Treatment with PPAR agonists and FGFR4 inhibitors

Abstract

The present invention provides a method of treating a subject in need of anti-fibroblast growth factor receptor 4 (anti-FGFR4) treatment. The method includes administering to the subject a therapeutically effective amount of a peroxisome proliferator-activated receptor (PPAR) alpha agonist in combination with anti-FGFR4 treatment. The method can include administering to the subject a therapeutically effective amount of a PPAR alpha agonist and a bile acid sequestrant. The present invention also provides a composition comprising an FGFR4 inhibitor, a PPAR alpha agonist, and optionally a bile acid sequestrant. The method reduces dysregulation of bile acid biosynthesis caused by FGFR4 inhibition by anti-FGFR4 treatment. The method reduces the severity and / or incidence of adverse events associated with anti-FGFR4 treatment.

Description

[Technical field]

[0001] Described herein are methods of treating a subject in need of anti-fibroblast growth factor receptor 4 (anti-FGFR4) treatment with a peroxisome proliferator-activated receptor (PPAR) alpha agonist. [Background technology]

[0002] One of the normal physiological roles of FGFR4 is to regulate bile acid synthesis. Stimulation of FGFR4 with its ligand, fibroblast growth factor 19 (FGF19), suppresses downstream cholesterol 7α-hydroxylase (CYP7A1) expression. CYP7A1 encodes cytochrome P4507A1 (also known as Cyp7a1, cholesterol 7-alpha-monooxygenase), which catalyzes the rate-limiting step in bile acid synthesis. Inhibition of FGFR4 activity increases bile acid synthesis due to lack of inhibition of CYP7A1. High levels of bile acids can cause liver toxicity and gastrointestinal problems, including diarrhea. Hepatotoxicity, characterized by single-cell necrosis, increased bilirubin, severe diarrhea, and reduced food intake, was reported in monkeys administered anti-FGF19 antibodies. In dogs, bile acid sequestration with cholestyramine attenuated FGFR4 inhibition-induced elevation of alanine aminotransferase (ALT), an indicator of potential hepatotoxicity.

[0003] FGFR4 inhibitors are being developed for the treatment of cancers such as hepatocellular carcinoma (HCC). Amplification of FGFR4 ligand FGF19 and high expression of FGFR4 are frequently reported in HCC cases, and both are shown to promote the progression of HCC. FGFR4 is also thought to be involved in other diseases, such as left ventricular hypertrophy (LVH), a complication of chronic kidney disease (CKD). Chronic kidney disease is associated with a significant increase in the risk of cardiovascular death. Bile acid dysregulation by FGFR4 inhibition may complicate or limit treatment.

[0004] There remains a need to regulate bile acid synthesis in therapies utilizing FGFR4 inhibitors. The present disclosure addresses these needs. Summary of the Invention

[0005] To meet these needs, the present disclosure provides a method for treating a subject in need of anti-FGFR4 therapy, comprising administering to the subject a therapeutically effective amount of a PPAR alpha agonist in combination with anti-FGFR4 therapy.

[0006] The present disclosure also provides a method of treating a subject in need of anti-FGFR4 therapy comprising administering to the subject an anti-FGFR4 therapy in combination with a therapeutically effective amount of a PPAR alpha agonist and a bile acid sequestrant.

[0007] The present disclosure also provides a composition comprising an FGFR4 inhibitor, a PPAR alpha agonist, and a pharma- ceutically acceptable excipient.

[0008] The present disclosure also provides a composition comprising an FGFR4 inhibitor, a PPAR alpha agonist, a bile acid sequestrant, and a pharma- ceutically acceptable excipient.

[0009] The present disclosure also provides a kit comprising an FGFR4 inhibitor and a PPAR alpha agonist.

[0010] The summary and the following detailed description will be better understood when read in conjunction with the accompanying drawings, in which: For the purpose of illustrating the disclosed invention, exemplary embodiments of the method are shown in the drawings, but the disclosed method is not limited to the exemplary embodiments of the method. [Brief description of the drawings]

[0011] [Figure 1A] FIG. 1 shows a negative feedback mechanism in bile acid (BA) synthesis. [Figure 1B]Negative feedback mechanism in bile acid (BA) synthesis. In response to increased BA, the intestinal hormone FGF19 in humans (FGF15 in mice) is produced, which then acts on the liver to bind to FGFR4 and activate its downstream signaling, thereby inhibiting cholesterol 7α-hydroxylase (CYP7A1), the rate-limiting enzyme in the classical pathway of BA synthesis. [Figure 1C] FIG. 1 shows the results of using a selective FGFR4 inhibitor, altering signaling-induced CYP7A1 downregulation, resulting in increased CYP7A1 expression and subsequently BA biosynthesis. [Figure 1D] Figure 1 shows that antibody-mediated inhibition of FGF19 increases Cyp7α1 and elevates bile acid synthesis, resulting in enhanced bile acid efflux and reduced uptake into hepatocytes. Increased bile acids alter solute transporters in enterocytes and disrupt enterohepatic recirculation of bile acids, subsequently causing diarrhea and hepatotoxicity. Abbreviations: apical sodium-dependent bile acid transporter (ASBT), bile salt export pump (BSEP), ileal bile acid-binding protein (IBABP), multidrug resistance protein (MRP) 2, 3, 4, organic anion transporter (OAT), organic solute transporter alpha-beta (OST-α and OST-β), sodium taurocholate cotransporting polypeptide (NTCP). Quoted from Pai et al., Toxicological Sciences 126(2), 446-456(2012). [Figure 1E] FIG. 1 shows the effect of a PPAR alpha agonist in combination with a selective FGFR4 inhibitor; the PPAR alpha agonist (e.g., a fibrate) counteracts FGFR4 inhibitor-induced CYP7A1 overexpression and attenuates bile acid dysregulation caused by FGFR4 inhibition. [Figure 1F] FIG. 1 is a schematic diagram showing the bile acid biosynthetic pathway, the rate-limiting enzyme being CYP7A1, and the stable intermediate C4 that is produced by increasing CYP7A1 expression or activity. [Figure 2A]Figure 1 is a bar graph showing the change in CYP7A1 expression (relative mRNA expression / actin) in Hep3B cells following 18 hour treatment with increasing concentrations of the selective FGFR4 inhibitor fisogatinib (BLU554). Treatment of cells with BLU554 resulted in a dose-dependent increase in CYP7A1 expression in Hep3B as measured using qPCR. Treatment of Hep3B with increasing concentrations of BLU-554 resulted in an increase in CYP7A1 expression (relative expression / actin). [Figure 2B] 1 is a bar graph showing the change in CYP7A1 expression (relative mRNA expression / actin) in Hep3B cells following 18-hour treatment with increasing concentrations of the selective FGFR4 inhibitor lobritinib (FGF-401). Treatment of Hep3B with increasing concentrations of FGF-401 resulted in increased CYP7A1 expression (relative expression / actin). [Figure 2C] 1 is a bar graph showing the change in CYP7A1 expression (relative mRNA expression / actin) in Hep3B cells following 18-hour treatment with increasing concentrations of the selective FGFR4 inhibitor erdafitinib, a potent tyrosine kinase inhibitor of FGFR1-4. Treatment of Hep3B with increasing concentrations of erdafitinib resulted in increased CYP7A1 expression (relative expression / actin). [Figure 2D] 1 is a bar graph showing the change in CYP7A1 expression (relative mRNA expression / actin) in Hep3B cells following 18-hour treatment with increasing concentrations of the selective FGFR4 inhibitor futibatinib (a potent and selective covalent inhibitor of FGFR1-4). Treatment of Hep3B with increasing concentrations of futibatinib resulted in increased CYP7A1 expression (relative expression / actin). [Figure 3A] 1 is a bar graph showing the change in CYP7A1 expression (relative mRNA expression / actin) in HuH-7 cells upon treatment for 18 hours with increasing concentrations of the selective FGFR4 inhibitor fisogatinib (BLU554). Treatment of the cells with the inhibitor resulted in increased expression of CYP7A1. [Figure 3B]1 is a bar graph showing the change in CYP7A1 expression (relative mRNA expression / actin) in HuH-7 cells upon treatment for 18 hours with increasing concentrations of the selective FGFR4 inhibitor lobritinib (FGF-401). Treatment of the cells with the inhibitor resulted in increased expression of CYP7A1. [Figure 3C] 1 is a bar graph showing the change in CYP7A1 expression (relative mRNA expression / actin) in HuH-7 cells upon treatment for 18 hours with increasing concentrations of erdafitinib, a selective FGFR4 inhibitor. Treatment of the cells with the inhibitor resulted in increased expression of CYP7A1. [Figure 3D] 1 is a bar graph showing the change in CYP7A1 expression (relative mRNA expression / actin) in HuH-7 cells upon treatment for 18 hours with increasing concentrations of the selective FGFR4 inhibitor, futibatinib. Treatment of the cells with the inhibitor resulted in increased expression of CYP7A1. [Figure 4A] 1 is a bar graph showing the change in CYP7A1 expression (relative mRNA expression / actin) in Hep3B cells after treatment of the cells with BLU554 (30 nM) in combination with fenofibrate, which reversed the increase in CYP7A1 expression caused by blockade of FGFR4 signaling in Hep3B cells. [Figure 4B] 1 is a bar graph showing the change in CYP7A1 expression (relative mRNA expression / actin) in Hep3B cells after treatment of the cells with FGF-401 (30 nM) in combination with fenofibrate, which reversed the increase in CYP7A1 expression caused by blockade of FGFR4 signaling in Hep3B cells. [Figure 4C] 1 is a bar graph showing the change in CYP7A1 expression (relative mRNA expression / actin) in Hep3B cells after treatment of the cells with erdafitinib (10 nM) in combination with fenofibrate, which reversed the increase in CYP7A1 expression caused by blockade of FGFR4 signaling in Hep3B cells. [Figure 4D]1 is a bar graph showing the change in CYP7A1 expression (relative mRNA expression / actin) in Hep3B cells after treatment of the cells with futibatinib (200 nM) in combination with fenofibrate, which reversed the increase in CYP7A1 expression caused by blockade of FGFR4 signaling in Hep3B cells. [Figure 5A] 1 is a bar graph showing the change in CYP7A1 expression (relative mRNA expression / actin) in HuH7 cells after treatment of the cells with BLU554 (30 nM) in combination with fenofibrate. In all cases except for futibatinib in HuH-7 cells, combination treatment with fenofibrate reduced CYP7A1 levels compared to FGFR inhibitor treatment alone. [Figure 5B] 1 is a bar graph showing the change in CYP7A1 expression (relative mRNA expression / actin) in HuH7 cells after treatment of the cells with FGF-401 (30 nM) in combination with fenofibrate. In all cases except for futibatinib in HuH-7 cells, combination treatment with fenofibrate reduced CYP7A1 levels compared to FGFR inhibitor treatment alone. [Figure 5C] 1 is a bar graph showing the change in CYP7A1 expression (relative mRNA expression / actin) in HuH7 cells after treatment of the cells with erdafitinib (10 nM) in combination with fenofibrate. In all cases except for futibatinib in HuH-7 cells, combination treatment with fenofibrate reduced CYP7A1 levels compared to FGFR inhibitor treatment alone. [Figure 5D] 1 is a bar graph showing the change in CYP7A1 expression (relative mRNA expression / actin) in HuH7 cells following treatment of the cells with futibatinib (200 nM) in combination with fenofibrate. [Figure 6A]1 is a bar graph showing the change in CYP7A1 expression (relative expression / actin) in Hep3B cells co-treated with fenofibric acid and an FGFR4 inhibitor, BLU554 (50 nM). Fenofibrate forms the active metabolite fenofibric acid. Co-treatment of Hep3B cells with fenofibric acid reduced the FGFR4 inhibitor-induced increase in CYP7A1 expression, as shown by BLU554. [Figure 6B] 1 is a bar graph showing the change in CYP7A1 expression (relative expression / actin) in Hep3B cells co-treated with fenofibric acid and an FGFR4 inhibitor, FGF-401 (30 nM). Fenofibrate forms the active metabolite fenofibric acid. Co-treatment of Hep3B cells with fenofibric acid reduced the FGFR4 inhibitor-induced increase in CYP7A1 expression, as shown by FGF-401. [Figure 7A] 1 is a bar graph showing the change in CYP7A1 expression (relative expression / actin) in Hep3B cells co-treated with ciprofibrate and the FGFR4 inhibitor BLU-554 (50 nM). [Figure 7B] 1 is a bar graph showing the change in CYP7A1 expression (relative expression / actin) in Hep3B cells co-treated with ciprofibrate and the FGFR4 inhibitor FGF-401 (30 nM). [Figure 7C] 1 is a bar graph showing the change in CYP7A1 expression (relative expression / actin) in Hep3B cells co-treated with ciprofibrate and the FGFR4 inhibitor erdafitinib (10 nM). [Figure 7D] 1 is a bar graph showing the change in CYP7A1 expression (relative expression / actin) in Hep3B cells co-treated with ciprofibrate and the FGFR4 inhibitor futibatinib (200 nM). [Figure 8A] 1 is a bar graph showing the change in CYP7A1 expression (relative expression / actin) in HuH7 cells co-treated with ciprofibrate and the FGFR4 inhibitor BLU-554 (50 nM). [Figure 8B] 1 is a bar graph showing the change in CYP7A1 expression (relative expression / actin) in HuH7 cells co-treated with ciprofibrate and the FGFR4 inhibitor FGF-401 (30 nM). [Figure 8C] 1 is a bar graph showing the change in CYP7A1 expression (relative expression / actin) in HuH7 cells co-treated with ciprofibrate and the FGFR4 inhibitor erdafitinib (10 nM). [Figure 8D] 1 is a bar graph showing the change in CYP7A1 expression (relative expression / actin) in HuH7 cells co-treated with ciprofibrate and the FGFR4 inhibitor futibatinib (200 nM). [Figure 9A] 1 is a bar graph showing the change in CYP7A1 expression (relative expression / actin) in Hep3B cells co-treated with gemfibrozil and the FGFR4 inhibitor BLU-554 (50 nM). [Figure 9B] 1 is a bar graph showing the change in CYP7A1 expression (relative expression / actin) in Hep3B cells co-treated with gemfibrozil and the FGFR4 inhibitor FGF-401 (30 nM). [Figure 9C] 1 is a bar graph showing the change in CYP7A1 expression (relative expression / actin) in Hep3B cells co-treated with gemfibrozil and the FGFR4 inhibitor erdafitinib (10 nM). [Figure 9D] 1 is a bar graph showing the change in CYP7A1 expression (relative expression / actin) in Hep3B cells co-treated with gemfibrozil and the FGFR4 inhibitor futibatinib (200 nM). [Figure 10A] 1 is a bar graph showing the change in CYP7A1 expression (relative expression / actin) in HuH7 cells co-treated with gemfibrozil and the FGFR4 inhibitor BLU-554 (50 nM). [Figure 10B]1 is a bar graph showing the change in CYP7A1 expression (relative expression / actin) in HuH7 cells co-treated with gemfibrozil and the FGFR4 inhibitor FGF-401 (30 nM). [Figure 10C] 1 is a bar graph showing the change in CYP7A1 expression (relative expression / actin) in HuH7 cells co-treated with gemfibrozil and the FGFR4 inhibitor erdafitinib (10 nM). [Figure 10D] 1 is a bar graph showing the change in CYP7A1 expression (relative expression / actin) in HuH7 cells co-treated with gemfibrozil and the FGFR4 inhibitor futibatinib (200 nM). [Figure 11A] 1 is a bar graph showing the change in CYP7A1 expression (relative expression / actin) in Hep3B cells co-treated with pemafibrate and the FGFR4 inhibitor BLU-554 (50 nM). [Figure 11B] 1 is a bar graph showing the change in CYP7A1 expression (relative expression / actin) in Hep3B cells co-treated with pemafibrate and the FGFR4 inhibitor FGF-401 (30 nM). [Figure 11C] 1 is a bar graph showing the change in CYP7A1 expression (relative expression / actin) in Hep3B cells co-treated with pemafibrate and the FGFR4 inhibitor erdafitinib (10 nM). [Figure 11D] 1 is a bar graph showing the change in CYP7A1 expression (relative expression / actin) in Hep3B cells co-treated with pemafibrate and the FGFR4 inhibitor futibatinib (200 nM). [Figure 12A] 1 is a bar graph showing the change in CYP7A1 expression (relative expression / actin) in HuH7 cells co-treated with pemafibrate and the FGFR4 inhibitor BLU-554 (50 nM). [Figure 12B] 1 is a bar graph showing the change in CYP7A1 expression (relative expression / actin) in HuH7 cells co-treated with pemafibrate and the FGFR4 inhibitor FGF-401 (30 nM). [Figure 12C] 1 is a bar graph showing the change in CYP7A1 expression (relative expression / actin) in HuH7 cells co-treated with pemafibrate and the FGFR4 inhibitor erdafitinib (10 nM). [Figure 12D] 1 is a bar graph showing the change in CYP7A1 expression (relative expression / actin) in HuH7 cells co-treated with pemafibrate and the FGFR4 inhibitor futibatinib (200 nM). [Figure 13A] 1 is a bar graph showing increased serum C4 levels (ng / ml) in mice treated with anti-FGFR4 therapy, either FGF401 at 30 mg / kg or H3B-6527 at 300 mg / kg. [Figure 13B] 1 is a bar graph showing increased serum C4 levels (ng / ml) in mice treated with 100 mg / kg BLU554 anti-FGFR4 therapy. [Figure 14A] FIG. 1 is a bar graph showing the change in bile acid (BA) levels (fold change vs. control) in liver of athymic nude (Nu / Nu) mice treated for 3 weeks with oral doses of vehicle ((1) 0.5% MC / Tween), H3B-6527 ((2) 300 mg / kg, BID) or FGF401 / Lobritinib ((3) 30 mg / kg, BID). Mice were euthanized 4 hours after the last dose and plasma, liver and gallbladder samples were collected to measure bile acid levels. Full names of bile acids measured: LCA: lithocholic acid, DCA: deoxycholic acid, TLCA: taurolithocholic acid, TDCA: taurodeoxycholic acid, CDCA: chenodeoxycholic acid, UDCA: ursodeoxycholic acid, CA: cholic acid, aMCA: α-muricholic acid, bMCA: β-muricholic acid, wMCA: ω-muricholic acid, GCA: glycodeoxycholic acid, TCDCA: taurochenodeoxycholic acid, TUDCA: tauroursodeoxycholic acid, TCA: taurocholic acid. [Figure 14B]FIG. 1 is a bar graph showing the change in plasma bile acid (BA) levels (fold change vs. control) in athymic nude (Nu / Nu) mice treated for 3 weeks with oral doses of vehicle ((1) 0.5% MC / Tween), H3B-6527 ((2) 300 mg / kg, BID) or FGF401 / Lobritinib ((3) 30 mg / kg, BID). Mice were euthanized 4 hours after the last dose and plasma, liver and gallbladder samples were collected to measure bile acid levels. Full names of bile acids measured: LCA: lithocholic acid, DCA: deoxycholic acid, TLCA: taurolithocholic acid, TDCA: taurodeoxycholic acid, CDCA: chenodeoxycholic acid, UDCA: ursodeoxycholic acid, CA: cholic acid, aMCA: α-muricholic acid, bMCA: β-muricholic acid, wMCA: ω-muricholic acid, GCA: glycodeoxycholic acid, TCDCA: taurochenodeoxycholic acid, TUDCA: tauroursodeoxycholic acid, TCA: taurocholic acid. [Figure 14C] FIG. 1 is a bar graph showing the change in bile acid (BA) levels (fold change vs. control) in the gallbladder of athymic nude (Nu / Nu) mice treated for 3 weeks with oral doses of vehicle ((1) 0.5% MC / Tween), H3B-6527 ((2) 300 mg / kg, BID) or FGF401 / Lobritinib ((3) 30 mg / kg, BID). Mice were euthanized 4 hours after the last dose and plasma, liver and gallbladder samples were collected to measure bile acid levels. Full names of bile acids measured: LCA: lithocholic acid, DCA: deoxycholic acid, TLCA: taurolithocholic acid, TDCA: taurodeoxycholic acid, CDCA: chenodeoxycholic acid, UDCA: ursodeoxycholic acid, CA: cholic acid, aMCA: α-muricholic acid, bMCA: β-muricholic acid, wMCA: ω-muricholic acid, GCA: glycodeoxycholic acid, TCDCA: taurochenodeoxycholic acid, TUDCA: tauroursodeoxycholic acid, TCA: taurocholic acid. [Figure 15A]1 is a bar graph showing the change in serum C4 (ng / ml) with treatment with BLU554 (100 mg / kg) and the indicated doses of fenofibrate (mg / kg). Fenofibrate administered at the indicated concentrations (mg / kg) ameliorated the increase in serum C4 levels induced by BLU-554 (100 mg / kg) in vivo. [Figure 15B] 1 is a bar graph showing changes in Cyp7a1 mRNA expression (%Actb) by treatment with BLU554 (100 mg / kg) and the indicated doses of fenofibrate (mg / kg). Fenofibrate administered at the indicated concentrations (mg / kg) ameliorated the elevation of serum C4 levels induced by BLU-554 (100 mg / kg) in vivo. FIG. 1 shows that co-administration of fenofibrate with BLU-554 dose-dependently reversed BLU-554-induced Cyp7a1 upregulation in mouse liver. [Figure 16A] FIG. 1 is a graph showing the change in serum C4 (ng / ml) in athymic nude mice bearing HuH-7 tumors and treated for 2 weeks with fenofibrate (40 mg / kg QD), FGF401 (30 or 100 mg / kg BID), BLU554 (100 mg / kg BID (100 mpk)), fenofibrate (40 mg / kg QD) plus FGF401 (30 or 100 mg / kg BID) or fenofibrate (40 mg / kg QD) plus BLU554 (100 mg / kg BID (100 mpk)). [Figure 16B]FIG. 16B is a graph showing the change in tumor volume (mm3) in athymic nude mice bearing HuH-7 tumors and treated for 2 weeks with fenofibrate (40 mg / kg QD), FGF401 (30 or 100 mg / kg BID), BLU554 (100 mg / kg BID (100 mpk)), fenofibrate (40 mg / kg QD) plus FGF401 (30 or 100 mg / kg BID) or fenofibrate (40 mg / kg QD) plus BLU554 (100 mg / kg BID (100 mpk). FIG. 16C shows the change in tumor volume (mm3) over time (days) in mice treated as shown in FIG. 16A. Data points represent mean tumor volume (n=6 per group) and error bars represent standard error of the mean. [Figure 16C] FIG. 16B is a graph showing the change in tumor volume (mm3) over time (days) in mice treated as shown in FIG. 16A. Athymic nude mice bearing HuH-7 tumors and treated for 2 weeks with fenofibrate (40 mg / kg QD), FGF401 (30 or 100 mg / kg BID), BLU554 (100 mg / kg BID (100 mpk)), fenofibrate (40 mg / kg QD) plus FGF401 (30 or 100 mg / kg BID) or fenofibrate (40 mg / kg QD) plus BLU554 (100 mg / kg BID (100 mpk)). Data points represent mean tumor volume (n=6 per group) and error bars represent standard error of the mean. [Figure 17A] 1 is a bar graph showing CYP7A1 mRNA expression (relative expression / actin) in Hep3B cells treated with anti-FGF19 antibody alone. Anti-FGF19 antibody neutralizes FGF19 ligand, blocks the signaling pathway that binds FGF19 ligand to FGFR4, and then activates downstream signaling that primarily downregulates CYP7A1 expression in hepatocytes. [Figure 17B]1 is a bar graph showing CYP7A1 mRNA expression (relative expression / actin) in HuH-7 cells treated with anti-FGF19 antibody alone. Anti-FGF19 antibody neutralizes FGF19 ligand, blocks the signaling pathway that binds FGF19 ligand to FGFR4, and then activates downstream signaling that primarily downregulates CYP7A1 expression in hepatocytes. [Figure 17C] 1 is a bar graph showing CYP7A1mRNA expression (relative expression / actin) in Hep3B cells treated with anti-FGF19 antibody and fenofibrate in combination.Anti-FGF19 antibody neutralizes FGF19 ligand, blocks the signaling pathway that connects FGF19 ligand to FGFR4, and then activates downstream signaling, which mainly downregulates CYP7A1 expression in hepatocytes.Figure 1 shows that fenofibrate can reverse the increase in CYP7A1 expression induced by FGFR4 inhibition caused by neutralization of FGF19 using anti-FGF19 antibody. [Figure 17D] 1 is a bar graph showing CYP7A1mRNA expression (relative expression / actin) in HuH-7 cells treated with anti-FGF19 antibody and fenofibrate in combination.Anti-FGF19 antibody neutralizes FGF19 ligand, blocks the signaling pathway that connects FGF19 ligand to FGFR4, and then activates downstream signaling, which mainly downregulates CYP7A1 expression in hepatocytes.Figure 1 shows that fenofibrate can reverse the increase in CYP7A1 expression induced by FGFR4 inhibition caused by neutralization of FGF19 using anti-FGF19 antibody. [Figure 17E] shows exemplary images from a Western blot assay detecting the decrease in phosphorylated FGFR4 (Tyr642) following anti-FGF19 antibody treatment. [Figure 18A]1 is a bar graph showing the change in CYP7A1 upregulation in Hep3B cells when cells are treated with 30 nM of the FGFR4 inhibitor FGF401 in combination with the PPAR alpha / delta agonist elafibranor at the indicated concentrations (μM). Data shows the relative mRNA expression of CYP7A1 to actin. [Figure 18B] 1 is a bar graph showing the change in CYP7A1 upregulation in HuH-7 cells when cells are treated with 30 nM of the FGFR4 inhibitor FGF401 in combination with the PPAR alpha / delta agonist elafibranor at the indicated concentrations (μM). Data shows the relative mRNA expression of CYP7A1 to actin. [Figure 19] FIG. 1 is a bar graph showing the change in serum C4 levels (ng / ml) in C57BL / 6 mice orally dosed for 6 days with vehicle (1), fenofibrate ((2) 100 mg / kg QD), gemfibrozil ((3) 100 mg / kg QD), BLU554 ((4) 100 mg / kg BID), a combination of BLU554 and fenofibrate, or a combination of BLU554 and gemfibrozil (30 mg / kg QD, 100 mg / kg QD, 150 mg / kg QD, or 300 mg / kg QD). Prior to the last day of the study, mice were fasted overnight and serum was collected 4 hours after the last dose for C4 analysis. The vehicle for fenofibrate and gemfibrozil was 0.5% methylcellulose / 0.5% Tween 80, and the vehicle for BLU554 was 80% PEG400 / 4% hydroxypropyl-β-cyclodextrin. In the combination treatment groups, the compounds were formulated and administered separately. Columns represent the mean C4 values ​​(n=3-4 per group) and error bars represent the standard error of the mean. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] The disclosed compositions and methods may be more readily understood by reference to the following detailed description, which is provided in conjunction with the accompanying drawings, which form a part of this disclosure: It is to be understood that the disclosed compositions and methods are not limited to the specific methods described and / or illustrated herein, and that the terminology used herein is for the purpose of describing particular embodiments by way of example only, and is not intended to be a limitation of the claimed compositions and methods.

[0013] Unless specifically stated otherwise, any explanation of a possible mechanism or mode of action, or reason for an improvement, is intended to be illustrative only, and the disclosed compositions and methods are not limited by the accuracy or inaccuracy of any such proposed mechanism or mode of action, or reason for an improvement.

[0014] Throughout this specification, the description refers to compositions and methods of using the compositions. When this disclosure describes or claims features or embodiments related to a composition, such features or embodiments are equally applicable to methods of using the composition. Similarly, when this disclosure describes or claims features or embodiments related to methods of using a composition, such features or embodiments are equally applicable to the composition.

[0015] It should be understood that certain features of the disclosed compositions and methods that are, for clarity, described herein in the context of separate embodiments, can also be provided in combination in a single embodiment. Conversely, for brevity, various features of the disclosed compositions and methods that are described in the context of a single embodiment can also be provided separately or in any subcombination.

[0016] Throughout the specification and claims, various terms relating to the aspects of the present specification are used. Such terms are to be given their ordinary meaning in the art unless otherwise indicated. Other specifically defined terms are to be interpreted in a manner consistent with the definitions provided herein.

[0017] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. In case of conflict, the present specification, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing. The materials, methods, and examples disclosed herein are illustrative only and are not intended to be limiting.

[0018] The terms "substantial" or "substantially" as used herein refer to a degree of similarity, difference, increase, or decrease in comparison to a known value. Substantial can include a similarity, difference, increase, or decrease of at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% in comparison to a known value.

[0019] It is understood that amounts, sizes, formulations, parameters, and other quantities and characteristics are not, and need not be, exact, but may be approximate and / or larger or smaller, as desired, to reflect tolerances, conversion factors, rounding, measurement errors, and the like, and other factors known to those of skill in the art. In general, an amount, size, formulation, parameter, or other quantity or characteristic is "about" or "approximate," whether or not it is expressly stated as such. When "about" is used before a quantitative value, it is understood that the parameter also includes the particular quantitative value itself, unless specifically stated otherwise. The term "about," as used herein when referring to a measurable value, such as an amount, a temporal duration, and the like, is meant to encompass a variation of ±10%, ±5%, ±1%, or ±0.1% from the specified value, when such variation is appropriate to carry out the disclosed method. The modifier "about" should also be considered to disclose a range defined by the absolute values ​​of the two endpoints. For example, the phrase "about 2 to about 4" also discloses a range of "2 to 4." The term "about" may refer to plus or minus 10% of a given number. For example, "about 10%" may indicate a range of 9% to 11%, and "about 1" may mean 0.9 to 1.1. Other meanings of "about" may be apparent from the context, such as rounding, so, for example, "about 1" may also mean 0.5 to 1.4.

[0020] As used herein, approximation language may be applied to modify any quantitative expression that can be varied without resulting in a change in the basic function to which it is related. All ranges are combinable.

[0021] Furthermore, the term "comprising" should be understood to have its open meaning of "including," but this term also includes the limiting meaning of the term "consisting. For example, a composition containing components A and B can be a composition containing A, B, and other components, but can also be a composition consisting of only A and B.

[0022] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to a "cell" includes a combination of two or more cells, and the like.

[0023] As used herein, the terms "individual," "patient," and "subject" are used interchangeably to refer to members of any animal species, including, but not limited to, birds, humans and other primates, and other mammals, including commercially important mammals or animal models such as mice, rats, monkeys, cows, pigs, horses, sheep, cats, and dogs. Preferably, the subject is a human.

[0024] As used herein, the terms "treat", "treatment", and the like refer to a method or step taken to reduce or alleviate the number, severity, and / or frequency of one or more symptoms of a disease in a subject. As used herein, "treat" and "treatment" may include prevention, management, prophylactic treatment, and / or inhibition or reduction of the number, severity, and / or frequency of one or more symptoms of a disease in a subject. As used herein, "treat" and "treatment" may also refer to prevention, management, prophylactic treatment, and / or inhibition or reduction of the number, severity, and / or frequency of one or more adverse events resulting from an anti-FGFR4 treatment.

[0025] The terms "effective amount" and "therapeutically effective amount" are used interchangeably herein and refer to an amount of a drug effective to achieve a particular biological or therapeutic result, such as, but not limited to, amelioration of one or more symptoms of a disease, reduction in the number, severity, and / or frequency of one or more symptoms of a disease in a subject. The terms "effective amount" and "therapeutically effective amount" are used interchangeably herein and refer to an amount of a drug effective to achieve a particular biological or therapeutic result, such as, but not limited to, amelioration of one or more adverse events resulting from anti-FGFR4 treatment. The therapeutically effective amount of a drug may vary depending on factors such as the disease state, age, sex, body surface area, and weight of the subject, and the ability of the drug to induce a desired response in the subject.

[0026] As used herein, the term "in need of" in the context of a subject "in need of" refers to the need for treatment for the treatment of a disease or condition or for the treatment of an adverse event arising from anti-FGFR treatment.

[0027] As used herein, the term "small molecule" refers to a molecule having a molecular weight of less than 1000 grams / mole.

[0028] Combination therapy The present specification provides a method for treating a subject that requires anti-FGFR4 treatment.The method includes administering a therapeutically effective amount of a PPAR alpha agonist to the subject in combination with anti-FGFR4 treatment.The present specification also provides a method for treating a subject, including administering a therapeutically effective amount of a PPAR alpha agonist to the subject in combination with a means for anti-FGFR4 treatment.The means for anti-FGFR4 treatment are known in the art, and examples include, for example, FGFR4 inhibitors.

[0029] In some embodiments, anti-FGFR4 treatment or means for anti-FGFR4 treatment is an FGFR4 inhibitor. FGFR4 inhibitor can include direct FGFR4 inhibitor. Direct FGFR4 inhibitor can directly contact, interact, bind, or otherwise change (e.g., reduce) the level of FGFR4 activity. In some embodiments, anti-FGFR4 treatment or means for anti-FGFR4 treatment can be a direct FGFR4 inhibitor. Examples of direct FGFR4 inhibitor include small molecule FGFR4 inhibitor, small molecule pan-FGFR inhibitor, or anti-FGFR4 antibody or its binding fragment.

[0030] FGFR4 inhibitors can include indirect FGFR4 inhibitors. Indirect FGFR4 inhibitors do not directly contact, interact, bind, or otherwise change (e.g., reduce) the level of FGFR4 activity. Indirect FGFR4 inhibitors indirectly inhibit FGFR4 function, for example, by contacting, interacting, or binding with FGF19, Klotho beta (also referred to herein as Klotho-β, KLβ, or KLB), or other molecules in the FGFR4 signaling pathway. Indirect FGFR4 inhibitors can include FGFR4 signaling inhibitors. Exemplary FGFR4 signaling inhibitors are inhibitors that inhibit or reduce the level of signaling molecules that function upstream or downstream of FGFR4 in the FGFR4 signaling pathway. In some embodiments, anti-FGFR4 therapy or a means for anti-FGFR4 therapy can be an indirect FGFR4 inhibitor. Examples of indirect FGFR4 inhibitors include anti-FGF19 antibodies or binding fragments thereof, and anti-Klotho beta antibodies or binding fragments thereof. Anti-FGF19 antibodies are described at least in U.S. Patent Nos. 7,678,373, 8,293,241, 8,409,579, and 9,266,955. Anti-Kloso beta antibodies are described at least in U.S. Patent Application Publication No. US / 2022 / 0089780. Other examples of anti-Kloso beta antibodies or binding fragments thereof include anti-human Kloso beta antibodies or binding fragments thereof obtained from Novus Biologicals (catalog numbers: NBP3-09315, MAB58891, MAB5889, and AF5889), Affinity Biosciences (catalog number DF14991), Thermo Fisher Scientific (catalog numbers: PA5-119246 and PA5-44023) or R&D Systems (catalog numbers: AF2619 and MAB3738).

[0031] In some embodiments, the anti-FGFR4 treatment or the means for anti-FGFR4 treatment is a direct FGFR4 inhibitor and / or an indirect FGFR4 inhibitor. The FGFR4 inhibitor can include a small molecule FGFR4 inhibitor, or an anti-FGFR4 antibody or a binding fragment thereof. The FGFR4 signaling inhibitor can include a small molecule FGFR4 inhibitor, an anti-FGFR4 antibody or a binding fragment thereof, an anti-FGF19 antibody or a binding fragment thereof, or an anti-Klothobeta antibody or a binding fragment thereof. Small molecule FGFR4 inhibitors include, but are not limited to, robritinib (FGF401), H3B-6527, ICP-105, fisogatinib (BLU554), INCB062079, erdafitinib, futibatinib, pemigatinib, infigratinib, and combinations thereof.

[0032] The FGFR4 inhibitor may include an anti-FGFR4 antibody or a binding fragment thereof. The anti-FGFR4 antibody may include a U3-1784 antibody or a binding fragment thereof. The U3-1784 antibody or a binding fragment thereof includes a heavy chain variable region and a light chain variable region of the following amino acid sequence (Bartz et al., Mol Cancer Ther 2019;18:1832-43, the complementarity determining regions (CDRs) in the heavy chain variable region and the light chain variable region are underlined).

[0033] SEQ ID NO:1 [ka]

[0034] SEQ ID NO:2 [ka]

[0035] The FGFR4 signaling inhibitor may comprise an anti-FGF19 antibody or a binding fragment thereof. The anti-FGF19 antibody may comprise an FGF19 neutralizing antibody.

[0036] Anti-FGFR4 treatment or means for anti-FGFR4 treatment can include a combination of an FGFR4 inhibitor and a second chemotherapeutic agent. In some embodiments, the second chemotherapeutic agent is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is an antibody or a binding fragment of an antibody. The immune checkpoint inhibitor can include an antibody or a binding fragment of an antibody that binds to programmed cell death 1 (PD1), programmed cell death ligand 1 (PD-L1), programmed cell death ligand 2 (PD-L2), or cytotoxic T-lymphocyte antigen 4 (CTLA4).

[0037] In some embodiments, the PPAR-alpha agonist is a small molecule. In some embodiments, the PPAR-alpha agonist is fenofibrate, fenofibric acid, ciprofibrate, gemfibrozil, bezafibrate, elafibranor, pemafibrate, or a combination thereof. Fenofibrate is a prodrug that is hydrolyzed by tissue and plasma esterases after absorption to its main active metabolite, fenofibric acid. Elafibranor is a dual PPAR alpha / delta agonist. The chemical structures of fenofibrate, fenofibric acid, ciprofibrate, gemfibrozil, bezafibrate, elafibranor, and pemafibrate are shown below. [ka] [ka]

[0038] The disclosed methods can include administering the FGFR4 inhibitor in one or more doses in an amount between about 0.5 mg and about 3000 mg per day. For example, the disclosed methods can include administering the FGFR4 inhibitor in one or more doses in an amount between about 0.5 mg and about 3000 mg, between about 1 mg and about 2500 mg, between about 5 mg and about 2000 mg, between about 10 mg and about 3000 mg, between about 15 mg and about 3000 mg, between about 20 mg and about 3000 mg, between about 25 mg and about 3000 mg, between about 30 mg and about 3000 mg, between about 35 mg and about 3000 mg, between about 50 mg and about 3000 mg, between about 45 mg and about 3000 mg, or between about 50 mg and about 3000 mg per day. For example, the disclosed methods can include administering an FGFR4 inhibitor in one or more doses in an amount between about 0.5 mg and about 2500 mg, between about 1 mg and about 2000 mg, between about 5 mg and about 1500 mg, between about 10 mg and about 1000 mg, between about 15 mg and about 500 mg, between about 20 mg and about 250 mg, between about 25 mg and about 200 mg, between about 30 mg and about 150 mg, between about 35 mg and about 100 mg, between about 40 mg and about 100 mg, between about 45 mg and about 100 mg, or between about 50 mg and about 100 mg per day.

[0039] The disclosed methods can include administering the FGFR4 inhibitor in an amount between about 0.01 mg / kg and about 50 mg / kg per day. The FGFR4 inhibitor can be administered in one or more doses. For example, the disclosed methods can include administering the FGFR4 inhibitor in an amount between about 0.01 mg / kg and about 50 mg / kg per day, between about 0.05 mg / kg and about 50 mg / kg, between about 0.1 mg / kg and about 50 mg / kg, between about 0.5 mg / kg and about 50 mg / kg, between about 1 mg / kg and about 50 mg / kg, between about 5 mg / kg and about 50 mg / kg, between about 10 mg / kg and about 50 mg / kg, between about 15 mg / kg and about 50 mg / kg, between about 20 mg / kg and about 50 mg / kg, between about 25 ... This may include administering the FGFR4 inhibitor in one or more doses in an amount of between about 50 mg / kg, between about 20 mg / kg and about 50 mg / kg, between about 25 mg / kg and about 50 mg / kg, between about 30 mg / kg and about 50 mg / kg, between about 35 mg / kg and about 50 mg / kg, between about 40 mg / kg and about 50 mg / kg, or between about 45 mg / kg and about 50 mg / kg.

[0040] In some embodiments, the disclosed methods comprise orally administering an FGFR4 inhibitor to a subject in a fed or fasted state.

[0041] In some embodiments, the disclosed methods include administering the FGFR4 inhibitor by injection. In some embodiments, the disclosed methods include administering the FGFR4 inhibitor by intravenous injection.

[0042] The disclosed methods can include administering a PPAR alpha agonist in an amount between about 0.05 mg and about 3000 mg per day in one or more doses. For example, the disclosed methods can include administering a PPAR alpha agonist in one or more doses in an amount between about 0.05 mg and about 3000 mg, between about 0.1 mg and about 3000 mg, between about 1 mg and about 2500 mg, between about 5 mg and about 2000 mg, between about 10 mg and about 3000 mg, between about 15 mg and about 3000 mg, between about 20 mg and about 3000 mg, between about 25 mg and about 3000 mg, between about 30 mg and about 3000 mg, between about 35 mg and about 3000 mg, between about 50 mg and about 3000 mg, between about 45 mg and about 3000 mg, or between about 50 mg and about 3000 mg per day. For example, the disclosed methods can include administering a PPAR alpha agonist in one or more doses in an amount between about 0.05 mg and about 2500 mg, between about 0.1 mg and about 2000 mg, between about 1 mg and about 2000 mg, between about 5 mg and about 1500 mg, between about 10 mg and about 1000 mg, between about 15 mg and about 500 mg, between about 20 mg and about 250 mg, between about 25 mg and about 200 mg, between about 30 mg and about 150 mg, between about 35 mg and about 100 mg, between about 40 mg and about 100 mg, between about 45 mg and about 100 mg, or between about 50 mg and about 100 mg per day.

[0043] The disclosed methods can include administering a PPAR alpha agonist in an amount between about 0.001 mg and about 50 mg per day. For example, the disclosed methods can include administering a PPAR alpha agonist in an amount between about 0.001 mg / kg and about 50 mg / kg, between about 0.005 mg / kg and about 50 mg / kg, between about 0.01 mg / kg and about 50 mg / kg, between about 0.05 mg / kg and about 50 mg / kg, between about 0.1 mg / kg and about 50 mg / kg, between about 0.5 mg / kg and about 50 mg / kg, between about 1 mg / kg and about 50 mg / kg, between about 5 mg / kg and about 50 mg / kg, between about 1 ... and about 50 mg / kg, between about 15 mg / kg and about 50 mg / kg, between about 20 mg / kg and about 50 mg / kg, between about 25 mg / kg and about 50 mg / kg, between about 30 mg / kg and about 50 mg / kg, between about 35 mg / kg and about 50 mg / kg, between about 40 mg / kg and about 50 mg / kg, or between about 45 mg / kg and about 50 mg / kg.

[0044] In some embodiments, the method further comprises administering to the subject a bile acid sequestrant. In some embodiments, the bile acid sequestrant is cholestyramine, colestipol, colesevelam, or a combination thereof. In some embodiments, the bile acid sequestrant is cholestyramine.

[0045] The disclosed methods can include administering a PPAR alpha agonist before, simultaneously, or after administration of an anti-FGFR4 therapy. In these methods, a bile acid sequestrant is further administered and can be administered before, simultaneously, or after administration of a PPAR alpha agonist. In these methods, a bile acid sequestrant is further administered and can be administered before, simultaneously, or after administration of an anti-FGFR4 therapy.

[0046] Also disclosed are methods of treating a subject in need of anti-FGFR4 therapy, comprising administering to the subject an anti-FGFR4 therapy in combination with a therapeutically effective amount of a PPAR alpha agonist and a bile acid sequestrant. The disclosed methods can include administering the PPAR alpha agonist and the bile acid sequestrant before, simultaneously, or after administration of the anti-FGFR4 therapy.

[0047] Examples of combination therapies are shown in Tables 1 and 2. [Table 1-1] [Table 1-2] [Table 2-1] [Table 2-2]

[0048] The disclosed method is administered to a subject in need of treatment for a proliferative disease, a metabolic disease, a cardiovascular disease, or a renal disease. The subject can be in need of treatment for a proliferative disease that is an FGFR4-mediated cancer, hepatocellular carcinoma, cholangiocarcinoma, or a solid tumor. The subject can be in need of treatment for a metabolic disease, such as nonalcoholic steatohepatitis (NASH) or diabetes. The subject can be in need of treatment for type 2 diabetes. The subject can be in need of treatment for concentric hypertrophy. The subject can be in need of treatment for cardiovascular disease. The subject can be in need of treatment for chronic renal disease. The subject can be in need of treatment for left ventricular hypertrophy.

[0049] The disclosed methods can provide one or more of: a reduction in the number of adverse events associated with anti-FGFR4 treatment, a reduction in the frequency of adverse events associated with anti-FGFR4 treatment, a reduction in the severity of adverse events associated with anti-FGFR4 treatment, an extension of the duration of anti-FGFR4 treatment, an increase in the daily dose of anti-FGFR4 treatment, and an increase in the subject's patient compliance with anti-FGFR4 treatment. In some embodiments, the adverse event is diarrhea, nausea, vomiting, an increase in aspartate transaminase (AST) level, an increase in alanine transaminase (ALT) level, an increase in gamma-glutamyltransferase (GGT) level, an increase in serum bilirubin level, an increase in prothrombin time (PT), or a combination thereof.

[0050] In some embodiments, the method provides for a reduction in serum levels of C4 (7-alpha-hydroxy-4-cholesten-3-one), bile acids, or a combination thereof. The method can reduce serum levels of C4 in a subject by between about 5% and about 95% compared to serum levels in a subject receiving anti-FGFR4 therapy without a PPAR alpha agonist. For example, the method can reduce serum levels of C4 in a subject by between about 5% and about 10%, between about 5% and about 15%, between about 5% and about 20%, between about 5% and about 25%, between about 5% and about 30%, between about 5% and about 35%, between about 5% and about 40%, between about 5% and about 45%, between about 5% and about 50%, between 5% and about 55%, between about 5% and about 60%, between about 5% and about 65%, between about 5% and about 70%, between about 5% and about 75%, between about 5% and about 80%, between about 5% and about 85%, between about 5% and about 90%, or between about 5% and about 95%, compared to serum C4 levels in a subject receiving anti-FGFR4 therapy without PPAR alpha.

[0051] composition Also disclosed is a composition comprising an FGFR4 inhibitor, a PPAR alpha agonist and a pharma- ceutically acceptable excipient. The composition may comprise the FGFR4 inhibitor and the PPAR alpha agonist in a single unit dosage form.

[0052] The present specification also discloses a composition comprising an FGFR4 inhibitor, a PPAR alpha agonist, a bile acid sequestrant, and a pharma- ceutically acceptable excipient. The disclosed composition can comprise an FGFR4 inhibitor, a PPAR alpha agonist, a bile acid sequestrant, and a pharma- ceutically acceptable excipient in a single unit dosage form.

[0053] In some embodiments, the FGFR4 inhibitor in the composition is a small molecule FGFR4 inhibitor. In some embodiments, the small molecule FGFR4 inhibitor is robritinib (FGF401), H3B-6527, ICP-105, fisogatinib (BLU554), INCB062079, erdafitinib, futibatinib, pemigatinib, infigratinib, or a combination thereof.

[0054] In some embodiments, the FGFR4 inhibitor in the composition is an anti-FGFR4 antibody or a binding fragment thereof, or an anti-FGR19 antibody or a binding fragment thereof. In some embodiments, the anti-FGFR4 antibody is U3-1784 or a binding fragment thereof.

[0055] In some embodiments, the PPAR-alpha agonist in the composition is fenofibrate, fenofibric acid, ciprofibrate, gemfibrozil, bezafibrate, elafibranor, pemafibrate, or a combination thereof. In some embodiments, the bile acid sequestrant is cholestyramine, colestipol, colesevelam, or a combination thereof.

[0056] kit The present specification also provides a kit comprising an FGFR4 inhibitor and a PPAR alpha agonist.The kit can comprise an FGFR4 inhibitor as a single unit dosage form and a PPAR alpha as a single unit dosage form.The kit can comprise an FGFR4 inhibitor and a PPAR alpha on the same blister pack.The kit can further comprise a bile acid sequestrant.

[0057] The kit may include instructions for use and a dosage chart providing dosage and regimen recommendations. EXAMPLES

[0058] FGFR4 inhibitors are being developed for the treatment of cancers such as hepatocellular carcinoma (HCC), solid tumors, CKD, and cardiovascular disease. Dysregulation of bile acids by FGFR4 inhibition may complicate or even limit anti-FGFR4 treatment in subjects.

[0059] Example 1. FGFR4 inhibitors increase CYP7A1 expression and BA biosynthesis, which is attenuated by the PPAR alpha agonists fenofibrate and fenofibric acid An exemplary negative feedback mechanism for bile acid (BA) synthesis is shown in Figures 1A and 1B. Selective inhibition of FGFR4 with an inhibitor increases BA biosynthesis (Figures 1C and 1D). PPAR alpha agonists counter this increase (Figure 1E).

[0060] Materials and Methods The Examples detail the use of FGFR4 inhibitors with or without PPAR alpha agonists, demonstrating that PPAR alpha agonists attenuate BA dysregulation by FGFR4 inhibitors in in vitro cell studies and in vivo treatments.

[0061] Treatment of HCC cell lines (Hep3B and HuH-7) with the selective FGFR4 inhibitors BLU-554 (30 nM) or FGF401 (30 nM) or the pan-FGFR inhibitors erdafitinib (10 nM) or futibatinib (200 nM) in combination with increasing concentrations of fenofibrate or fenofibric acid (2, 5, or 10 μM) caused a reversal of CYP7A1 upregulation induced by FGFR4 signaling inhibition (Figures 4A-6B).

[0062] For in vivo experiments, male C57BL / 6 mice were orally administered vehicle, fenofibrate, FGFR4 inhibitor, or a combination of fenofibrate and FGFR4 inhibitor at the indicated doses. Before the last day of the study, animals were fasted overnight and serum was collected 4 hours after the last dose. Serum C4 (7-alpha-hydroxy-4-cholesten-3-one) analysis was performed on an Agilent 6495 triple quadrupole mass spectrometer equipped with a jet stream source coupled to an Agilent 1290 UPLC stack. Data were processed using Waters TargetLynx data processing software. Calibration curves were generated using a reference standard (7A4C; Sigma-Aldrich). The calibration range was 0.5ng / ml-200ng / ml with an internal deuterated standard (7A4C-D7; Avanti Polar Lipids) concentration of 100ng / ml. The vehicle for fenofibrate, FGF401, and H3B-6527 was 0.5% methylcellulose / 0.5% Tween 80 (MC / Tween), and the vehicle for BLU554 was 80% PEG400 / 4% hydroxypropyl-β-cyclodextrin (HPBCD). When combination treatments were tested, the vehicle treatment group received both vehicles formulated separately. In the combination treatment group, the compounds were formulated and administered separately. The average C4 values ​​for each group of four mice are shown, and error bars represent the standard error of the mean.

[0063] result As shown in Figures 2A-2D and 3A-3D, treatment of cells with increasing concentrations of selective FGFR4 inhibitors for 18 hours increased CYP7A1 expression (relative expression / actin) in two hepatocellular carcinoma (HCC) cell lines, Hep3B cells (Figures 2A-2D) and HuH-7 cells (Figures 3A-3D). Treatment of cells with the FGFR4-specific inhibitors BLU-554 and FGF-401, and the pan-FGFR inhibitors erdafitinib and futibatinib, increased CYP7A1 expression in Hep3B (Figures 2A-2D) and HuH-7 cells (Figures 3A-3D), as measured using qPCR. Treatment of cells with BLU-554 (30 nM) in combination with fenofibrate resulted in the reversal of the increased CYP7A1 expression caused by FGFR4 signaling blockade using BLU-554 in both Hep3B (Figure 4A) and HuH-7 cells (Figure 5A). Treatment of cells with FGFR4-specific inhibitor FGF401 (Figures 4B and 5B) and pan-FGFR inhibitors erdafitinib (Figures 4C and 5C) and futibatinib (Figures 4D and 5D) in combination with fenofibrate also resulted in the reversal of the increased CYP7A1 expression, except in the case of futibatinib in HuH-7 cells. Hep3B cells were also treated with fenofibric acid, the active metabolite of fenofibrate. Co-treatment of BLU554 (Figure 6A) or FGF401 (Figure 6B) to treat cells with fenofibric acid also reduced CYP7A1 levels. Fenofibrate or fenofibric acid was added simultaneously at concentrations of 2, 5, and 10 μM in combination with 30 nM BLU-554 in growth medium (DMEM supplemented with 10% fetal bovine serum) for 18 hours. mRNA was then isolated and CYP7A1 expression was measured using TaqMan® qPCR assays (CYP7A1 assay ID: Hs00167982_m1, ACTNB assay ID: Hs99999903_m1).

[0064] Figures 7A-7D are bar graphs showing changes in CYP7A1 expression (relative expression / actin) in Hep3B cells co-treated with ciprofibrate and the FGFR4 inhibitors BLU-554 (50 nM, Figure 7A), FGF-401 (30 nM, Figure 7B), erdafitinib (10 nM, Figure 7C), and futibatinib (200 nM, Figure 7D).

[0065] Figures 8A-8D are bar graphs showing changes in CYP7A1 expression (relative expression / actin) in HuH7 cells co-treated with ciprofibrate and the FGFR4 inhibitors BLU-554 (50 nM, Figure 8A), FGF-401 (30 nM, Figure 8B), erdafitinib (10 nM, Figure 8C), and futibatinib (200 nM, Figure 8D).

[0066] Figures 9A-9D are bar graphs showing changes in CYP7A1 expression (relative expression / actin) in Hep3B cells co-treated with gemfibrozil and FGFR4 inhibitors BLU-554 (50 nM, Figure 9A), FGF-401 (30 nM, Figure 9B), erdafitinib (10 nM, Figure 9C), and futibatinib (200 nM, Figure 9D).

[0067] Figures 10A-10D are bar graphs showing changes in CYP7A1 expression (relative expression / actin) in HuH7 cells co-treated with gemfibrozil and FGFR4 inhibitors BLU-554 (50 nM, Figure 10A), FGF-401 (30 nM, Figure 10B), erdafitinib (10 nM, Figure 10C), and futibatinib (200 nM, Figure 10D).

[0068] Figures 11A-11D are bar graphs showing changes in CYP7A1 expression (relative expression / actin) in Hep3B cells co-treated with pemafibrate and the FGFR4 inhibitors BLU-554 (50 nM, Figure 11A), FGF-401 (30 nM, Figure 11B), erdafitinib (10 nM, Figure 11C), and futibatinib (200 nM, Figure 11D).

[0069] Figures 12A-12D are bar graphs showing changes in CYP7A1 expression (relative expression / actin) in HuH7 cells co-treated with pemafibrate and the FGFR4 inhibitors BLU-554 (50 nM, Figure 12A), FGF-401 (30 nM, Figure 12B), erdafitinib (10 nM, Figure 12C), and futibatinib (200 nM, Figure 12D).

[0070] Figures 13A and 13B are bar graphs showing the change in serum C4 levels (ng / ml) in mice treated with anti-FGFR4 therapy: athymic nude (Nu / Nu) mice were treated with H3B-6527 (300 mg / Kg) or FGF401 (30 mg / Kg) (Figure 13A) and C57BL / 6 mice were treated with BLU-554 (100 mg / kg) (Figure 13B).

[0071] Figures 14A-14C are bar graphs showing the change in bile acid (BA) levels (fold change vs. control) in liver (Figure 14A), plasma (Figure 14B), and gallbladder (Figure 14C) of athymic nude (Nu / Nu) mice treated for 3 weeks with vehicle (0.5% MC / Tween), H3B-6527 (300 mg / kg, BID), or FGF401 / Lobritinib (30 mg / kg, BID). Four hours after the last dose, mice were euthanized and plasma, liver, and gallbladder samples were collected to measure bile acid levels. Bile acids were measured at the University of Kansas Medical Center Metabolomics Core Facility according to established protocols. Treatment with either H3B-6527 or FGF-401 increased bile acid levels as measured in plasma, liver, and gallbladder when compared to vehicle (control) treated mice.

[0072] Figure 15A shows the changes in serum C4 levels in mice treated with BLU-554 (100 mg / kg) alone or in combination with different doses of the PPAR alpha agonist fenofibrate for 6 days, and shows that fenofibrate reduced the increase in C4 caused by BLU554. The data in Figure 15B show the changes in hepatic Cyp7a1 mRNA expression (%Actb) under the different test conditions, which correlate with the serum C4 levels in Figure 15A.

[0073] Example 2. Fenofibrate attenuates FGFR4 inhibitor-induced C4 increase in tumor-bearing mice Materials and Methods Compound BLU554, a highly covalent and specific FGFR4 inhibitor, was tested in the HuH-7 xenograft mouse model, a human hepatocellular carcinoma cell line driven by FGFR4 via FGF19 amplification.

[0074] Athymic nude Nu / Nu mice were injected subcutaneously in the abdomen with the FGF19-amplified HuH-7 human hepatocellular carcinoma cell line. Tumors grew to approximately 150 mm 3Once the mice were in the 100-mL dose group, they were dosed with vehicle, fenofibrate (40 mg / kg QD), FGF401 (30 or 100 mg / kg BID), BLU554 (100 mg / kg BID), fenofibrate (40 mg / kg, QD) plus FGF401 (30 or 100 mg / kg BID), or fenofibrate (40 mg / kg QD) plus BLU554 (100 mg / kg BID) for 14 days. Animals were fasted overnight and serum was collected 4 hours after the last dose. Serum C4 (7-alpha-hydroxy-4-cholesten-3-one) analysis was performed on an Agilent 6495 triple quadrupole mass spectrometer equipped with a jet stream source coupled to an Agilent 1290 UPLC stack. Data were processed using Waters TargetLynx data processing software. Calibration curves were generated using a reference standard (7A4C; Sigma-Aldrich). The calibration range was 0.5ng / ml-200ng / ml with an internal deuterated standard (7A4C-D7; Avanti Polar Lipids) concentration of 100ng / ml. The vehicle for fenofibrate and FGF401 was 0.5% methylcellulose / 0.5% Tween 80, and the vehicle for BLU554 was 80% PEG400 / 4% hydroxypropyl-β-cyclodextrin. Vehicle treatment groups received both vehicles formulated separately. In the combination treatment groups, the compounds were formulated and administered separately. The mean C4 values ​​for each group of six mice are shown, with error bars representing the standard error of the mean.

[0075] result As shown in FIG. 16A, BLU554 and FGF401 monotherapy significantly increased the levels of 7alpha-hydroxy-4-cholesten-3-one (C4), a peripheral marker of Cyp7a1 activity, serving as an indirect measure of hepatic bile acid synthesis. In contrast, co-treatment of BLU554-treated animals with the PPAR alpha agonist fenofibrate significantly attenuated the elevation of C4. Co-treatment of FGF401-treated animals with fenofibrate attenuated the elevation of C4 only at the 30 mg / kg BID dose, but not at the 100 mg / kg BID dose. These results demonstrate how co-treatment or co-administration of FGFR4 inhibitors with PPAR alpha agonists limits the toxicity induced by FGFR4 inhibitors. FIG. 16B-16C show the tumor volume (mm ) over time (days) in mice treated as shown in FIG. 16A. 3 ). Data points represent mean tumor volume (n=6 per group) and error bars represent standard error of the mean.

[0076] Example 3. Fenofibrate ameliorates neutralizing anti-FGF19 antibody-induced CYP7A1 upregulation in FGFR4-dependent / FGF19-amplified hepatocellular carcinoma cell lines Materials and Methods Hep3B and HUH-7 cells were treated with mouse isotype monoclonal IgG control (R&D-MAB002) at 10 μg / ml or hFGF19 antibody (R&D-AF969) at 2 μg / ml, 5 μg / ml and 10 μg / ml for 24 hours. Then, mRNA was isolated and CYP7A1 expression was measured using qPCR. Data shown represent the fold change in CYP7A1 mRNA levels compared to vehicle (DMSO) treated cells, and actin was used as an internal control.

[0077] Hep3B and HUH-7 cells were treated with 10 μg / ml hFGF19 antibody (R&D-AF969) in combination with 2 μM, 5 μM, or 10 μM fenofibrate for 24 hours. Two control groups included untreated cells and cells treated with 10 μg / ml mouse isotype monoclonal IgG (R&D-MAB002) for 24 hours.

[0078] result Treatment of cells with neutralizing anti-FGF19 antibody increased CYP7A1 expression in a dose-dependent manner in Hep3B (Figure 17A) and HuH-7 (Figure 17B). Treatment of cells with neutralizing anti-FGF19 (10 μg / ml) in combination with fenofibrate resulted in the reversal of the increase in CYP7A1 expression caused by FGFR4 signaling blockade using neutralizing anti-FGF19 antibody in both Hep3B (Figure 17C) and HuH-7 cells (Figure 17D).

[0079] Example 4. Effect of PPAR agonist elafibranor on FGF401-induced CYP7A1 expression Figures 18A and 18B are bar graphs showing changes in CYP7A1 upregulation in Hep3B cells (Figure 18A) and HuH-7 cells (Figure 18B) when cells are treated with 30 nM of the FGFR4 inhibitor FGF401 and the indicated concentrations (μM) of the PPAR alpha / delta agonist elafibranor. Data show the relative expression of CYP7A1 to actin.

[0080] Elafibranor did not attenuate the increased CYP7A1 upregulation in this assay when tested at the concentrations indicated.

[0081] Example 5. PPAR agonist gemfibrozil and its effect on serum C4 in vivo C57BL / 6 mice were dosed for 6 days with vehicle, fenofibrate (100 mg / kg QD), gemfibrozil (100 mg / kg QD), BLU554 (100 mg / kg BID), a combination of BLU554 and fenofibrate, or a combination of BLU554 and gemfibrozil (30 mg / kg QD, 100 mg / kg QD, 150 mg / kg QD, or 300 mg / kg QD). Before the last day of the study, mice were fasted overnight and serum was collected 4 hours after the last dose for C4 analysis. The vehicle for fenofibrate and gemfibrozil was 0.5% methylcellulose / 0.5% Tween 80, and the vehicle for BLU554 was 80% PEG400 / 4% hydroxypropyl-β-cyclodextrin. In the combination treatment groups, the compounds were formulated and administered separately. Columns represent mean C4 values ​​(n = 3–4 per group) and error bars represent standard error of the mean. Serum C4 (7-alpha-hydroxy-4-cholesten-3-one) analysis was performed on an Agilent 6495 triple quadrupole mass spectrometer equipped with a jet stream source coupled to an Agilent 1290 UPLC stack. Data were processed using Waters TargetLynx data processing software. Calibration curves were generated using a reference standard (7A4C; Sigma-Aldrich). The calibration range was 0.5 ng / ml-200 ng / ml with an internal deuterated standard (7A4C-D7; Avanti Polar Lipids) concentration of 100 ng / ml.

[0082] The results are shown in Figure 19.

Claims

1. A composition for use in the treatment of a subject requiring anti-fibroblast growth factor receptor 4 (anti-FGFR4) therapy, comprising an anti-FGFR4 therapeutic agent, which is administered in combination with a therapeutically effective amount of a peroxisome proliferator-activated receptor (PPAR) alpha agonist.

2. The anti-FGFR4 therapeutic agent is an FGFR4 inhibitor comprising a small molecule FGFR4 inhibitor (a small molecule is a molecule having a molecular weight of less than 1000 g / mol), an anti-FGFR4 antibody or its conjugated fragment, an anti-FGF19 antibody or its conjugated fragment, or an anti-closobeta antibody or its conjugated fragment, which can be optionally used. The aforementioned small molecule FGFR4 inhibitors are robritinib (FGF401), H3B-6527, ICP-105, fisogatinib (BLU554), INCB062079, erdafitinib, futivatinib, pemigatinib, infiglatinib, or a combination thereof. The anti-FFFR4 antibody is a humanized anti-FFFR4 antibody or its conjugated fragment, and is optionally selected. The aforementioned humanized anti-FGFR4 antibody is either the U3-1784 antibody or its conjugated fragment. The composition according to claim 1.

3. The aforementioned anti-FGFR4 therapeutic agent comprises a combination of an FGFR4 inhibitor and a second chemotherapeutic agent, optionally, The second chemotherapeutic agent is an immune checkpoint inhibitor, and is administered selectively. The aforementioned immune checkpoint inhibitor is an antibody or an antibody-binding fragment, and is optionally, The composition according to claim 1 or 2, wherein the antibody or the antibody-binding fragment binds to programmed cell death 1 (PD1), programmed cell death ligand 1 (PD-L1), programmed cell death ligand 2 (PD-L2), or cytotoxic T lymphocyte antigen 4 (CTLA4).

4. The PPAR-alpha agonist is a small molecule (a small molecule is a molecule having a molecular weight of less than 1000 grams / mol), and / or The composition according to claim 1 or 2, wherein the PPAR-alpha agonist is fenofibrate, fenofibric acid, cyprofibrate, gemfibrozil, bezafibrate, ellafibranol, pemafibrate, or a combination thereof.

5. The FGFR4 inhibitor described above is A dose between approximately 0.5 mg and approximately 3000 mg per day, and / or A dose between approximately 0.01 mg / kg and approximately 50 mg / kg per day, and / or In a state of feeding or fasting, the subject is given orally or by injection. The composition according to claim 2, which is administered.

6. The PPAR-alpha agonist is administered in doses between approximately 0.05 mg and approximately 3000 mg per day, and / or The composition according to claim 1 or 2, administered in doses of approximately 0.001 mg / kg and approximately 50 mg / kg per day.

7. The use further comprises administering a bile acid chelating agent to the subject, optionally, The bile acid chelating agent is cholestyramine, colestipol, coleseveram, or a combination thereof, or the bile acid chelating agent is cholestyramine and / or The composition according to claim 1 or 2, wherein the PPAR alpha agonist and the bile acid chelating agent are administered before, simultaneously with, or after the administration of the anti-FGFR4 therapeutic agent.

8. The composition according to claim 1 or 2, wherein the PPAR alpha agonist is administered before, simultaneously with, or after the administration of the anti-FGFR4 therapeutic agent.

9. A composition for use in the treatment of a subject requiring anti-fibroblast growth factor receptor 4 (anti-FGFR4) therapy, comprising an anti-FGFR4 therapeutic agent, which is administered in combination with a therapeutically effective amount of a peroxisome proliferator-activated receptor (PPAR) alpha agonist and a bile acid chelating agent.

10. The aforementioned subjects are those who require treatment for proliferative disorders, metabolic disorders, cardiovascular disorders, or kidney disorders, or The subjects mentioned above are those who require treatment for proliferative disorders that are FFFR4-mediated cancers, hepatocellular carcinomas, cholangiocarcinomas, or solid tumors, or The aforementioned subjects are those who require treatment for metabolic diseases including non-alcoholic steatohepatitis (NASH) or diabetes, or The aforementioned subjects are those who require treatment for type 2 diabetes, or The aforementioned subjects are those who require treatment for concentric cardiac hypertrophy, or The aforementioned subjects are those who require treatment for cardiovascular disease, or The aforementioned subjects require treatment for chronic kidney disease, or The aforementioned subjects require treatment for left ventricular hypertrophy. The composition according to any one of claims 1, 2, and 9.

11. The use provides one or more of the following: a reduction in the number of adverse events associated with anti-FGFR4 therapy, a reduction in the frequency of adverse events associated with anti-FGFR4 therapy, a reduction in the severity of adverse events associated with anti-FGFR4 therapy, an extension of the duration of anti-FGFR4 therapy, an increase in the daily dose of anti-FGFR4 therapy, and an increase in patient compliance to anti-FGFR4 therapy, optionally, The aforementioned adverse events include diarrhea, nausea, vomiting, elevated aspartate transaminase (AST) levels, elevated alanine transaminase (ALT) levels, elevated gamma-glutamyltransferase (GGT) levels, elevated serum bilirubin levels, increased prothrombin time (PT), or a combination thereof, and / or The aforementioned use results in a decrease in serum levels of C4 (7-alpha-hydroxy-4-cholesten-3-one), bile acids, or combinations thereof, and / or The composition according to any one of claims 1, 2, and 9, wherein the use results in a reduction of serum levels of C4 (7-alpha-hydroxy-4-cholesten-3-one) in a subject between about 5% and about 95% compared to the serum levels of the subject receiving anti-FGFR4 treatment without the PPAR alpha-agonist.

12. A composition comprising a fibroblast growth factor receptor 4 (FGFR4) inhibitor, a peroxisome proliferator-activated receptor (PPAR) alpha agonist, and a pharmaceutically acceptable excipient, wherein the FGFR4 inhibitor and the PPAR alpha agonist are optionally provided as a single unit dosage form.

13. The FGFR4 inhibitor comprises a small molecule FGFR4 inhibitor (a small molecule is a molecule having a molecular weight of less than 1000 g / mol), an anti-FGFR4 antibody or its conjugated fragment, an anti-FGF19 antibody or its conjugated fragment, or an anti-closobeta antibody or its conjugated fragment, optionally selected from the above. The small molecule FGFR4 inhibitors are robritinib (FGF401), H3B-6527, ICP-105, fisogatinib (BLU554), INCB062079, erdafitinib, futivatinib, pemigatinib, infiglatinib, or a combination thereof, and / or The composition according to claim 12, wherein the PPAR-alpha agonist is fenofibrate, fenofibric acid, cyprofibrate, gemfibrozil, bezafibrate, ellafibranol, pemafibrate, or a combination thereof.

14. A composition comprising a fibroblast growth factor receptor 4 (FGFR4) inhibitor, a peroxisome proliferator-activated receptor (PPAR) alpha agonist, a bile acid chelating agent, and a pharmaceutically acceptable excipient, wherein the composition comprises optionally The FGFR4 inhibitor and the PPAR alpha agonist are provided as a single unit dosage form and / or The FGFR4 inhibitor comprises a small molecule FGFR4 inhibitor (a small molecule is a molecule having a molecular weight of less than 1000 g / mol), an anti-FGFR4 antibody or its conjugated fragment, an anti-FGF19 antibody or its conjugated fragment, or an anti-closobeta antibody or its conjugated fragment, and / or The small molecule FGRR4 inhibitors are robritinib (FGF401), H3B-6527, ICP-105, fisogatinib (BLU554), INCB062079, erdafitinib, futivatinib, pemigatinib, infiglatinib, or a combination thereof, and / or The PPAR-alpha agonist is fenofibrate, fenofibric acid, cyprofibrate, gemfibrozil, bezafibrate, ellafibranol, pemafibrate, or a combination thereof, and / or A composition wherein the bile acid chelating agent is cholestyramine, colestipol, coleseveram, or a combination thereof, or the bile acid chelating agent is cholestyramine.

15. A kit comprising a fibroblast growth factor receptor 4 (FGFR4) inhibitor and a peroxisome proliferator-activated receptor (PPAR) alpha agonist, wherein optionally, The FGFR4 inhibitor is provided as a single unit dosage form, and the PPAR alfa is provided as a single unit dosage form, and / or The FGFR4 inhibitor and the PPAR alpha agonist are provided in the same blister pack, and / or The kit further comprises a bile acid chelating agent.

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