms associated with HCC; increased survival or progression-free survival, and / or increased treatment options, delayed time from surgery to recurrence, reduced jaundice, inhibited liver invasion, reduced pain, improved appetite, improved digestion, reduced gallbladder size, and reduced incidence of blood clots.

[0270] The terms "inhibit," "reduce," and "decrease" with respect to tumor or cancer growth or progression refer to a measurable inhibition of tumor or cancer growth, spread, or metastasis using methods known in the art. Tumor or cancer growth, progression, or spread is inhibited, reduced, or decreased if the tumor burden is reduced by at least about 10%, 20%, 30%, 50%, 80%, or 100%.

[0271] In some embodiments, the methods provided provide HCC biomarkers (e.g., EpCam, VEGF, EGFR, FLT1, theophylline, HCC-22-5, KRT23, AHSG, FTL, C16Cer, C16DHC, C18DHC, S1P, C24DHC, C24:1DHC, C18Cer, C20Cer, C24Cer, C24:1Cer, sphingosine, and SA1P, CTSD, HYOU1, PSAP, and LAMP-2). In some embodiments, one or more symptoms of hepatocellular carcinoma are alleviated by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% compared to a control subject or compared to a subject prior to treatment with a HIF1-α pathway inhibitor and a PFKFB3 inhibitor.

[0272] In some embodiments, the level of one, two, three, four, five or more hepatocellular carcinoma biomarkers (e.g., EpCam, VEGF, EGFR, FLT1, theophylline, HCC-22-5, KRT23, AHSG, FTL, C16Cer, C16DHC, C18DHC, S1P, C24DHC, C24:1DHC, C18Cer, C20Cer, C24Cer, C24:1Cer, sphingosine, and SA1P, CTSD, HYOU1, PSAP, and LAMP-2) in the subject's biological sample is reduced by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% compared to a control subject or compared to the subject prior to treatment with a HIF1-α pathway inhibitor and a PFKFB3 inhibitor.

[0273] In one embodiment, the subject achieves a complete response. In one embodiment, the subject achieves a partial response. In one embodiment, the subject achieves a stable state of disease. In one embodiment, the subject achieves a slowly progressive state of disease.

[0274] In additional embodiments, the methods provided include further administering an additional therapeutic agent to the subject.

[0275] The disclosures of each of U.S. Patent Application Nos. 63 / 189,204, 63 / 189,205, 63 / 189,206, and 63 / 189,207, each filed May 16, 2021, are incorporated herein by reference in their entirety. All references, articles, publications, patents, patent publications, and patent applications cited herein are incorporated by reference in their entirety for all purposes. However, the mention of any references, articles, publications, patents, patent publications, and patent applications cited herein is not, and should not be construed as, an admission or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world.

Claims

1. A composition for use in treating a pancreatic or hepatic disease in a subject in need of such treatment, said use comprising: (a) administering to the subject a composition comprising an effective amount of a HIF1-α pathway inhibitor and an effective amount of a PFKFB3 inhibitor; (b) administering to the subject a composition comprising an effective amount of a HIF1-α pathway inhibitor, wherein the subject has previously been administered a PFKFB3 inhibitor; or (c) administering to the subject a composition comprising an effective amount of a PFKFB3 inhibitor, wherein the subject has previously been administered a HIF1-α pathway inhibitor; The composition, wherein the PFKFB3 inhibitor does not inhibit the PI3K / AKT / mTOR pathway or HIF1-α.

2. The composition described in claim 1, wherein the composition is administered as a preventive treatment for the pancreatic or liver disease.

3. 2. The composition of claim 1, wherein the pancreatic disease is pancreatic steatosis or pancreatic cancer and the liver disease is nonalcoholic fatty liver disease (NAFLD) or hepatocellular carcinoma.

4. The administered HIF1-α pathway inhibitor may be an antibody or an antigen-binding antibody fragment (e.g., a single chain antibody, a single domain antibody (e.g., VHH), a Fab fragment, an F(ab') ２ The composition of claim 1, which is a polypeptide that is a polypeptide that is a polypeptide of the present invention, the polypeptide of the present invention being a polypeptide of the present invention.

5. The composition of claim 1, wherein the administered HIF1-α pathway inhibitor is silibinin, PX-478, YC-1, ganetespib (ST-9090), phenethyl isothiocyanate, BAY-87-2243, or a salt thereof.

6. The administered HIF1-α pathway inhibitor is a HIF1-α inhibitor, and optionally is an antibody or antigen-binding antibody fragment (e.g., a single chain antibody, a single domain antibody (e.g., VHH), a Fab fragment, a F(ab') ２ The composition of claim 1, wherein the antibody is selected from a polypeptide, a polypeptide chain, a polypeptide fragment, an Fd fragment, an Fv fragment, an scFv, a dAb fragment, or another engineered molecule, such as diabodies, triabodies, tetrabodies, minibodies, and minimal recognition units), a nucleic acid molecule (e.g., an aptamer, an antisense molecule, a ribozyme, MiRNA, dsRNA, ssRNA, and shRNA), a peptibody, a nanobody, a HIF1-α binding polypeptide, and a small molecule HIF1-α inhibitor.

7. The composition of claim 6, wherein the administered HIF1-α inhibitor is the antisense oligonucleotide EZN-2968, nanobody AG-1, AG-2, AG-3, AG-4, AG-5, VHH212 or AHPC.

8. The administered PFKFB3 inhibitor may be an antibody or an antigen-binding antibody fragment (e.g., a single chain antibody, a single domain antibody, a Fab fragment, an F(ab') ２ The composition of claim 1, which is a PFKFB3 binding polypeptide, or a small molecule PFKFB3 inhibitor, which is a PFKFB3 fragment, Fd fragment, Fv fragment, scFv, dAb fragment, or another engineered molecule such as a diabody, triabody, tetrabody, minibody, and minimal recognition unit), a nucleic acid molecule (e.g., an aptamer, an antisense molecule, ribozyme, MiRNA, dsRNA, ssRNA, and shRNA), a peptibody, a nanobody, a PFKFB3 binding polypeptide, or a small molecule PFKFB3 inhibitor.

9. The PFKFB3 inhibitor administered is (a) AZ67 or a salt thereof, (b) BrAcNHEtOP (N-bromoacetylethanolamine phosphate), PFK15 (1-(4-pyridinyl)-3-(2-quinolinyl)-2-propen-1-one), or PFK-158 ((E)-1-(4-pyridinyl)-3-[7-(trifluoromethyl)-2-quinolinyl]-2-propen-1-one), or a salt thereof, 1A-1C or 1D, or a salt thereof; or (e) the structure of Formula AZ44-Formula AZ70 or Formula AZ71, or a salt thereof, as shown in FIG. 1E.

10. The composition of claim 1, wherein the HIF1-α pathway inhibitor and the PFKFB3 inhibitor are co-administered to the subject, optionally prior to or after onset of one or more symptoms of the pancreatic or liver disease, and optionally administration is oral, parenteral, orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal, intranasal, intratumoral, or intravenous.

11. The composition described in claim 1, wherein the use further comprises administering an additional therapeutic agent to the subject.

12. The composition described in any one of claims 1 to 11, wherein the pancreatic disease is pancreatic steatosis, optionally fatty infiltration of the pancreas, fatty infiltration of the pancreas accompanied by pancreatic inflammation, or fatty infiltration of the pancreas accompanied by the development of pancreatic fibrosis.

13. The composition of any one of claims 1 to 11, wherein the pancreatic disease is pancreatic cancer, optionally an exocrine pancreatic cancer (e.g., adenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma, or colloid carcinoma).

14. The composition described in any one of claims 1 to 11, wherein the liver disease is non-alcoholic fatty disease (e.g., non-alcoholic fatty liver (NAFL), non-alcoholic steatohepatitis (NASH), or NAFLD-associated liver fibrosis).

15. The composition described in any one of claims 1 to 11, wherein the pancreatic disease is hepatocellular carcinoma.

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