Compositions and methods of treatment for severe hypertriglyceridemia

Abstract

Compositions, methods and treatment regimens for mutant fibroblast growth factor-21 (FGF-21) peptide conjugates for the treatment of severe hypertriglyceridemia are provided.

Description

[Technical Field]

[0001] <Related Applications> This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 355,397, filed June 24, 2022, U.S. Provisional Patent Application No. 63 / 399,165, filed August 18, 2022, U.S. Provisional Patent Application No. 63 / 373,594, filed August 26, 2022, U.S. Provisional Patent Application No. 63 / 386,202, filed December 6, 2022, U.S. Provisional Patent Application No. 63 / 485,641, filed February 17, 2023, and U.S. Utility Patent Application No. 18 / 340,421, filed June 23, 2023, the disclosures of each of which are incorporated herein by reference in their entirety.

[0002] <Sequence Listing> This application contains a sequence listing containing the file "180234-011806.xml," created on June 22, 2023, and having a size of 40,014 bytes, the contents of which are incorporated herein by reference.

[0003] <Technical field to which the invention pertains> The present invention relates to compositions, methods, and treatment regimens for mutant Fibroblast Growth Factor-21 (FGF-21) peptide conjugates for the treatment of severe hypertriglyceridemia, comprising a polyethylene glycol (PEG) moiety attached to the mutant FGF-21 peptide via a glycosyl moiety. [Background technology]

[0004] FGF-21 is an endocrine hormone that occurs in nature as a monomeric, non-glycosylated protein. FGF-21, along with FGF-19 and FGF-23, belongs to the endocrine-acting subfamily, whereas the remaining 18 mammalian FGF ligands are classified into five paracrine-acting subfamilies. Summary of the Invention

[0005] Provided herein are methods for treating severe hypertriglyceridemia in a subject in need thereof. In some embodiments, the disclosed methods comprise administering a glycoPEGylated FGF21 analog to a subject in need thereof. In some embodiments, the disclosed methods comprise administering to a subject in need thereof a pharmaceutical composition comprising a mutant fibroblast growth factor-21 (FGF-21) peptide conjugate and a pharmaceutically acceptable carrier, wherein the mutant FGF-21 peptide conjugate comprises: i) a mutant FGF-21 peptide comprising the amino acid sequence of SEQ ID NO:2; ii) a glycosyl moiety; and iii) a 20 kDa polyethylene glycol (PEG), wherein the mutant FGF-21 peptide is attached to the glycosyl moiety by a covalent bond between the threonine at amino acid position 173 of SEQ ID NO:2 and a first site on the glycosyl moiety, and the glycosyl moiety is attached to the 20 kDa PEG by a covalent bond between a second site on the glycosyl moiety and the 20 kDa PEG.

[0006] An embodiment of the present disclosure is a method of treating severe hypertriglyceridemia (SHTG) in a subject in need thereof, comprising administering to the subject once weekly a pharmaceutical composition comprising 9 mg to 30 mg of a mutant fibroblast growth factor-21 (FGF-21) peptide conjugate and a pharmaceutically acceptable carrier, wherein the mutant FGF-21 peptide conjugate comprises: i) a mutant FGF-21 peptide comprising the amino acid sequence of SEQ ID NO:2; ii) a glycosyl moiety; and iii) a 20 kDa polyethylene glycol (PEG), wherein the mutant FGF-21 peptide is attached to the glycosyl moiety by a covalent bond between the threonine at amino acid position 173 of SEQ ID NO:2 and a first site of the glycosyl moiety, and wherein the glycosyl moiety is attached to the 20 kDa PEG by a covalent bond between a second site of the glycosyl moiety and the 20 kDa PEG, wherein administration of the pharmaceutical composition results in a reduction of triglyceride levels by at least 20% from baseline. In some embodiments, administration of the pharmaceutical composition results in a reduction in median triglyceride levels of at least 30% from baseline.

[0007] In some embodiments, administration of the pharmaceutical composition results in a reduction in median triglyceride levels of at least 30% from baseline.

[0008] In some embodiments, administration of the pharmaceutical composition results in a median decrease in triglyceride levels of at least 40% from baseline.

[0009] In some embodiments, the administration results in normalization of triglyceride levels to 150 mg / dl or less.

[0010] In some embodiments, the administration results in at least a 10% decrease in non-HDL cholesterol levels from baseline, at least a 10% decrease in apoB levels from baseline, at least a 10% decrease in apoC3 levels from baseline, or a combination thereof.

[0011] In some embodiments, the administration results in at least a 10% increase in HDL cholesterol levels from baseline, at least a 10% increase in adiponectin levels from baseline, or a combination thereof.

[0012] In some embodiments, the administration results in a decrease in the production of TG-rich lipoproteins.

[0013] In some embodiments, the administration results in improved clearance of TG-rich lipoproteins.

[0014] In some embodiments, the administration results in improved insulin sensitivity.

[0015] In some embodiments, the subject in need thereof has baseline hepatic steatosis.

[0016] In some embodiments, administration results in a greater than 30% reduction in liver fat.

[0017] In some embodiments, the methods of the present disclosure comprise administering the pharmaceutical composition to a subject in need thereof for 8 weeks or more.

[0018] In some embodiments, the subject in need thereof is a human subject. In some embodiments, the pharmaceutical composition is administered subcutaneously.

[0019] In some embodiments, the subject in need thereof has a fasting triglyceride (TG) of ≧500 mg / dL and ≦2000 mg / dL.

[0020] In some embodiments, the pharmaceutical composition comprises 9 mg of the mutant FGF-21 peptide conjugate.

[0021] In some embodiments, the pharmaceutical composition comprises 15 mg to 18 mg of the mutant FGF-21 peptide conjugate.

[0022] In some embodiments, the pharmaceutical composition comprises 27 mg to 30 mg of the mutant FGF-21 peptide conjugate. In some embodiments, administration results in at least a 10% decrease in an alanine transaminase (ALT) marker, at least a 10% decrease in an aspartate aminotransferase (AST) marker, at least a 10% median decrease in high-sensitivity C-reactive protein (hsCRP), or a combination thereof. In some embodiments, administration results in at least a 10% decrease in fasting plasma glucose, at least a 0.2% decrease in HBA1c, or a combination thereof.

[0023] In some embodiments, the subject in need of administration is undergoing background lipid-modifying therapy (LMT). In some embodiments, the LMT comprises a statin, prescription fish oil, a fibrate, or a combination thereof. In some embodiments, the subject in need of administration is on background LMT, and administration results in a reduction in non-HDL cholesterol levels of at least 10% from baseline. In some embodiments, the subject in need of administration is on background LMT, and administration results in a reduction in apoB cholesterol levels of at least 10% from baseline.

[0024] An embodiment of the present disclosure is a method of treating severe hypertriglyceridemia (SHTG) in a subject in need thereof, comprising administering to the subject in need thereof once weekly a pharmaceutical composition comprising 27 mg to 30 mg of a mutant fibroblast growth factor-21 (FGF-21) peptide conjugate and a pharmaceutically acceptable carrier, wherein the mutant FGF-21 peptide conjugate comprises: i) a mutant FGF-21 peptide comprising the amino acid sequence of SEQ ID NO:2; ii) a glycosyl moiety; and iii) a 20 kDa polyethylene glycol (PEG), wherein the mutant FGF-21 peptide is attached to the glycosyl moiety by a covalent bond between the threonine at amino acid position 173 of SEQ ID NO:2 and a first site of the glycosyl moiety; and the glycosyl moiety is attached to the 20 kDa polyethylene glycol (PEG) by a covalent bond between a second site of the glycosyl moiety and the 20 kDa PEG. The pharmaceutical composition is linked to PEG, and administration of the pharmaceutical composition results in a reduction in triglyceride levels of at least 20% from baseline, and administration of the pharmaceutical composition results in one or more of the following: at least a 10% reduction in alanine aminotransferase (ALT) markers from baseline, at least a 10% reduction in aspartate aminotransferase (AST) markers from baseline, at least a 10% median reduction in high sensitivity C-reactive protein (hsCRP) from baseline, at least a 10% reduction in fasting plasma glucose from baseline, at least a 0.3% reduction in HBA1c from baseline, at least a 10% reduction in non-HDL cholesterol levels from baseline, at least a 10% reduction in apoB levels from baseline, at least a 10% reduction in apoC3 levels from baseline, at least a 10% increase in HDL cholesterol levels from baseline, at least a 10% increase in adiponectin levels from baseline, and a greater than 30% reduction in liver fat from baseline.

[0025] Another aspect of the present disclosure is a method of treating severe hypertriglyceridemia (SHTG) in a subject in need thereof, comprising administering to the subject in need thereof once every two weeks a pharmaceutical composition comprising 31 mg to 44 mg of a mutant fibroblast growth factor-21 (FGF-21) peptide conjugate and a pharmaceutically acceptable carrier, wherein the mutant FGF-21 peptide conjugate comprises: i) a mutant FGF-21 peptide comprising the amino acid sequence of SEQ ID NO:2; ii) a glycosyl moiety; and iii) a 20 kDa polyethylene glycol (PEG), wherein the mutant FGF-21 peptide is attached to the glycosyl moiety by a covalent bond between the threonine at amino acid position 173 of SEQ ID NO:2 and a first site of the glycosyl moiety, and the glycosyl moiety is attached to the 20 kDa PEG by a covalent bond between a second site of the glycosyl moiety and the 20 kDa PEG, wherein administration of the pharmaceutical composition results in a reduction of triglyceride levels by at least 20% from baseline.

[0026] In some embodiments, administration of the pharmaceutical composition results in a reduction in median triglyceride levels of at least 30% from baseline.

[0027] In some embodiments, administration of the pharmaceutical composition results in a median decrease in triglyceride levels of at least 40% from baseline.

[0028] In some embodiments, the administration results in normalization of triglyceride levels to 150 mg / dl or less.

[0029] In some embodiments, the administration results in at least a 10% decrease in non-HDL cholesterol levels from baseline, at least a 10% decrease in apoB levels from baseline, at least a 10% decrease in apoC3 levels from baseline, or a combination thereof.

[0030] In some embodiments, the administration results in at least a 10% increase in HDL cholesterol levels from baseline, at least a 10% increase in adiponectin levels from baseline, or a combination thereof.

[0031] In some embodiments, the administration results in a decrease in the production of TG-rich lipoproteins.

[0032] In some embodiments, the administration results in improved clearance of TG-rich lipoproteins.

[0033] In some embodiments, the administration results in improved insulin sensitivity.

[0034] In some embodiments, the subject in need thereof has baseline hepatic steatosis.

[0035] In some embodiments, administration results in a greater than 30% reduction in liver fat.

[0036] In some embodiments, the methods of the present disclosure comprise administering the pharmaceutical composition to a subject in need thereof for 8 weeks or more.

[0037] In some embodiments, the subject in need thereof is a human subject. In some embodiments, the pharmaceutical composition is administered subcutaneously.

[0038] In some embodiments, the subject in need thereof has a fasting triglyceride (TG) of ≧500 mg / dL and ≦2000 mg / dL.

[0039] In some embodiments, the pharmaceutical composition comprises 36 mg to 44 mg of the mutant FGF-21 peptide conjugate. In some embodiments, administration results in a median decrease in hsCRP of at least 10%.

[0040] In some embodiments, the subject in need of administration is undergoing background lipid-modifying therapy (LMT). In some embodiments, the LMT comprises a statin, prescription fish oil, a fibrate, or a combination thereof. In some embodiments, the subject in need of administration is on background LMT, and administration results in a reduction in non-HDL cholesterol levels of at least 10% from baseline. In some embodiments, the subject in need of administration is on background LMT, and administration results in a reduction in apoB cholesterol levels of at least 10% from baseline.

[0041] An embodiment of the present disclosure is a method of treating severe hypertriglyceridemia (SHTG) in a subject in need thereof, comprising administering to the subject in need thereof once every two weeks a pharmaceutical composition comprising 36 mg to 44 mg of a mutant fibroblast growth factor-21 (FGF-21) peptide conjugate and a pharmaceutically acceptable carrier, wherein the mutant FGF-21 peptide conjugate comprises: i) a mutant FGF-21 peptide comprising the amino acid sequence of SEQ ID NO:2; ii) a glycosyl moiety; and iii) a 20 kDa polyethylene glycol (PEG), wherein the mutant FGF-21 peptide is attached to the glycosyl moiety by a covalent bond between the threonine at amino acid position 173 of SEQ ID NO:2 and a first site of the glycosyl moiety; and the glycosyl moiety is attached to the 20 kDa polyethylene glycol (PEG) by a covalent bond between a second site of the glycosyl moiety and the 20 kDa PEG. The pharmaceutical composition is linked to PEG, and administration of the pharmaceutical composition results in a reduction in triglyceride levels of at least 20% from baseline, and administration of the pharmaceutical composition results in one or more of the following: at least a 10% reduction in median high-sensitivity C-reactive protein (hsCRP) from baseline, at least a 10% reduction in non-HDL cholesterol from baseline, at least a 10% reduction in apoB from baseline, at least a 10% reduction in apoC3 from baseline, at least a 10% increase in HDL cholesterol from baseline, at least a 10% increase in adiponectin from baseline, and more than a 30% reduction in liver fat from baseline.

[0042] In some embodiments, the glycosyl moiety of the mutant FGF-21 peptide conjugate comprises at least one of an N-acetylgalactosamine (GalNAc) residue, a galactose (Gal) residue, a sialic acid (Sia) residue, a 5-amine analog of a Sia residue, a mannose (Man) residue, mannosamine, a glucose (Glc) residue, an N-acetylglucosamine (GlcNAc) residue, a fucose residue, a xylose residue, or a combination thereof.

[0043] In some embodiments, the glycosyl moiety of the mutant FGF-21 peptide conjugate comprises at least one N-acetylgalactosamine (GalNAc) residue, at least one galactose (Gal) residue, at least one sialic acid (Sia) residue, or a combination thereof. In some embodiments, at least one Sia residue is a nine-carbon carboxylated sugar. In some embodiments, at least one Sia residue is N-acetyl-neuraminic acid (2-keto-5-acetamido-3,5-dideoxy-D-glycero-D-galactonunulopyranose-1-onic acid (Neu5Ac), N-glycolylneuraminic acid (Neu5Gc), 2-keto-3-deoxy-nonulosonic acid (KDN), or a 9-substituted sialic acid. In some embodiments, the 9-substituted sialic acid is 9-O-lactyl-Neu5Ac, 9-O-acetyl-Neu5Ac, 9-deoxy-9-fluoro-Neu5Ac, or 9-azido-9-deoxy-Neu5Ac.

[0044] In some embodiments, the glycosyl moiety of the mutant FGF-21 peptide conjugate comprises the structure -GalNAc-Sia-.

[0045] In some embodiments, the 20 kDa PEG moiety of the mutant FGF-21 peptide conjugate is attached to the glycosyl moiety by a covalent bond to a linker, and the linker comprises at least one amino acid residue. In some embodiments, the at least one amino acid residue is glycine (Gly).

[0046] In some embodiments, the mutant FGF-21 peptide conjugate comprises the structure -GalNAc-Sia-Gly-PEG(20 kDa).

[0047] In some embodiments, the 20 kDa PEG of the FGF-21 peptide conjugate is a linear or branched PEG. In some embodiments, the 20 kDa PEG is a 20 kDa methoxy-PEG.

[0048] In some embodiments, the mutant FGF-21 peptide conjugate comprises the following structure: [ka] Here, n is an integer selected from 450 to 460.

[0049] An embodiment of the present disclosure relates to use of a pharmaceutical composition for the treatment of severe hypertriglyceridemia (SHTG), comprising administering once weekly to a subject in need thereof a pharmaceutical composition comprising 9 mg to 30 mg of a mutant fibroblast growth factor-21 (FGF-21) peptide conjugate and a pharmaceutically acceptable carrier, wherein the pharmaceutical composition is administered once weekly, the mutant FGF-21 peptide conjugate comprising: i) a mutant FGF-21 peptide comprising the amino acid sequence of SEQ ID NO:2; ii) a glycosyl moiety; and iii) a 20 kDa polyethylene glycol (PEG), wherein the mutant FGF-21 peptide is attached to the glycosyl moiety by a covalent bond between the threonine at amino acid position 173 of SEQ ID NO:2 and a first site of the glycosyl moiety, and the glycosyl moiety is attached to the 20 kDa PEG by a covalent bond between a second site of the glycosyl moiety and the 20 kDa PEG.

[0050] An embodiment of the present disclosure relates to the use of a pharmaceutical composition for reducing triglyceride levels by at least 20% from baseline in a subject with severe hypertriglyceridemia (SHTG), the pharmaceutical composition comprising 9 mg to 30 mg of a mutant fibroblast growth factor-21 (FGF-21) peptide conjugate and a pharmaceutically acceptable carrier, the pharmaceutical composition being administered once weekly, the mutant FGF-21 peptide conjugate comprising: i) a mutant FGF-21 peptide comprising the amino acid sequence of SEQ ID NO:2; ii) a glycosyl moiety; and iii) a 20 kDa polyethylene glycol (PEG), wherein the mutant FGF-21 peptide is attached to the glycosyl moiety by a covalent bond between the threonine at amino acid position 173 of SEQ ID NO:2 and a first site of the glycosyl moiety, and the glycosyl moiety is attached to the 20 kDa PEG by a covalent bond between a second site of the glycosyl moiety and the 20 kDa PEG. In some embodiments, the pharmaceutical composition reduces triglyceride levels by at least 30% or at least 40% from baseline in subjects with severe hypertriglyceridemia.

[0051] In some embodiments, the pharmaceutical composition comprises 27 mg to 30 mg of a mutant fibroblast growth factor-21 (FGF-21) peptide conjugate and a pharmaceutically acceptable carrier.

[0052] An embodiment of the present disclosure relates to the use of a pharmaceutical composition for the treatment of severe hypertriglyceridemia (SHTG), the pharmaceutical composition comprising 31 mg to 44 mg of a mutant fibroblast growth factor-21 (FGF-21) peptide conjugate and a pharmaceutically acceptable carrier, the pharmaceutical composition being administered once every two weeks, the mutant FGF-21 peptide conjugate comprising: i) a mutant FGF-21 peptide comprising the amino acid sequence of SEQ ID NO:2; ii) a glycosyl moiety; and iii) a 20 kDa polyethylene glycol (PEG), wherein the mutant FGF-21 peptide is attached to the glycosyl moiety by a covalent bond between the threonine at amino acid position 173 of SEQ ID NO:2 and a first site of the glycosyl moiety, and the glycosyl moiety is attached to the 20 kDa PEG by a covalent bond between a second site of the glycosyl moiety and the 20 kDa PEG.

[0053] An embodiment of the present disclosure relates to the use of a pharmaceutical composition for reducing triglyceride levels by at least 20% from baseline in a subject with severe hypertriglyceridemia (SHTG), the pharmaceutical composition comprising 9 mg to 30 mg of a mutant fibroblast growth factor-21 (FGF-21) peptide conjugate and a pharmaceutically acceptable carrier, the pharmaceutical composition being administered once every two weeks, the mutant FGF-21 peptide conjugate comprising: i) a mutant FGF-21 peptide comprising the amino acid sequence of SEQ ID NO:2; ii) a glycosyl moiety; and iii) a 20 kDa polyethylene glycol (PEG), wherein the mutant FGF-21 peptide is attached to the glycosyl moiety by a covalent bond between the threonine at amino acid position 173 of SEQ ID NO:2 and a first site of the glycosyl moiety, and the glycosyl moiety is attached to the 20 kDa PEG by a covalent bond between a second site of the glycosyl moiety and the 20 kDa PEG.

[0054] In some embodiments, the pharmaceutical composition reduces triglyceride levels by at least 30% or at least 40% from baseline in subjects with severe hypertriglyceridemia.

[0055] In some embodiments, the pharmaceutical composition comprises 36 mg to 44 mg of a mutant fibroblast growth factor-21 (FGF-21) peptide conjugate and a pharmaceutically acceptable carrier. [Brief explanation of the drawings]

[0056] [Figures 1A-1F] Figures 1A-1F are graphs showing the effect of pegozafermin on serum triglycerides. Figure 1A: Primary endpoint—median percent change in TG from baseline to week 8. Figure 1B: Percentage of subjects achieving a TG response of <500 mg / dL, <150 mg / dL, or ≥50% reduction from baseline to week 8. Figure 1C: TG subgroup analysis among subjects not receiving background lipid-modifying therapy. Figure 1D: TG subgroup analysis among subjects receiving background lipid-modifying therapy. Figure 1E: TG subgroup analysis among subjects without T2DM. Figure 1F: TG subgroup analysis among subjects with T2DM. Data are based on the full analysis set population; pooled pegozafermin groups were analyzed by van Elteren test, and individual pegozafermin dose groups were analyzed by Wilcoxon rank-sum test. QW, once weekly; Q2W, once every two weeks.

[0057] [Figures 2A-2E] Figures 2A-2E are graphs showing the effects of pegosafermin on serum lipids. LS mean (+ / - SE) percent change from baseline to week 8 for non-HDL-C (Figure 2A), apolipoprotein B (Figure 2B), apolipoprotein C3 (Figure 2C), LDL cholesterol (Figure 2D), and HDL cholesterol (Figure 2E). Data are based on the full analysis set population and analyzed with MMRM. QW, once weekly; Q2W, once every 2 weeks.

[0058] [Figures 3A-3E] Figures 3A-3E show the effects of pegosafermin on markers of liver health and metabolic dysregulation. Figure 3A: LS mean (+ / - SE) percent change from baseline to week 8 in liver fat fraction, as assessed by MRI-PDFF. Figure 3B: MRI proton density fat fraction (MRI-PDFF) images showing changes in liver fat fraction in a representative subject with a baseline liver fat fraction of >25%. Figure 3C: Percentage of subjects achieving normalization of liver fat (<5% by MRI-PDFF), a relative reduction in liver fat of ≥30%, or ≥50% after 8 weeks. Figure 3D: LS mean (+ / - SE) percent change in adiponectin. Figure 3E: Median fasting insulin in insulin-naive subjects. Data are based on the full analysis set population; pooled pegosafermin groups were analyzed by MMRM or van Elteren test, and individual pegosafermin dose groups by Wilcoxon rank-sum test. The image in Figure 3B was generated using a common color scale for all subjects. MRI‐PDFF, magnetic resonance imaging‐whole liver proton density fat fraction; QW, once weekly; Q2W, once every 2 weeks.

[0059] [Figure 4] FIG. 4 shows that the primary study cohort was randomized 1:1:1:1:1 to one of four doses of pegosafermin (9 mg QW, 18 mg QW, 27 mg QW, or 36 mg Q2W) or placebo, and the fibrate cohort was randomized 1:1 to either pegosafermin 27 mg QW or placebo QW for 8 weeks.

[0060] [Figure 5] Figure 5 shows a set of patient placement and population analyses.

[0061] [Figure 6A] Figure 6A shows the MRI-PDFF images of all individuals.

[0062] [Figure 6B] FIG. 6B is a graph showing the treatment response for all individuals.

[0063] [Figure 7A] FIG. 7A is a graph showing the median percent change in triglycerides from baseline at week 8 among subjects receiving no background therapy.

[0064] [Figure 7B] Figure 7B is a graph showing the median percent change in triglycerides from baseline at week 8 among subjects receiving background therapy. Pegozafermin resulted in a significant reduction in triglycerides among subjects receiving background therapy. The background therapy was combination lipid-modifying therapy (LMT).

[0065] [Figure 8] 1 is a graph showing the median percent change in triglycerides from baseline among subjects receiving statins, prescription fish oil, and fibrates at week 8. Pegozafermin resulted in a significant reduction in triglycerides among subjects receiving statins, prescription fish oil, and fibrates.

[0066] [Figure 9A] Figure 9A is a graph showing percent change in triglycerides from baseline in the overall population. p-values for change from baseline vs. placebo are based on van Elteren test for pooled pegosafermin and Wilcoxon rank-sum test for individual pegosafermin groups. ; Full analysis set; *p<0.05; ***p<0.001 vs. placebo. (QW: weekly; Q2W: every 2 weeks)

[0067] [Figure 9B]Figure 9B is a graph showing the percent change in triglycerides from baseline among subjects taking a background high-intensity statin. High-intensity statin is defined as a daily dose of atorvastatin 40-80 mg or rosuvastatin 20-40 mg. Pegozafermin resulted in a reduction in triglycerides among subjects receiving a background high-intensity statin. pegosafermin vs. placebo p-values are based on van Elteren test for pooled pegosafermin and Wilcoxon rank-sum test for individual pegosafermin groups. ; full analysis set; *p<0.05; ***p<0.001 vs. placebo. (QW: weekly; Q2W: every 2 weeks)

[0068] [Figure 10] Figure 10 is a graph showing that pegosafermin-treated patients reach their initial treatment goals, regardless of background treatment. p-values for change from baseline vs. placebo are based on Cochran-Mantel-Haenszel tests for pooled pegosafermin and Wilcoxon rank-sum tests for individual pegosafermin groups; full analysis set; *p<0.05; **p<0.01; ***p<0.001 vs. placebo).

[0069] [Figure 11] Figure 11 is a graph showing that pegosafermin treatment resulted in improvements in non-HDL cholesterol, regardless of background treatment. The results are consistent with data from patients receiving background treatment with statins or statin combinations, prescription omega-3 fatty acids, and fibrates. Least squares means data based on MMRM analysis. Post-hoc analysis; full analysis set.

[0070] [Figure 12]Figure 12 is a graph showing that pegosafermin treatment resulted in improvements in apolipoprotein B, regardless of background treatment. Results are consistent with data from patients receiving background treatment with statins or statin combinations, prescription omega-3 fatty acids, and fibrates. Least squares means data based on MMRM analysis. Post-hoc analysis; full analysis set.

[0071] [Figure 13] Figure 13 is a graph showing no significant change in LDL cholesterol regardless of background treatment. The results are consistent with data from patients receiving background treatment with statins or statin combinations, prescription omega-3 fatty acids, and fibrates. Least squares means data based on MMRM analysis. Post-hoc analysis; full analysis set.

[0072] [Figure 14] Figure 14 is a graph showing that pegosafermin treatment resulted in improvements in HDL cholesterol regardless of background treatment. The results are consistent with data from patients receiving background treatment with statins or statin combinations, prescription omega-3 fatty acids, and fibrates. Least squares means data based on MMRM analysis. Post-hoc analysis; full analysis set.

[0073] [Figure 15] Figure 15 is a patient flow diagram. QW, once weekly; Q2W, once every two weeks.

[0074] [Figures 16A-16F]Figures 16A-16F are graphs showing the effect of pegosafermin on serum triglycerides. Figure 16A: Median percent change in TG from baseline through week 8 (primary endpoint). Figure 16B: Percentage of subjects achieving a TG response of <500 mg / dL, <150 mg / dL, or ≥50% reduction from baseline to week 8. TG subgroup analysis among subjects (no background lipid-modifying therapy (Figure 16C), on background lipid-modifying therapy (Figure 16D), without T2DM (Figure 16E), and with T2DM (Figure 16F)). Data are based on the full analysis set population (defined as all randomized subjects who received at least one study treatment and had baseline and at least one post-baseline TG value) and were analyzed using the van Elteren test for pooled pegosafermin groups and the Wilcoxon rank-sum test for individual pegosafermin treatment groups. N represents independent subjects examined at baseline and four post-baseline time points for TG-related graphs. All p-values are two-sided and based on comparison with placebo. QW, once weekly; Q2W, once every 2 weeks; PBO, placebo; PGZ, pegosafermin.

[0075] [Figures 17A-17E] Figures 17A-17E are graphs showing the effects of pegosafermin on serum lipids. The least squares (LS) mean (+ / - SE) or median (apoC3) percent change from baseline to week 8 is shown for non-HDL-C (Figure 17A), apolipoprotein B (Figure 17B), apolipoprotein C3 (Figure 17C), LDL cholesterol (Figure 17D), and HDL cholesterol (Figure 17E). Data are analyzed via MMRM based on the full analysis set population (defined as all randomized subjects who received at least one study treatment and had baseline and at least one post-baseline TG). N represents independent subjects examined at baseline and two post-baseline time points for TG-related graphs. All p-values are two-sided and based on comparisons with the placebo arm (QW, once weekly; Q2W, once every 2 weeks; PBO, placebo; PGZ, pegosafermin).

[0076] [Figures 18A-18C] Figures 18A-18C show the effect of pegosafermin on hepatic steatosis. Figure 18A: LS mean (+ / - SE) percent change from baseline in hepatic fat fraction at week 8, as assessed by MRI-PDFF. Figure 18B: MRI-PDFF images showing changes in hepatic fat fraction from a representative subject with elevated baseline hepatic fat fraction, defined as >25%. Figure 18C: Percentage of subjects achieving normalization of hepatic fat (<5% by MRI-PDFF), a relative reduction in hepatic fat of ≥30%, or ≥50% after 8 weeks. Data are based on the full analysis set population (defined as all randomized subjects who received at least one study treatment and had baseline and at least one post-baseline TG) and were analyzed using the MMRM or van Elteren test for pooled pegosafermin groups and the Wilcoxon rank-sum test for individual pegosafermin treatment groups. N represents independent subjects examined at baseline and one post-baseline time point for the hepatic fat graph. All individual MRI-PDFF images in Figure 18B were generated as 384 × 288 mm and color-corrected to a common color scale to allow direct comparison between images. MRI-PDFF, magnetic resonance imaging—whole liver proton density fat fraction; QW, once weekly; Q2W, once every two weeks; PBO, placebo; PGZ, pegosafermin.

[0077] [Figure 19] FIG. 19 is a graph showing that triglyceride reduction was comparable across all predefined groups. DETAILED DESCRIPTION OF THE INVENTION

[0078] <Definition> For clarity and readability, the following definitions are provided. The technical features described in these definitions are understood in all embodiments and in each embodiment of the present invention. Additional definitions and explanations may be specifically provided within the description of these embodiments. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature and laboratory procedures used herein in cell culture, molecular genetics, organic chemistry, nucleic acid chemistry, and hybridization are those widely known and commonly employed in the art. Standard techniques are used for nucleic acid and peptide synthesis. These techniques and procedures are performed according to conventional methods in the art and various general references (e.g., Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY), which references are provided herein.

[0079] Enzymes: Enzymes are catalytically active biomolecules that carry out biochemical reactions such as the transfer of a glycosyl moiety or modified glycosyl moiety from a respective glycosyl donor to another glycosyl moiety attached to an amino acid or peptide of FGF-21.

[0080] Protein: A protein typically comprises one or more peptides or polypeptides. Proteins are typically folded into a three-dimensional form necessary for the protein to perform its biological function. The sequence of a protein or peptide is generally understood to be its order, i.e., the succession of its amino acids.

[0081] Recombinant Protein: The term "recombinant protein" refers to a protein produced in a heterologous system, i.e., a protein produced in an organism that does not naturally produce such protein or a variant of such protein. That is, the protein or peptide is "recombinantly produced." Typically, heterologous systems used in the art to produce recombinant proteins are bacteria (e.g., Escherichia (E.) coli), yeast (e.g., Saccharomyces cerevisiae), or certain mammalian cell culture strains.

[0082] Expression host: An expression host is an organism used for recombinant protein production. Common expression hosts are bacteria such as E. coli, yeasts such as Saccharomyces cerevisiae or Pichia astoris, or mammalian cells such as human cells.

[0083] RNA, mRNA: RNA is the common abbreviation for ribonucleic acid. It is a nucleic acid molecule, i.e., a polymer made up of nucleotides. These nucleotides are usually monomers of adenosine monophosphate, uridine monophosphate, guanosine monophosphate, and cytidine monophosphate, which are linked together along a so-called backbone. The backbone is formed by a phosphodiester bond between the sugar, i.e., ribose, of the first adjacent monomer and the phosphate moiety of the second adjacent monomer. A particular sequence of monomers is called an RNA sequence.

[0084] DNA: DNA is the common abbreviation for deoxyribonucleic acid. It is a nucleic acid molecule, a polymer composed of nucleotide monomers. These nucleotides are typically monomers of deoxyadenosine monophosphate, deoxythymidine monophosphate, deoxyguanosine monophosphate, and deoxycytidine monophosphate, which consist of a sugar moiety (deoxyribose), a base moiety, and a phosphate moiety and are polymerized with a characteristic backbone structure. The backbone structure is typically formed by a phosphodiester bond between the sugar moiety of the nucleotide (deoxyribose) of one adjacent monomer and the phosphate moiety of the second adjacent monomer. The specific order of the monomers, i.e., the order of the bases attached to the sugar / phosphate backbone, is called the DNA sequence. DNA can be single-stranded or double-stranded. In the double-stranded form, the nucleotides of the first strand hybridize to the nucleotides of the second strand, for example, by A / T-base-pairing and G / C-base-pairing.

[0085] Sequence of a nucleic acid molecule / nucleic acid sequence: The sequence of a nucleic acid molecule is generally understood to be the specific individual order, i.e. the succession of its nucleotides.

[0086] Sequence of amino acid molecules / amino acid sequence: The sequence of a protein or peptide is generally understood to be its order, i.e. the succession of its amino acids.

[0087] Sequence identity: Two or more sequences are identical if they have the same length and order of nucleotides or amino acids. The percentage of identity typically represents the degree to which two sequences match, typically representing the percentage of nucleotides corresponding at sequence positions to identical nucleotides in a reference sequence, such as a native or wild-type sequence. The degree of identity is determined by comparing sequences to determine their length, i.e., the length of the longest sequence compared. This means that a first sequence consisting of 8 nucleotides / amino acids is 80% identical to a second sequence consisting of 10 nucleotides / amino acids that includes the first sequence. In other words, in this disclosure, sequence identity specifically relates to the percentage of nucleotides / amino acids in two or more sequences of the same length that share the same position. Gaps are typically considered non-identical positions, regardless of their actual position in the alignment.

[0088] Newly introduced amino acid: "Newly introduced amino acid" refers to an amino acid that is newly introduced into an amino acid sequence compared to a natural / wild-type amino acid sequence. Typically, the natural amino acid sequence is altered by mutation to have a specific amino acid side chain at a desired position within the amino acid sequence. In the present disclosure, the amino acid threonine is specifically introduced into the amino acid sequence C-terminally adjacent to the proline residue.

[0089] Functional group: This term is understood according to the general understanding of those skilled in the art and refers to a chemical moiety present on a molecule (e.g., a peptide, or an amino acid of a peptide or a glycosyl residue attached to a peptide) that participates in covalent or non-covalent attachment to other chemical molecules (e.g., allows for attachment of a glycosyl residue or PEG).

[0090] Native amino acid sequence: This term is understood according to the general understanding of those skilled in the art and refers to an amino acid sequence in its naturally occurring form without any human mutations or amino acid modifications. It is also referred to as a "wild-type sequence." "Native FGF-21" or "wild-type FGF-21" refers to FGF-21 having a naturally occurring amino acid sequence, such as the (unmutated) amino acid sequence of human FGF-21 shown in SEQ ID NO: 1. The presence or absence of an N-terminal methionine, depending on the expression host used, does not usually alter the state of the protein considered to have its native or native / wild-type sequence.

[0091] Mutation: This term should be understood according to the general understanding of those skilled in the art. An amino acid sequence is said to be "mutated" when it contains at least one added, deleted, or exchanged amino acid, i.e., an amino acid mutation, compared to its native or naturally occurring amino acid sequence. A mutated protein is also called a mutant. In the present disclosure, a mutant FGF-21 peptide is specifically a peptide having an amino acid exchange adjacent to a proline residue on the C-terminal side of the proline residue. A consensus sequence for O-linked glycosylation is thereby introduced into FGF-21, and as a result, the mutant FGF-21 peptide contains a newly introduced O-linked glycosylation site. An amino acid exchange is typically indicated as S172T, which means that the amino acid serine at position 172 in an amino acid sequence such as SEQ ID NO:1 is replaced with the amino acid threonine.

[0092] Pharmaceutically effective amount: A pharmaceutically effective amount in this disclosure is typically understood to be an amount sufficient to induce a medicinal effect.

[0093] Treatment / Treatment: The term "treatment" means to "treat" or "treat" a disease or condition, to inhibit a disease (slowing or preventing the progression of a disease), to relieve the symptoms or side effects of a disease (including palliative treatment), or to pallify a disease (causing regression of a disease).

[0094] Therapeutically effective amount: An amount of a compound sufficient to treat a disease or condition, inhibit a disease or condition, alleviate the symptoms or side effects of a disease, and / or cause regression of a disease or condition.

[0095] Half-life: The term "half-life" is used herein in the context of administration of mutant FGF-21 peptides and / or conjugates thereof and is defined as the time required for the plasma concentration of the drug, i.e., mutant FGF-21 peptides and / or conjugates, to decrease by half in a subject.

[0096] O-linked glycosylation: "O-linked glycosylation" occurs at serine or threonine residues (Tanner et al. Biophys. Acta. 906:81-91 (1987); and Hounsell et al. J. 13:19-26 (1996)). In the present disclosure, an O-linked glycosylation site is an amino acid motif in the amino acid sequence of a peptide that is recognized by a glycosyltransferase as an attachment point for a glycosyl residue and includes the amino acid motif proline-threonine (PT), which is not present in the native / wild-type amino acid sequence. In particular, a threonine residue is newly introduced adjacent to and C-terminal to a proline residue. A glycosyl moiety is then attached to the -OH group of the threonine residue by a glycosyltransferase.

[0097] Newly introduced O-linked glycosylation site: A "newly introduced O-linked glycosylation site" refers to an O-linked glycosylation site that was not present in native or wild-type FGF-21 prior to the introduction of a threonine C-terminal to the proline residue as described herein.

[0098] Adjacent: Adjacent refers to an amino acid that is immediately adjacent to another amino acid in an amino acid sequence, either N-terminal or C-terminal to the respective amino acid. In the present invention, for example, a newly introduced threonine residue is adjacent to a proline residue at the C-terminal end of the proline residue.

[0099] Glycosyl moiety: A glycosyl moiety is a moiety consisting of one or more identical or different glycosyl residues that binds a mutant FGF-21 peptide to polyethylene glycol (PEG), thereby forming a conjugate comprising the peptide, the glycosyl moiety, and PEG. The glycosyl moiety can be a mono-, di-, tri-, or oligoglycosyl moiety. The glycosyl moiety may contain one or more sialic acid residues, one or more N-acetylgalactosamine (GalNAc) residues, one or more galactose (Gal) residues, and others. The glycosyl moiety may be modified with, for example, PEG or methoxy-PEG (m-PEG), an alkyl derivative of PEG.

[0100] Glycoconjugation: As used herein, the term "glycoconjugation" refers to the enzymatically mediated conjugation of a PEG-modified glycosyl moiety to a glycosyl residue of an amino acid or (poly)peptide, e.g., a mutant FGF-21 of the present disclosure. A subgenus of "glycoconjugation" is "glyco-PEGylation," in which the modifying group of the modified glycosyl moiety is PEG or m-PEG. PEG can be either linear or branched. Typically, branched PEGs have a central branched core moiety and multiple linear polymer chains connected to the branched core moiety. PEG is commonly used in branched form and can be prepared by adding ethylene oxide to various polyols, such as glycerol, pentaerythritol, and sorbitol. The central branched moiety can also be derived from several amino acids, such as lysine. Branched PEGs can be represented by the general formula R(-PEG-OX) mwhere R represents a core moiety such as glycerol or pentaerythritol, X represents a capping or terminal group, and m represents the number of arms. The terms "glyco-PEG" and "glycosyl-PEG" are used interchangeably and refer to a chemical moiety consisting of PEG or methoxy-PEG (mPEG or m-PEG), one or more glycosyl residues (or glycosyl moieties), and, optionally, a linker between the PEG / methoxy-PEG and the glycosyl moiety, e.g., an amino acid such as glycine. An example of a glycosyl-PEG / glyco-PEG moiety is PEG-sialic acid (PEG-Sia). It should be noted that the terms "glyco-PEG" and "glycosyl-PEG," and the analogous terms for glyco-PEG moieties, "PEG-sialic acid" and "PEG-Sia," may or may not include a linker between the PEG and the glycosyl moiety. That is, "PEG-sialic acid" encompasses, for example, PEG-sialic acid, PEG-Gly-sialic acid, and mPEG-Gly-sialic acid in addition to PEG-sialic acid.

[0101] Sequence motif: A sequence motif refers to a short amino acid sequence (e.g., containing only two amino acids) that occurs at any position in a long amino acid sequence, such as the amino acid sequence of human FGF-21. For example, a sequence motif represented as P172T means that the proline at position 172 is immediately C-terminal to a threonine residue.

[0102] Sialic Acid: The term "sialic acid" or "Sia" refers to any member of a family of nine-carbon carboxylated sugars. The most common member of the sialic acid family is N-acetyl-neuraminic acid (2-keto-5-acetamido-3,5-dideoxy-D-glycero-D-galactonunulopyranos-1-onic acid (sometimes abbreviated as Neu5Ac, NeuAc, or NANA). A second member of the family is N-glycolylneuraminic acid (Neu5Gc or NeuGc), in which the N-acetyl group of NeuAc is hydroxylated. A third member of the sialic acid family is 2-keto-3-deoxy-nonulosonic acid (KDN) (Nadano et al. (1986) J. Biol. Chem. 261:11550-11557). Also included are 9-substituted sialic acids such as 9-O-C1-C6 acyl-Neu5Ac, such as 9-O-lactyl-Neu5Ac or 9-O-acetyl-Neu5Ac, 9-deoxy-9-fluoro-Neu5Ac, and 9-azido-9-deoxy-Neu5Ac. For more information on this sialic acid family, see, for example, Varki, Glycobiology 2:25-40 (1992).

[0103] Pharmaceutically acceptable excipient: A "pharmaceutically acceptable" excipient includes any material that, when combined with a mutant FGF-21 peptide conjugate of the present disclosure, retains the activity of the conjugate and is non-reactive with the subject's immune system. Examples include, but are not limited to, standard pharmaceutical excipients such as phosphate buffered saline, water, salts, emulsions such as oil / water emulsions, and various types of wetting agents.

[0104] Pharmaceutical container: A "pharmaceutical container" is a container suitable for carrying a liquid pharmaceutical composition, typically made of an inert material and sterile.

[0105] Administration: The term "administering" refers to oral administration, inhalation, administration as a suppository, topical contact, intravenous administration, intraperitoneal administration, intramuscular administration, intralesional administration, intranasal administration, or subcutaneous administration to a subject, or implantation of a sustained-release device, e.g., a mini-osmotic pump. Administration can be by any route, including parenteral and transmucosal (e.g., oral, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial administration. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, and the like.

[0106] Fibroblast growth factor 21 (FGF21) is an endogenous hormone that regulates lipid and glucose metabolism and energy expenditure.

[0107] Provided herein is a method for treating severe hypertriglyceridemia in a subject in need thereof. In some embodiments, the method of the present disclosure comprises administering to a subject in need thereof a glycoPEGylated FGF21 analog (also referred to herein as pegosafermin (PGZ)) designed to have a longer half-life than native FGF21. Provided herein is a method for treating severe hypertriglyceridemia in a subject in need thereof. In some embodiments, the methods of the disclosure comprise administering to a subject in need thereof a pharmaceutical composition comprising a mutant fibroblast growth factor-21 (FGF-21) peptide conjugate and a pharmaceutically acceptable carrier, wherein the mutant FGF-21 peptide conjugate comprises: i) a mutant FGF-21 peptide comprising the amino acid sequence of SEQ ID NO:2; ii) a glycosyl moiety; and iii) a 20 kDa polyethylene glycol (PEG), wherein the mutant FGF-21 peptide is attached to the glycosyl moiety by a covalent bond between the threonine at amino acid position 173 of SEQ ID NO:2 and a first site of the glycosyl moiety, and the glycosyl moiety is attached to the 20 kDa PEG by a covalent bond between a second site of the glycosyl moiety and the 20 kDa PEG.

[0108] <Mutant FGF-21 peptides and their conjugates> Native FGF-21 has a relatively short in vitro half-life, with reported circulating half-lives ranging from 0.5 to 4 hours in rodents and nonhuman primates, limiting its clinical applicability. Recombinant human FGF-21 has a half-life of 1 to 2 hours. Various half-life extension strategies have been developed to improve the pharmacokinetic properties of FGF-21.

[0109] See also WO2019 / 043457, the contents of which are incorporated herein in their entirety.

[0110] Some aspects of the present disclosure relate to FGF-21 conjugates for use in treating severe hypertriglyceridemia. In some embodiments, the methods of the present disclosure include administering to a subject in need thereof a glycoPEGylated FGF21 analog (also referred to herein as pegozafermin (PGZ)) designed to have a longer half-life than native FGF21. Provided herein are methods for treating severe hypertriglyceridemia in a subject in need thereof. In some embodiments, the methods of the disclosure comprise administering to a subject in need thereof a pharmaceutical composition comprising a mutant fibroblast growth factor-21 (FGF-21) peptide conjugate and a pharmaceutically acceptable carrier, wherein the mutant FGF-21 peptide conjugate comprises: i) a mutant FGF-21 peptide comprising the amino acid sequence of SEQ ID NO:2; ii) a glycosyl moiety; and iii) a 20 kDa polyethylene glycol (PEG), wherein the mutant FGF-21 peptide is attached to the glycosyl moiety by a covalent bond between the threonine at amino acid position 173 of SEQ ID NO:2 and a first site of the glycosyl moiety, and the glycosyl moiety is attached to the 20 kDa PEG by a covalent bond between a second site of the glycosyl moiety and the 20 kDa PEG.

[0111] In some embodiments, the 20 kDa PEG moiety is attached to the glycosyl moiety by covalent bonding to a linker, and the linker contains at least one amino acid residue. In some embodiments, the at least one amino acid residue is glycine (Gly). In some embodiments, the mutant FGF-21 peptide conjugate comprises the structure -GalNAc-Sia-Gly-PEG(20 kDa). In some embodiments, the mutant FGF-21 peptide conjugate comprises the following structure. [Chemical formula] Here, n is an integer selected from 450 to 460.

[0112] In some embodiments, the 20 kDa PEG is linear PEG. In other embodiments, the 20 kDa PEG is branched PEG. In some embodiments, the 20 kDa PEG is 20 kDa methoxy-PEG.

[0113] In some embodiments, the subject is a human subject.

[0114] In some embodiments, the pharmaceutical composition is administered subcutaneously.

[0115] In some embodiments, the liquid pharmaceutical composition comprises 9 - 48 mg / ml of the FGF-21 peptide conjugate, for example, 9 mg / ml, 18 mg / ml, 20 mg / ml, 28 mg / ml, 30 mg / ml, 36 mg / ml, 42 mg / ml, 44 mg / ml, 48 mg / ml of the FGF-21 peptide conjugate.

[0116] See U.S. Patent Nos. 10,407,479, 10874714, 11,596,669 and 11596669. These are hereby incorporated by reference in their entirety.

[0117] [PEGylation] In glycoPEGylation, a PEG moiety is transferred to an amino acid or glycosyl residue attached to an amino acid of a protein or peptide using a glycosyltransferase. A typical final structure is protein-glycosyl moiety-optional linker-PEG. A more specific final structure is protein-amino acid (N-, C-, or internal) of the protein-one or more glycosyl residues-optional linker (e.g., amino acid linker)-linear or branched PEG moiety of various lengths, where the glycosyl moiety may contain one or more glycosyl residues. The one or more glycosyl residues comprising at least a portion of the structure may be any glycosyl residue capable of linking the protein to a PEG moiety. Various methods for glycoPEGylating proteins are known in the art and are described in detail below.

[0118] In some embodiments, the fibroblast growth factor-21 (FGF-21) peptide conjugate comprises: i) a mutant FGF-21 peptide comprising at least one threonine (T) residue adjacent to the C-terminal side of at least one proline (P) residue, thereby forming at least one O-linked glycosylation site that is not present in a corresponding native FGF-21, wherein the corresponding native FGF-21 has an amino acid sequence that is at least 95% identical to SEQ ID NO:1; ii) a 20 kDa polyethylene glycol (PEG), wherein the 20 kDa PEG is covalently attached to the mutant FGF-21 peptide at at least one threonine residue via at least one glycosyl moiety; and Includes.

[0119] In certain embodiments, the mutant FGF-21 peptide conjugate comprises a mutant FGF-21 peptide comprising the amino acid sequence PT. In particular embodiments thereof, the mutant FGF-21 peptide comprises at least one amino acid sequence selected from the group consisting of P172T, P156T, P5T, P3T, P9T, P50T, P61T, P79T, P91T, P116T, P129T, P131T, P134T, P139T, P141T, P144T, P145T, P148T, P150T, P151T, P158T, P159T, P166T, P178T, and combinations thereof, wherein the positions of proline and threonine are based on the amino acid sequence set forth in SEQ ID NO:1. In more particular embodiments, the mutant FGF-21 peptide comprises at least one amino acid sequence selected from the group consisting of P172T, P156T, P5T, and combinations thereof, and in particular consists of P172T, P156T, and combinations thereof, wherein the positions of the proline and threonine are based on the amino acid sequence set forth in SEQ ID NO: 1. In even more particular embodiments, the proline residue is located between amino acid 145 and the C-terminus of the mutant FGF-21 peptide, wherein the position of amino acid 145 is based on the amino acid sequence set forth in SEQ ID NO: 1.

[0120] In another specific embodiment, the mutant FGF-21 peptide comprises the amino acid sequence P172T, wherein the positions of the proline and threonine are based on the amino acid sequence shown in SEQ ID NO:1.

[0121] In another particular embodiment, the mutant FGF-21 peptide comprises the mutation S173T and the mutation R176A, where the positions of amino acids S and R are based on the amino acid sequence shown in SEQ ID NO:1, and in particular, the mutant FGF-21 peptide comprises the amino acid sequence shown in SEQ ID NO:2.

[0122] In another particular embodiment, the mutant FGF-21 peptide comprises the mutation Q157T, where the position of the amino acid Q is based on the amino acid sequence shown in SEQ ID NO:1, and in particular, the mutant FGF-21 peptide comprises the amino acid sequence shown in SEQ ID NO:4.

[0123] In another particular embodiment, the mutant FGF-21 peptide comprises the mutation D6T, where the position of amino acid D is based on the amino acid sequence shown in SEQ ID NO:1, and in particular, the mutant FGF-21 peptide comprises the amino acid sequence shown in SEQ ID NO:5.

[0124] In other particular embodiments, the mutant FGF-21 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:2-28, particularly an amino acid sequence selected from the group consisting of SEQ ID NOs:2-5, more particularly an amino acid sequence selected from the group consisting of SEQ ID NOs:2-4, and most particularly, the mutant FGF-21 peptide comprises the amino acid sequence shown in SEQ ID NO:2.

[0125] In other particular embodiments, the mutant FGF-21 peptide conjugate comprises at least one glycosyl moiety comprising N-acetylgalactosamine (GalNAc), galactose (Gal), and / or sialic acid (Sia). In certain embodiments thereof, at least one glycosyl moiety comprises the structure -GalNAc-Sia-.

[0126] In other particular embodiments, the mutant FGF-21 peptide conjugate comprises a 20 kDa PEG moiety linked to at least one glycosyl moiety via an amino acid residue, particularly glycine (Gly). In even more particular embodiments, the mutant FGF-21 peptide conjugate comprises the structure -GalNAc-Sia-Gly-PEG(20 kDa). Even more specifically, the mutant FGF-21 peptide conjugate comprises the following structure: [ka] Here, n is an integer selected from 450 to 460.

[0127] In some embodiments, the mutant FGF-21 peptide conjugate comprises a 20 kDa PEG that is linear or branched PEG. In some embodiments, the mutant FGF-21 peptide conjugate comprises a 20 kDa PEG that is linear PEG. In some embodiments, the 20 kDa PEG is 20 kDa methoxy-PEG.

[0128] In an exemplary embodiment, the polymeric modifying group is PEG. In another exemplary embodiment, the PEG moiety has a molecular weight of 20-30 kDa. In an exemplary embodiment, the PEG moiety has a molecular weight of 17 kDa, 18 kDa, 19 kDa, 20 kDa, 21 kDa, 22 kDa, 23 kDa, 24 kDa, 25 kDa, 26 kDa, 27 kDa, 28 kDa, 29 kDa, 30 kDa, 31 kDa, 32 kDa, or 33 kDa. In another exemplary embodiment, the PEG moiety has a molecular weight of 20 kDa. In another exemplary embodiment, the PEG moiety has a molecular weight of 30 kDa. In another exemplary embodiment, the PEG moiety has a molecular weight of about 5 kDa. In another exemplary embodiment, the PEG moiety has a molecular weight of about 10 kDa. In another exemplary embodiment, the PEG moiety has a molecular weight of about 40 kDa.

[0129] In some embodiments, the glycosyl linking group is a linear 10 kDa-PEG-sialyl, and one or two of these glycosyl linking groups are covalently attached to the peptide.

[0130] In some embodiments, the glycosyl linking group is a linear 20 kDa-PEG-sialyl, and one or two of these glycosyl linking groups are covalently attached to the peptide. In an exemplary embodiment, the glycosyl linking group is a linear 30 kDa-PEG-sialyl, and one or two of these glycosyl linking groups are covalently attached to the peptide. In an exemplary embodiment, the glycosyl linking group is a linear 5 kDa-PEG-sialyl, and one, two, or three of these glycosyl linking groups are covalently attached to the peptide. In an exemplary embodiment, the glycosyl linking group is a linear 40 kDa-PEG-sialyl, and one or two of these glycosyl linking groups are covalently attached to the peptide.

[0131] In some embodiments, the mutant FGF-21 peptide is PEGylated according to the methods described herein. In some embodiments, the mutant FGF-21 peptide is PEGylated according to the methods described herein. 172 T and R 176 A is a nucleotide sequence selected from the group consisting of 1, 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, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 1 The glycosyl moiety not attached to a PEG moiety may be attached to the newly introduced threonine residue.

[0132] In some embodiments, the 20 kDa PEG moiety is attached to at least one glycosyl linker via a linker, e.g., an amino acid residue, e.g., a small amino acid such as alanine or glycine, e.g., glycine (Gly). Thus, the PEG or m-PEG moiety is attached to the amino acid, which is attached to a glycosyl moiety, such as Sia. The glycosyl moiety is attached to the amino acid linker (if present) and to the newly introduced threonine residue in the mutant FGF-21 amino acid sequence. The amino acid residue is attached to the PEG and glycosyl residue via the methods described in WO 03 / 031464, which is incorporated herein by reference.

[0133] In some embodiments, the mutant FGF-21 peptide (e.g., SEQ ID NO:2) conjugate comprises the structure -GalNAc-Sia-Gly-PEG(20 kDa), where GalNAc is attached, for example, to a newly introduced threonine residue and Sia. Sia is further attached via a glycine residue to a PEG of 17 kDa, 18 kDa, 19 kDa, 20 kDa, 21 kDa, 22 kDa, 23 kDa, 24 kDa, 25 kDa, 26 kDa, 27 kDa, 28 kDa, 29 kDa, 30 kDa, 31 kDa, 32 kDa, or 33 kDa.

[0134] In some embodiments, the mutant FGF-21 peptide (e.g., SEQ ID NO:2) conjugate comprises the structure -GalNAc-Sia-Gly-PEG(20 kDa), where GalNAc is attached, for example, to a newly introduced threonine residue and Sia. Sia is further attached via a glycine residue to a 20 kDa, 21 kDa, 22 kDa, 23 kDa, 24 kDa, 25 kDa, 26 kDa, 27 kDa, 28 kDa, 29 kDa, or 30 kDa PEG.

[0135] In some embodiments, the mutant FGF-21 peptide (e.g., SEQ ID NO:2) conjugate comprises the structure -GalNAc-Sia-Gly-PEG(20 kDa), where GalNAc is attached, for example, to a newly introduced threonine residue and Sia. Sia is further attached via a glycine residue to a 20 kDa, 25 kDa, or 30 kDa PEG.

[0136] In some embodiments, the mutant FGF-21 peptide (e.g., SEQ ID NO:2) conjugate comprises the structure -GalNAc-Sia-Gly-PEG(20 kDa), where GalNAc is attached, for example, to a newly introduced threonine residue and Sia. Sia is further attached to a 20 kDa or 30 kDa PEG via a glycine residue.

[0137] In some embodiments, the mutant FGF-21 peptide (e.g., SEQ ID NO:2) conjugate comprises the structure -GalNAc-Sia-Gly-PEG(20 kDa), where GalNAc is attached, for example, to a newly introduced threonine residue and Sia. Sia is further attached to a 20 kDa PEG via a glycine residue.

[0138] In some embodiments, the mutant FGF-21 peptide conjugate comprises the following structure: [ka] Here, n is an integer selected from 450 to 460.

[0139] The 20 kDa PEG may be linear or branched. In some embodiments, the 20 kDa PEG is linear 20 kDa PEG. Furthermore, the 20 kDa PEG may be 20 kDa methoxy-PEG (mPEG, m-PEG). PEGs and mPEGs of different molecular weights can be obtained from various suppliers, such as, for example, JenKem Technology USA, Plano, Texas, USA, or Merckle Biotec, Ulm, Germany. PEG 20 kDa means that the PEG residues are, on average, 20 kDa in size, and it is understood in the art that the majority of PEG residues are 20 kDa in size.

[0140] <Mutant FGF-21 peptides and their conjugates> As described herein, variants of fibroblast growth factor-21 (FGF-21) have been generated that have surprising properties, including variants with exceptionally long half-lives, which variants: i) a mutant FGF-21 peptide comprising at least one threonine (T) residue adjacent to the C-terminal side of at least one proline (P) residue, thereby forming at least one O-linked glycosylation site that is not present in a corresponding native FGF-21, wherein the corresponding native FGF-21 has an amino acid sequence that is at least 95% identical to SEQ ID NO:1; ii) a 20-30 kDa polyethylene glycol (PEG) covalently attached at at least one threonine residue of said mutant FGF-21 peptide via at least one glycosyl moiety; and is a peptide conjugate comprising:

[0141] To attach a 20-30 kDa PEG residue, a threonine residue is introduced into the amino acid sequence of native FGF-21 at a position adjacent to a proline residue already present in the amino acid sequence of native FGF-21, i.e., C-terminal to the native proline residue. For this purpose, (i) an additional threonine is introduced immediately adjacent to the native proline residue, or (ii) a native amino acid present in the native amino acid sequence of FGF-21 adjacent to the terminal side of the native proline residue is converted to a threonine residue. In the present invention, option (ii) is an exemplary embodiment. As described herein, multiple threonine residues may be introduced at positions adjacent to the C-terminus of the already present proline residue. Thus, the mutant FGF-21 of the present invention may contain both an additional threonine residue and a threonine residue introduced in place of the native amino acid.

[0142] The introduction of a new threonine residue C-terminal to and adjacent to the proline residue creates a consensus sequence for O-glycosylating enzymes. Because proline residues are typically present on the surface of proteins (e.g., in turns, kinks, loops, etc.), designs calling for O-glycosylation and PEGylation using a PEG-glycosyl moiety adjacent to a proline residue have the advantage of the relative accessibility of the target binding site to glycosyltransferases that transfer the glycosyl or glycol-PEG moiety, and the ability to accommodate the conjugated glycosyl and / or PEG structure without disrupting the protein structure.

[0143] Common methods for introducing a threonine residue into the native amino acid sequence of FGF-21 include those described in Sambrook and Russell, Molecular Cloning, A Laboratory Manual (3rd ed. 2001), Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990), and Ausubel et al., eds., Current Protocols in Molecular Biology (1994).

[0144] In some embodiments, the native FGF-21 amino acid sequence corresponds to the native amino acid sequence of human FGF-21 shown in SEQ ID NO:1.

[0145] In some embodiments, the variant FGF-21 peptide comprises the amino acid sequence PT, a threonine residue C-terminally adjacent to a proline residue. The sequence PT is not present in the native FGF-21 amino acid sequence.

[0146] Optionally, the mutant FGF-21 peptide comprises P 172 T (e.g., SEQ ID NO: 2 or 3), P 156 T (e.g., SEQ ID NO: 4), P 5 T (e.g., SEQ ID NO: 5), P 3 T (e.g., SEQ ID NO: 6), P 9 T (e.g., SEQ ID NO: 7), P 50 T (e.g., SEQ ID NO: 8), P 61 T (e.g., SEQ ID NO: 9), P 79 T (e.g., SEQ ID NO: 10), P 91 T (e.g., SEQ ID NO: 11), P 116 T (e.g., SEQ ID NO: 12), P 120 T (e.g., SEQ ID NO: 13), P 125 T (e.g., SEQ ID NO: 14), P 129 T (e.g., SEQ ID NO::15), P 131 T (e.g., SEQ ID NO::16), P 134 T (e.g., SEQ ID NO::17), P 139 T (e.g., SEQ ID NO::18), P 141 T (e.g., SEQ ID NO::19), P 144 T (e.g., SEQ ID NO::20), P 145 T (e.g., SEQ ID NO::21), P 148 T (e.g., SEQ ID NO::22), P 150T (e.g., SEQ ID NO::23), P 151 T (e.g., SEQ ID NO::24), P 158 T (e.g., SEQ ID NO::25), P 159 T (e.g., SEQ ID NO::26), P 166 T (e.g., SEQ ID NO: 27), P 178 In some embodiments, the mutant FGF-21 peptide comprises at least one amino acid sequence selected from the group consisting of P (e.g., SEQ ID NO:28), T (e.g., SEQ ID NO:29), and combinations thereof, wherein the positions of proline and threonine are based on the amino acid sequence set forth in SEQ ID NO:1. 172 T, P 156 T, P 5 In some embodiments, the mutant FGF-21 peptide comprises at least one amino acid sequence selected from the group consisting of P, T, and combinations thereof. 172 T, P 156 In some embodiments, the mutant FGF-21 peptide comprises at least one amino acid sequence selected from the group consisting of: P172T, ...

[0147] In some embodiments, the proline residue is located between amino acid 145 and the C-terminus of the mutant FGF-21 peptide, where the position of amino acid 145 is based on the amino acid sequence set forth in SEQ ID NO: 1. As demonstrated by the results presented herein, the C-terminus of FGF-21 surprisingly tolerates the attachment of PEG, particularly glycosyl-PEG moieties. This was unexpected, as literature reports that β-Klotho binding of FGF-21 requires an intact C-terminus.

[0148] In certain embodiments, the mutant FGF-21 peptide comprises a mutant S 172 T and R 176A, and the positions of amino acids S and R are based on the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the mutant FGF-21 peptide comprises the amino acid sequence set forth in SEQ ID NO: 2. Mutation R 176 A has been shown to be beneficial to the overall stability of the protein after the introduction of an O-linked glycosylation site at treninion 173. This mutation removes the relatively large arginine side chain and replaces it with a smaller alanine side chain, which is thought to interfere less with the large number of glycosyl-PEG moieties attached to the mutant FGF-21 peptide.

[0149] In an alternative embodiment, the mutant FGF-21 peptide comprises the mutant Q 157 In some embodiments, the mutant FGF-21 peptide comprises the amino acid shown in SEQ ID NO:4, i.e., D, T, and the position of the amino acid Q is based on the amino acid sequence shown in SEQ ID NO:1. 6 T, and the position of amino acid D is based on the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the mutant FGF-21 peptide comprises the amino acid sequence set forth in SEQ ID NO:5.

[0150] In some embodiments, the mutant FGF-21 peptide conjugate comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-28. In some embodiments, the mutant FGF-21 peptide conjugate comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-5. In some embodiments, the mutant FGF-21 peptide conjugate comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-4. In some embodiments, the mutant FGF-21 peptide conjugate comprises the amino acid sequence shown in SEQ ID NO:2.

[0151] In some embodiments, conjugates of modified sugars and mutant FGF-21 peptides are illustratively provided. In some embodiments, the mutant FGF-21 peptide conjugates produced comprise a mutant FGF peptide and at least one modified sugar, wherein a first modified sugar of the at least one modified sugar is linked to an amino acid of the peptide via a glycosyl linking group. As described herein, the amino acid to which the glycosyl linking group is attached is mutated to generate a site that is recognized by a glycosyltransferase.

[0152] In some embodiments, the mutant FGF-21 peptide conjugate may comprise a mutant FGF-21 peptide and a glycosyl group linked to a mutant amino acid residue of the mutant FGF-21 peptide.

[0153] In some embodiments, the glycosyl group is an intact glycosyl linking group. In another exemplary embodiment, the glycosyl group further comprises a modifying group. In another exemplary embodiment, the modifying group is a non-glycosidic modifying group. In another exemplary embodiment, the modifying group does not comprise a naturally occurring saccharide moiety.

[0154] <Pharmaceutical Composition> In some embodiments, the pharmaceutical compositions are suitable for use in various drug delivery systems. Formulations suitable for use in the present invention are described in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, PA, 17th ed. (1985). The pharmaceutical compositions are intended for parenteral, intranasal, topical, oral, or local administration, such as by subcutaneous injection, aerosol inhalation, or transdermal absorption, for prophylactic and / or therapeutic treatment. Generally, pharmaceutical compositions are administered parenterally, for example, subcutaneously or intravenously.

[0155] In some embodiments, the present invention provides compositions for parenteral administration, which comprise a mutant FGF-21 peptide conjugate dissolved or suspended in an acceptable carrier, particularly an aqueous carrier (e.g., water, buffered water, saline, phosphate-buffered saline (PBS), etc.). The composition may also include detergents such as Tween 20 and Tween 80; stabilizers such as mannitol, sorbitol, sucrose, trehalose, etc.; and preservatives such as EDTA and m-cresol. The composition may include pharmaceutically acceptable auxiliary substances necessary to approximate physiological conditions, such as pH adjusters, buffers, tonicity adjusters, wetting agents, detergents, etc.

[0156] In some embodiments, the pharmaceutical compositions may be sterilized by conventional sterilization techniques or sterile filtered. The resulting aqueous solutions may be packaged for ready use or lyophilized. The lyophilized formulation is combined with a sterile aqueous carrier prior to administration. Compositions containing FGF peptide conjugates may be administered for prophylactic and / or therapeutic treatments, particularly for the treatment of SHTG. In therapeutic applications, the compositions are administered to a subject already suffering from a disease or condition associated with SHTG in an amount sufficient to cure or at least partially arrest the symptoms of the disease and its complications. An amount sufficient to accomplish this is defined as a "therapeutically effective amount," which generally depends on the patient's health and weight.

[0157] In some embodiments, the therapeutic dosing regimen may be in the range of about 3 mg to about 44 mg, in the range of about 3 mg to about 36 mg; in the range of about 3 mg to about 30 mg; in the range of about 3 mg to about 27 mg; in the range of about 3 mg to about 18 mg; in the range of about 3 mg to about 15 mg; in the range of about 9 mg to about 44 mg, in the range of about 9 mg to about 36 mg; in the range of about 9 mg to about 30 mg; in the range of about 9 mg to about 27 mg; in the range of about 9 mg to about 18 mg; in the range of about 9 mg to about 15 mg; in the range of about 15 mg to about 44 mg, in the range of about 15 mg to about 36 mg, in the range of about 15 mg to about 30 mg, in the range of about 15 mg to about 27 mg, or in the range of about 15 mg to about 18 mg; range of about 18 mg to about 44 mg, range of about 18 mg to about 36 mg, range of about 18 mg to about 30 mg, range of 18 mg to 27 mg; range of about 15 mg to about 44 mg, range of about 15 mg to about 36 mg, range of about 15 mg to about 30 mg, range of 15 mg to 27 mg; range of 15 mg to 18 mg; range of about 3 mg to about 9 mg; range of about 9 mg to about 15 mg; range of about 9 mg to about 18 mg; range of about 18 mg to about 27 mg; range of about 27 mg to about 30 mg; range of about 18 mg to about 27 mg; range of about 3 mg to about 18 mg; range of about 18 mg to about 36 mg. In some embodiments, the therapeutic dosing regimen includes a range of about 3 mg to about 50 mg, about 5 mg to about 50 mg, about 10 mg to about 50 mg, about 20 mg to about 50 mg, about 30 mg to about 50 mg, or about 40 mg to about 50 mg, and any integer within any of the ranges shown. In some embodiments, the therapeutic dosing regimen includes a range of about 5 mg to about 40 mg, about 10 mg to about 40 mg, about 20 mg to about 40 mg, about 30 mg to about 40 mg, or about 35 mg to about 40 mg, and any integer within any of the ranges shown. In some embodiments, the therapeutic dosing regimen includes a range of about 5 mg to about 30 mg, about 10 mg to about 30 mg, about 20 mg to about 30 mg, or about 25 mg to about 30 mg, and any integer within any of the ranges shown. In some embodiments, the therapeutic dosing regimen includes ranges of about 10 mg to about 20 mg; about 15 mg to about 20 mg; and any integer within any of the ranges indicated.In some embodiments, the therapeutic dosing regimen comprises a dose of about 3 mg; about 9 mg; about 15 mg, about 18 mg; about 27 mg, about 30 mg, about 36 mg, or about 44 mg. In some embodiments, the therapeutic dosing regimen comprises a dose of about 3 mg; about 4 mg; about 5 mg; about 6 mg; about 7 mg; about 8 mg; about 9 mg; about 10 mg; about 11 mg; about 12 mg; about 13 mg; about 14 mg; about 15 mg, about 16 mg; about 17 mg; about 18 mg; about 19 mg; about 20 mg; about 21 mg; about 22 mg; about 23 mg; about 24 mg; about 25 mg; about 26 mg; about 27 mg, about 28 mg; about 29 mg; about 30 mg, about 31 mg; about 32 mg; about 33 mg; about 34 mg; about 35 mg; about 36 mg; about 37 mg; about 38 mg; about 39 mg; about 40 mg; about 41 mg; about 42 mg; about 43 mg; or about 44 mg. As used herein, the term "about" refers to an amount 10% greater or less than the specified amount. For example, about 10 mg refers to a range of 9 to 11 mg. In yet another specific embodiment, the therapeutic dosing regimen includes a dose of 9.1 mg; about 18.2 mg; or about 39 mg.

[0158] In some embodiments, the pharmaceutical composition is a liquid pharmaceutical composition comprising at least one mutant FGF-21 peptide conjugate and a pharmaceutically acceptable carrier. In some embodiments, the mutant FGF-21 peptide conjugate is present at a concentration ranging from 0.1 mg / mL to 50 mg / mL. In some embodiments, the mutant FGF-21 peptide conjugate is present at a concentration ranging from 10 mg / mL to 48 mg / mL. For example, the FGF-21 peptide conjugate is present at a concentration of about 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, 39, 40, 41, 42, 43, or 44 mg / mL. In some embodiments, the mutant FGF-21 peptide conjugate is present at a concentration of 26±4 mg / mL. For example, the FGF-21 peptide conjugate is present at a concentration of about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 mg / mL. In some embodiments, the mutant FGF-21 peptide conjugate is present at 36±6 mg / mL. For example, the FGF-21 peptide conjugate is present at a concentration of about 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42 mg / mL.

[0159] In some embodiments, the liquid pharmaceutical composition comprises 10 to 48 mg / ml of the peptide conjugate, e.g., 15 mg / ml, 18 mg / ml, 20 mg / ml, 28 mg / ml, 30 mg / ml, 36 mg / ml, 42 mg / ml, 44 mg / ml, 48 mg / ml of the FGF-21 peptide conjugate.

[0160] In some embodiments, the liquid pharmaceutical composition comprises or consists of about 10 mg / ml to about 48 mg / ml of a mutant fibroblast growth factor-21 (FGF-21) peptide conjugate, about 50 mM to about 500 mM arginine; about 0.01 to about 0.1% (w / v) polysorbate 80 (PS-80) or polysorbate 20 (PS-20); about 20 mM buffer, pH 7-8; and a pharmaceutically acceptable carrier. In some embodiments, the liquid pharmaceutical composition comprises or consists of about 10 mg / ml to about 48 mg / ml of a variant fibroblast growth factor-21 (FGF-21) peptide conjugate, about 150 mM to about 500 mM arginine, about 0.01 to about 0.1% (w / v) polysorbate 80 (PS-80) or polysorbate 20 (PS-20), about 20 mM buffer, pH 7-8, and a pharmaceutically acceptable carrier. In some embodiments, the formulation has an osmolality of between about 250 mOsmol / kg and about 510 mOsmol / kg. In some embodiments, the liquid formulation comprises 10 mg / ml to 48 mg / ml of a mutant fibroblast growth factor-21 (FGF-21) peptide conjugate comprising a mutant FGF-21 peptide comprising the amino acid sequence of SEQ ID NO:2, a glycosyl moiety, and a 20 kDa polyethylene glycol (PEG), wherein the mutant FGF-21 peptide is attached to the glycosyl moiety by a covalent bond between the threonine at amino acid position 173 of SEQ ID NO:2 and a first site on the glycosyl moiety, and the glycosyl moiety is attached to the 20 kDa PEG by a covalent bond between a second site on the glycosyl moiety and the 20 kDa PEG; 50 mM to 500 mM arginine; 0.01 to 0.1% (w / v) polysorbate 80 (PS-80) or polysorbate 20 (PS-20); 20 mM buffer solution at pH 7-8; and a pharmaceutically acceptable carrier.In some embodiments, the liquid formulation comprises 10 mg / ml to 48 mg / ml of a mutant fibroblast growth factor-21 (FGF-21) peptide conjugate comprising a mutant FGF-21 peptide comprising the amino acid sequence of SEQ ID NO:2, a glycosyl moiety, and a 20 kDa polyethylene glycol (PEG), wherein the mutant FGF-21 peptide is attached to the glycosyl moiety by a covalent bond between the threonine at amino acid position 173 of SEQ ID NO:2 and a first site on the glycosyl moiety, and the glycosyl moiety is attached to the 20 kDa PEG by a covalent bond between a second site on the glycosyl moiety and the 20 kDa PEG; 150 mM to 500 mM arginine; 0.01 to 0.1% (w / v) polysorbate 80 (PS-80) or polysorbate 20 (PS-20); 20 mM buffer at pH 7-8; and a pharmaceutically acceptable carrier. In some embodiments, the formulation has an osmolality of between about 250 mOsmol / kg and about 550 mOsmol / kg.In some embodiments, the liquid pharmaceutical composition is about 10 mg / ml to about 48 mg / ml of a mutant fibroblast growth factor-21 (FGF-21) peptide conjugate comprising a mutant FGF-21 peptide comprising the amino acid sequence of SEQ ID NO:2, a glycosyl moiety, and a 20 kDa polyethylene glycol (PEG), wherein the mutant FGF-21 peptide is attached to the glycosyl moiety by a covalent bond between the threonine at amino acid position 173 of SEQ ID NO:2 and a first site on the glycosyl moiety, and the glycosyl moiety is attached to the 20 kDa polyethylene glycol (PEG) by a covalent bond between a second site on the glycosyl moiety and the 20 kDa PEG. The composition comprises or consists of a peptide conjugate bound to PEG, 50 mM to 500 mM arginine, about 50 mM to about 250 mM alanine, about 50 mM to about 250 mM proline, about 50 mM to about 250 mM glycine, about 50 mM to about 250 mM MgCl2, about 1% to about 5% (v / v) glycerol, about 1% to about 5% (v / v) PEG 400, or a combination thereof, 0.01 to 0.1% (w / v) polysorbate 80 (PS-80) or polysorbate 20 (PS-20), about 20 mM buffer solution having a pH of 7 to 8, and a pharmaceutically acceptable carrier.In some embodiments, the liquid pharmaceutical composition is about 10 mg / ml to about 48 mg / ml of a mutant fibroblast growth factor-21 (FGF-21) peptide conjugate comprising a mutant FGF-21 peptide comprising the amino acid sequence of SEQ ID NO:2, a glycosyl moiety, and a 20 kDa polyethylene glycol (PEG), wherein the mutant FGF-21 peptide is attached to the glycosyl moiety by a covalent bond between the threonine at amino acid position 173 of SEQ ID NO:2 and a first site on the glycosyl moiety, and the glycosyl moiety is attached to the 20 kDa polyethylene glycol (PEG) by a covalent bond between a second site on the glycosyl moiety and the 20 kDa PEG. The composition comprises or consists of a peptide conjugate bound to PEG, 150 mM to 500 mM arginine, about 50 mM to about 250 mM alanine, about 50 mM to about 250 mM proline, about 50 mM to about 250 mM glycine, about 50 mM to about 250 mM MgCl2, about 1% to about 5% (v / v) glycerol, about 1% to about 5% (v / v) PEG 400, or a combination thereof, 0.1 to 0.1% (w / v) polysorbate 80 (PS-80) or polysorbate 20 (PS-20), about 20 mM buffer solution having a pH of 7 to 8, and a pharmaceutically acceptable carrier. In some embodiments, the weight ratio of variant FGF-21 to arginine is about 0.6 to about 0.7, about 0.6 to about 0.8, about 0.6 to about 0.9, about 0.6 to about 1, e.g., about 0.6, 0.7, 0.8, 0.9, 0.1. In some embodiments, the molar ratio of variant FGF-21 to arginine is about 0.006 to about 0.008, about 0.006 to about 0.009, about 0.006 to about 0.010, about 0.007 to about 0.008, about 0.007 to about 0.009, about 0.007 to about 0.010, e.g., about 0.006, about 0.007, about 0.008, about 0.009.

[0161] In some embodiments, the liquid formulation has an osmolality of about 250 mOsmol / kg to about 550 mOsmol / kg.

[0162] In some embodiments, the liquid pharmaceutical composition comprises 20 mg / mL PEG-FGF21 in 20 mM Tris, 150 mM arginine, and 0.02% (w / v) PS-80, at a pH of 7.5. In some embodiments, the liquid pharmaceutical formulation comprises 20 mg / mL PEG-FGF21 in 20 mM phosphate, 150 mM arginine, and 0.02% (w / v) PS-80, at a pH of 7.5. In some embodiments, the composition has an osmolality of between about 250 mOsm / kg and about 380 mOsm / kg. In some embodiments, the composition has an osmolality of about 300 mOsm / kg. In some embodiments, the liquid pharmaceutical composition comprises 28 mg / mL PEG-FGF21 in 20 mM Tris, 275 mM arginine, and 0.02% (w / v) PS-80, at a pH of 7-8. In some embodiments, the composition has an osmolality of about 505 mOsm / kg. In some embodiments, the liquid pharmaceutical formulation comprises 18-44 mg / mL PEG-FGF21 in 20 mM Tris, 200-350 mM arginine, and 0.02% (w / v) PS-80, at a pH of 7.0-7.5. In some embodiments, the liquid pharmaceutical composition comprises about 20 mg / mL PEG-FGF21, about 150 mM arginine HCl, about 20 mM Tris, and 0.02% (w / v) PS-80, has a pH of about 7.5, and an osmolality of about 300 mOsm / kg. In some embodiments, the liquid pharmaceutical composition comprises about 28 mg / mL PEG-FGF21, about 260 mM arginine HCl, about 20 mM Tris, and about 0.02% (w / v) PS80, and has a pH of about 7.1. In some embodiments, the liquid pharmaceutical composition comprises about 28 mg / mL PEG-FGF21, about 260 mM arginine HCl, about 20 mM Tris, and about 0.02% (w / v) PS80, has a pH of about 7.1, and has an osmolality of about 505 mOsm / kg. In some embodiments, the liquid pharmaceutical composition comprises about 36 mg / mL PEG-FGF21, about 270 mM arginine hydrochloride, about 20 mM Tris, about 0.02% (w / v) PS80, and has a pH of about 7.1.In some embodiments, the liquid pharmaceutical composition comprises about 36 mg / mL PEG-FGF21, about 270 mM arginine HCl, about 20 mM Tris, and about 0.02% (w / v) PS80, has a pH of about 7.1, and an osmolality of about 530 mOsm / kg. In some embodiments, the liquid pharmaceutical composition comprises 36 mg / mL PEG-FGF21, 200 mM arginine, 20 mM Tris, and 0.02% (w / v) PS80, has a pH of about 7.1. In some embodiments, the liquid pharmaceutical composition comprises 36 mg / mL PEG-FGF21, 200 mM arginine HCl, 20 mM Tris, and 0.02% (w / v) PS80, has a pH of about 7.1, and an osmolality of about 421 mOsm / kg. In some embodiments, the liquid pharmaceutical composition comprises about 42 mg / mL PEG-FGF21, about 270 mM arginine HCl, about 20 mM Tris, about 0.02% (w / v) PS80, and has a pH of about 7.1. In some embodiments, the liquid pharmaceutical composition comprises about 42 mg / mL PEG-FGF21, about 270 mM arginine HCl, about 20 mM Tris, about 0.02% (w / v) PS80, and has a pH of about 7.1 and an osmolality of about 528 mOsm / kg. In some embodiments, the liquid pharmaceutical composition comprises 44 mg / mL mutant FGF21, 200 mM arginine HCl, 20 mM Tris, 0.02% (w / v) PS80, and has a pH of 7.1. In some embodiments, the liquid pharmaceutical composition comprises 44 mg / mL PEG-FGF21, 200 mM arginine HCl, 20 mM Tris, 0.02% (w / v) PS80, a pH of 7.1, and an osmolality of about 455 mOsm / kg. In some embodiments, the liquid pharmaceutical composition comprises 44 mg / mL PEG-FGF21, 230 mM arginine HCl, 20 mM Tris, 0.02% (w / v) PS80, a pH of 7.1. In some embodiments, the liquid pharmaceutical composition comprises 44 mg / mL PEG-FGF21, 230 mM arginine HCl, 20 mM Tris, 0.02% (w / v) PS80, a pH of 7.1, and an osmolality of about 485 mOsm / kg.

[0163] In some embodiments, the liquid composition further comprises a surfactant. In some embodiments, the surfactant comprises cetrimonium bromide, sodium gluconate, or a combination thereof. In some embodiments, the liquid formulation comprises about 0.05% to about 0.1% (w / v) cetrimonium bromide, about 0.05% to about 0.1% (w / v) sodium gluconate, or a combination thereof.

[0164] In some embodiments, the liquid pharmaceutical composition further comprises one or more active agents. In some embodiments, the PEG-FGF21 is co-formulated with one or more active agents.

[0165] The buffering agent may be present at a concentration of 1 mM to 100 mM. In some embodiments, the buffering agent is present at a concentration ranging from 2 mM to 75 mM, 5 mM to 50 mM, 10 mM to 25 mM, or 14 mM to 22 mM. In some embodiments, the buffering agent is present at a concentration of about 14, 16, 18, 20, 22, 24, 26, 30, 32, 34, 36, 38, 40 mM, or greater. For example, the buffering agent is present at a concentration of about 20 mM. The pH ranges from 6.0 to 8.5, 6.5 to 8.0, 6.75 to 8.0, or 7.1 to 8. The buffering agent may be a Tris-phosphate buffer. For example, the buffering agent may have a pH of 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.

[0166] The liquid pharmaceutical composition may further comprise a tonicity modifying agent. Suitable tonicity modifying agents include glycerol, amino acids, sodium chloride, proteins, or sugars and sugar alcohols. For example, the tonicity modifying agent may include arginine, such as arginine HCl or arginine sulfate. The tonicity modifying agent is present at a concentration of 50 mM to 500 mM. For example, the modifying agent (e.g., arginine HCl) may include 150 mM to 500 mM, 150 mM to 275 mM, or 245 mM to 275 mM arginine. In some embodiments, the tonicity modifying agent includes arginine, such as arginine HCl or arginine sulfate, and is present at a concentration between 31.6 mg / ml (150 mM) and 54.8 mg / ml (260 mM).

[0167] The liquid pharmaceutical composition may further comprise a non-ionic surfactant. The non-ionic surfactant may be a polysorbate-based non-ionic surfactant, particularly polysorbate 20 or polysorbate 80, more particularly polysorbate 80. The non-ionic surfactant may be present at a concentration of 0.01% (w / v) to 1% (w / v). For example, the non-ionic surfactant may be present at a concentration of 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1% (w / v).

[0168] In some embodiments, the liquid pharmaceutical composition may further comprise cetrimonium bromide, sodium gluconate, or a combination thereof. For example, the composition may comprise 0.05% to 0.1% (w / v) cetrimonium bromide, 0.05% to 0.1% (w / v) sodium gluconate, or a combination thereof.

[0169] In one embodiment, the liquid pharmaceutical composition comprises 10 mg / mL to 50 mg / mL of a mutant FGF-21 peptide conjugate, 1 mM to 100 mM of a buffer (e.g., Tris buffer), 150 mM to 500 mM of tonicity arginine, and 0.02% to 1% (w / v) of a polysorbate-based nonionic surfactant (particularly polysorbate 80), and has a pH of 7.0 to 8.0.

[0170] In some embodiments, the liquid formulation comprises 0.02% (w / v) PS80 (0.2 mg / ml). In some embodiments, the buffer is Tris buffer or phosphate buffer. In some embodiments, the liquid formulation comprises 20 mM Tris buffer. In some embodiments, the liquid formulation comprises 28 mg / ml of variant FGF-21. In some embodiments, the liquid formulation comprises 36 mg / ml of variant FGF-21. In some embodiments, the liquid formulation comprises 44 mg / ml of variant FGF-21. In some embodiments, the liquid formulation comprises 150 mM to 275 mM arginine. In some embodiments, the arginine is arginine HCl or arginine sulfate. In some embodiments, the pH is 7.1.

[0171] In some embodiments, the liquid pharmaceutical composition comprises about 20 mg / mL variant FGF21, about 150 mM arginine HCl, about 20 mM Tris, 0.02% (w / v) PS-80, and has a pH of about 7.5. In some embodiments, the liquid pharmaceutical composition comprises about 28 mg / mL variant FGF21, about 260 mM arginine HCl, about 20 mM Tris, about 0.02% (w / v) PS80, and has a pH of about 7.1. In some embodiments, the liquid pharmaceutical composition comprises about 36 mg / mL variant FGF21, about 270 mM arginine HCl, about 20 mM Tris, about 0.02% (w / v) PS80, and has a pH of about 7.1. In some embodiments, the liquid pharmaceutical composition comprises 36 mg / mL mutant FGF21, 200 mM arginine HCl, 20 mM Tris, 0.02% (w / v) PS80, and has a pH of about 7.1. In some embodiments, the liquid pharmaceutical composition comprises about 42 mg / mL mutant FGF21, about 270 mM arginine HCl, about 20 mM Tris, and about 0.02% (w / v) PS80, and has a pH of about 7.1. In some embodiments, the liquid pharmaceutical composition comprises 44 mg / mL mutant FGF21, 200 mM arginine HCl, 20 mM Tris, and 0.02% (w / v) PS80, and has a pH of 7.1. In some embodiments, the liquid pharmaceutical composition comprises 44 mg / mL mutant FGF21, 230 mM arginine HCl, 20 mM Tris, 0.02% (w / v) PS80, and a pH of 7.1.

[0172] In some embodiments, the liquid pharmaceutical composition comprises: (a) 10 mg / ml to 48 mg / ml of a mutant fibroblast growth factor-21 (FGF-21) peptide conjugate comprising a mutant FGF-21 peptide comprising the amino acid sequence of SEQ ID NO:2, a glycosyl moiety, and a 20 kDa polyethylene glycol (PEG), wherein the mutant FGF-21 peptide is attached to the glycosyl moiety by a covalent bond between the threonine at amino acid position 173 of SEQ ID NO:2 and a 0.01 position on the glycosyl moiety, and the glycosyl moiety is attached to the 20 kDa polyethylene glycol (PEG) by a covalent bond between a second position on the glycosyl moiety and the 20 kDa PEG. (b) a peptide conjugate linked to PEG; (b) 50 mM to 500 mM arginine; (c) 0.01% to 0.1% (w / v) polysorbate 80 (PS-80) or polysorbate 20 (PS-20); (d) a 5 mM to 25 mM buffer solution having a pH of 7 to 8; and (e) a pharmaceutically acceptable carrier.

[0173] In some embodiments, the liquid pharmaceutical composition comprises: (a) 10 mg / ml to 48 mg / ml of a mutant fibroblast growth factor-21 (FGF-21) peptide conjugate comprising a mutant FGF-21 peptide comprising the amino acid sequence of SEQ ID NO:2, a glycosyl moiety, and a 20 kDa polyethylene glycol (PEG), wherein the mutant FGF-21 peptide is attached to the glycosyl moiety by a covalent bond between the threonine at amino acid position 173 of SEQ ID NO:2 and a 0.01 position on the glycosyl moiety, and the glycosyl moiety is attached to the 20 kDa polyethylene glycol (PEG) by a covalent bond between a second position on the glycosyl moiety and the 20 kDa PEG. (b) a peptide conjugate linked to PEG; (b) 150 mM to 500 mM arginine; (c) 0.01% to 0.1% (w / v) polysorbate 80 (PS-80) or polysorbate 20 (PS-20); (d) a 5 mM to 25 mM buffer solution having a pH of 7 to 8; and (e) a pharmaceutically acceptable carrier.

[0174] In some embodiments, the liquid pharmaceutical composition comprises: (a) 10 mg / ml to 48 mg / ml of a mutant fibroblast growth factor-21 (FGF-21) peptide conjugate, the mutant FGF-21 peptide comprising the amino acid sequence of SEQ ID NO:2; a glycosyl moiety; and a 20 kDa polyethylene glycol (PEG), the glycosyl moiety being attached by a covalent bond between the threonine at amino acid position 173 of SEQ ID NO:2 and a first site of the glycosyl moiety; and the glycosyl moiety being attached by a covalent bond between a second site of the glycosyl moiety and the 20 kDa PEG. (b) a peptide conjugate linked to PEG; (b) 150 mM to 500 mM arginine, 50 mM to 250 mM alanine, 50 mM to 250 mM proline, 50 mM to 250 mM glycine, 50 mM to 250 mM MgCl2, 1 to 5% (v / v) glycerol, 1 to 5% (v / v) PEG 400, or a combination thereof; (c) 0.01% to 0.1% (w / v) polysorbate 80 (PS-80) or polysorbate 20 (PS-20); (d) a buffer solution of pH 7 to 8; and (e) a pharmaceutically acceptable carrier.

[0175] In some embodiments, the liquid formulation further comprises a surfactant. In some embodiments, the surfactant comprises cetrimonium bromide, sodium gluconate, or a combination thereof. In some embodiments, the liquid formulation comprises 0.05% to 0.1% (w / v) cetrimonium bromide, 0.05% to 0.1% (w / v) sodium gluconate, or a combination thereof.

[0176] In some embodiments, the buffer is Tris buffer or phosphate buffer. In some embodiments, the liquid formulation comprises 20 mM Tris buffer. In some embodiments, the liquid formulation has a pH of 7.0 to 7.5.

[0177] In some embodiments, the liquid pharmaceutical composition comprises 20 to 44 mg / ml of the mutant FGF-21 peptide conjugate.

[0178] In some embodiments, the liquid pharmaceutical composition comprises 150 mM to 275 mM arginine. In some embodiments, the arginine in the liquid pharmaceutical composition comprises arginine HCl, arginine sulfate, or a combination thereof. In some embodiments, the weight ratio of the mutant FGF-21 peptide conjugate to arginine is 0.6 to 0.9. In some embodiments, the molar ratio of the mutant FGF-21 peptide conjugate to arginine is about 0.006 to about 0.009.

[0179] In some embodiments, the liquid pharmaceutical composition comprises about 28 mg / mL of a mutant FGF-21 peptide conjugate, about 260 mM arginine HCl, and about 20 mM Tris, 0.02% (w / v) PS-80, and has a pH of about 7.1.

[0180] In some embodiments, the liquid pharmaceutical composition comprises about 20 mg / mL of a mutant FGF-21 peptide conjugate, about 150 mM arginine HCl, about 20 mM Tris, 0.02% (w / v) PS-80, and has a pH of about 7.5.

[0181] In some embodiments, the liquid pharmaceutical composition comprises about 36 mg / mL of a mutant FGF-21 peptide conjugate, about 200 mM arginine HCl, about 20 mM Tris, and 0.02% (w / v) PS-80, and has a pH of about 7.1.

[0182] In some embodiments, the liquid pharmaceutical composition comprises about 44 mg / mL of a mutant FGF-21 peptide conjugate, about 200 mM arginine HCl, about 20 mM Tris, and 0.02% (w / v) PS-80, and has a pH of about 7.1.

[0183] In some embodiments, the liquid pharmaceutical composition comprises about 44 mg / mL of a mutant FGF-21 peptide conjugate, about 230 mM arginine HCl, about 20 mM Tris, and 0.02% (w / v) PS-80, and has a pH of about 7.1.

[0184] In some embodiments, the liquid formulation has an osmolality of about 250 mOsmol / kg to about 550 mOsmol / kg.

[0185] In some embodiments, the liquid formulation is a liquid formulation described in U.S. Patent Application Publication No. 2022-0296678, which is incorporated by reference in its entirety.

[0186] Also encompassed herein, in some embodiments, is a pharmaceutical container containing any one or at least one of the mutant FGF-21 peptide conjugates described herein or a pharmaceutical composition comprising same. Exemplary such pharmaceutical containers include, but are not limited to, a syringe, an autoinjector, a vial, an infusion bottle, an ampoule, a carpoule, a syringe with a needle protection system, or a carpoule in an injection pen.

[0187] <Treatment for severe hypertriglyceridemia> Pegozafermin (PGZ) is a glycoPEGylated FGF21 analog with an N-terminal methionine, two point mutations, and a single 20 kDa linear polyethylene glycol covalently attached via a glycosyl moiety. PGZ data from a Phase 1b / 2a proof-of-concept study in subjects with NASH demonstrated global metabolic benefits with improvements in lipids (TG, LDL, non-HDL, and HDL), insulin resistance, HbA1c, body weight, and liver fat.

[0188] Severe hypertriglyceridemia (SHTG; ≥ 500 mg / dL) increases the risk of acute pancreatitis and cardiovascular disease. Current therapies rarely lower TG levels to desirable levels, highlighting the need for new therapies. In some embodiments, the pharmaceutical compositions and methods provided herein reduce triglyceride levels by at least 20% from baseline, at least 25% from baseline, at least 30% from baseline, at least 35% from baseline, or at least 40% from baseline.

[0189] In some embodiments, its administration significantly reduced TG, non-HDL-C, ApoB, and liver fat, increased HDL-C with minimal changes in LDL-C, and improved liver transaminases.

[0190] In some embodiments, the administration results in a decrease in the production of TG-rich lipoproteins.

[0191] In some embodiments, the administration results in improved clearance of TG-rich lipoproteins.

[0192] In some embodiments, the administration results in improved insulin sensitivity.

[0193] In some embodiments, administration results in normalization of triglyceride levels to 150 mg / dl or less.

[0194] In some embodiments, administration results in at least a 10% decrease in fasting plasma glucose, at least a 0.2% decrease in HBA1c, or a combination thereof.

[0195] In some embodiments, administration results in at least a 10% decrease in an alanine transaminase (ALT) marker, at least a 10% decrease in an aspartate aminotransferase (AST) marker, at least a 10% median decrease in high sensitivity C-reactive protein (hsCRP), or a combination thereof.

[0196] SHTG is commonly associated with obesity, metabolic syndrome, insulin resistance, type 2 diabetes, and nonalcoholic fatty liver disease (NAFLD). An ideal treatment would not only lower TG levels but also provide benefits for other metabolic comorbidities.

[0197] According to some aspects of the present disclosure, administration of mutant FGF-21 peptide conjugates significantly reduced TG and other atherogenic lipids in patients with SHTG. These results were consistent in patients receiving background lipid-modifying therapy (LMT), whether it be a statin, a combination of statins, prescription fish oil, or a fibrate. See Figures 7A-14. In some embodiments, administration of mutant FGF-21 peptide conjugates results in improved cardiometabolism (e.g., blood glucose regulation and liver fat reduction).

[0198] In some embodiments, the methods of the present disclosure comprise administering a mutant FGF-21 peptide conjugate and background lipid-modifying therapy (LMT) to a patient in need thereof.

[0199] An embodiment of the present disclosure is a method of treating severe hypertriglyceridemia (SHTG) in a subject in need thereof, comprising administering to the subject in need thereof once weekly a pharmaceutical composition comprising about 9 mg to about 30 mg of a mutant fibroblast growth factor-21 (FGF-21) peptide conjugate and a pharmaceutically acceptable carrier, wherein the mutant FGF-21 peptide conjugate comprises: i) a mutant FGF-21 peptide comprising the amino acid sequence of SEQ ID NO:2; ii) a glycosyl moiety; and iii) a 20 kDa polyethylene glycol (PEG), wherein the mutant FGF-21 peptide is attached to the glycosyl moiety by a covalent bond between the threonine at amino acid position 173 of SEQ ID NO:2 and a first site of the glycosyl moiety, and the glycosyl moiety is attached to the 20 kDa PEG by a covalent bond between a second site of the glycosyl moiety and the 20 kDa PEG, wherein administration of the pharmaceutical composition results in a reduction of triglyceride levels by at least 20% from baseline. In some embodiments, administration of the pharmaceutical composition results in a median reduction in triglyceride levels of at least 30% from baseline, hi some embodiments, administration of the pharmaceutical composition results in a median reduction in triglyceride levels of at least 40% from baseline.

[0200] In some embodiments, administration results in normalization of triglyceride levels to 150 mg / dl or less.

[0201] In some embodiments, the administration results in at least a 10% decrease in non-HDL cholesterol levels from baseline, at least a 10% decrease in apoB levels from baseline, at least a 10% decrease in apoC3 levels from baseline, or a combination thereof.

[0202] In some embodiments, administration results in at least a 10% increase in HDL cholesterol levels from baseline, at least a 10% increase in adiponectin levels from baseline, or a combination thereof.

[0203] In some embodiments, the administration results in a decrease in the production of TG-rich lipoproteins.

[0204] In some embodiments, the administration results in improved clearance of TG-rich lipoproteins.

[0205] In some embodiments, the administration results in improved insulin sensitivity.

[0206] In some embodiments, a subject in need thereof has baseline hepatic steatosis.

[0207] In some embodiments, administration results in greater than a 30% reduction in liver fat.

[0208] In some embodiments, the method comprises administering the pharmaceutical composition to a subject in need thereof for 8 weeks or more.

[0209] In some embodiments, the pharmaceutical composition is administered subcutaneously.

[0210] In some embodiments, the subject in need thereof is a human. In some embodiments, the subject in need thereof has fasting triglycerides (TG) of ≧500 mg / dL and ≦2000 mg / dL.

[0211] In some embodiments, the pharmaceutical composition comprises about 9 mg to about 30 mg of the mutant FGF-21 peptide conjugate. For example, the pharmaceutical composition comprises about 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 21 mg, 22 mg, 23 mg, 24 mg, 25 mg, 26 mg, 27 mg, 28 mg, 29 mg, or 30 mg of the mutant FGF-21 peptide conjugate.

[0212] In some embodiments, the pharmaceutical composition comprises from about 9 mg to about 14 mg, from about 15 mg to about 18 mg, from about 19 mg to about 26 mg, or from about 27 mg to about 30 mg.

[0213] In some embodiments, the pharmaceutical composition comprises about 9 mg of the mutant FGF-21 peptide conjugate.

[0214] In some embodiments, the pharmaceutical composition comprises about 15 mg to about 18 mg of the mutant FGF-21 peptide conjugate. In some embodiments, the pharmaceutical composition comprises about 15 mg of the mutant FGF-21 peptide conjugate. In some embodiments, the pharmaceutical composition comprises about 18 mg of the mutant FGF-21 peptide conjugate.

[0215] In some embodiments, the pharmaceutical composition comprises about 27 mg to about 30 mg of the mutant FGF-21 peptide conjugate. In some embodiments, the pharmaceutical composition comprises about 27 mg of the mutant FGF-21 peptide conjugate. In some embodiments, the pharmaceutical composition comprises about 30 mg of the mutant FGF-21 peptide conjugate. In some embodiments, administration results in at least a 10% decrease in an alanine transaminase (ALT) marker, at least a 10% decrease in an aspartate aminotransferase (AST) marker, at least a 10% median decrease in high-sensitivity C-reactive protein (hsCRP), or a combination thereof. In some embodiments, administration results in at least a 10% decrease in fasting plasma glucose, at least a 0.2% decrease in HBA1c, or a combination thereof.

[0216] In some embodiments, the subject in need thereof is undergoing background lipid-modifying therapy (LMT). In some embodiments, the LMT comprises a statin, prescription fish oil, a fibrate, or a combination thereof. In some embodiments, the subject in need thereof is on background LMT and administration results in a reduction in non-HDL cholesterol levels of at least 10% from baseline. In some embodiments, the subject in need thereof is on background LMT and administration results in a reduction in apoB cholesterol levels of at least 10% from baseline.

[0217] An embodiment of the present disclosure is a method of treating severe hypertriglyceridemia (SHTG) in a subject in need thereof, comprising administering to the subject in need thereof once weekly about 27 mg to about 30 mg of a pharmaceutical composition comprising a mutant fibroblast growth factor-21 (FGF-21) peptide conjugate and a pharmaceutically acceptable carrier, wherein the mutant FGF-21 peptide conjugate comprises: i) a mutant FGF-21 peptide comprising the amino acid sequence of SEQ ID NO:2; ii) a glycosyl moiety; and iii) a 20 kDa polyethylene glycol (PEG), wherein the mutant FGF-21 peptide is and a glycosyl moiety attached to a 20 kDa PEG by a covalent bond between the threonine at amino acid position 173 of NO:2 and a first site of the glycosyl moiety, wherein the glycosyl moiety is attached to a 20 kDa PEG by a covalent bond between a second site of the glycosyl moiety and the 20 kDa PEG, wherein administration of the pharmaceutical composition results in a reduction of triglyceride levels by at least 20% from baseline, and wherein administration of the pharmaceutical composition results in one or more of the following: at least a 10% reduction from baseline in an alanine transaminase (ALT) marker, at least a 10% reduction from baseline in aspartate aminotransferase (AST), high blood pressure, and / or kidney function. A median decrease from baseline in sensitivity C-reactive protein (hsCRP) of at least 10%, a decrease from baseline in fasting plasma glucose of at least 10%, a decrease from baseline in HBA1c of at least 0.3%, a decrease from baseline in non-HDL cholesterol levels of at least 10%, a decrease from baseline in apoB of at least 10%, a decrease from baseline in apoC3 of at least 10%, an increase from baseline in HDL cholesterol levels of at least 10%, an increase from baseline in adiponectin levels of at least 10%, and a decrease from baseline in liver fat of more than 30%.

[0218] Another aspect of the present disclosure is a method of treating severe hypertriglyceridemia (SHTG) in a subject in need thereof, comprising administering once weekly to a subject in need thereof a pharmaceutical composition comprising about 31 mg to about 44 mg of a mutant fibroblast growth factor-21 (FGF-21) peptide conjugate and a pharmaceutically acceptable carrier, wherein the mutant FGF-21 peptide conjugate comprises: i) a mutant FGF-21 peptide comprising the amino acid sequence of SEQ ID NO:2; ii) a glycosyl moiety; and iii) a 20 kDa polyethylene glycol (PEG), wherein the mutant FGF-21 peptide is attached to the glycosyl moiety by a covalent bond between the threonine at amino acid position 173 of SEQ ID NO:2 and a first site of the glycosyl moiety, and the glycosyl moiety is attached to the 20 kDa PEG by a covalent bond between a second site of the glycosyl moiety and the 20 kDa PEG, wherein administration of the pharmaceutical composition results in a reduction of triglyceride levels by at least 20% from baseline.

[0219] In some embodiments, administration of the pharmaceutical composition results in a reduction in median triglyceride levels of at least 30% from baseline.

[0220] In some embodiments, administration of the pharmaceutical composition results in a median decrease in triglyceride levels of at least 40% from baseline.

[0221] In some embodiments, the pharmaceutical composition comprises about 31 mg to about 44 mg of mutant FGF-21 peptide conjugate. In some embodiments, the pharmaceutical composition comprises about 31 mg to about 35 mg of mutant FGF-21 peptide conjugate. In some embodiments, the pharmaceutical composition comprises about 36 mg to about 44 mg of mutant FGF-21 peptide conjugate. For example, the pharmaceutical composition may comprise about 31 mg, 32 mg, 33 mg, 34 mg, 35 mg, 36 mg, 37 mg, 38 mg, 39 mg, 40 mg, 41 mg, 42 mg, 43 mg, or 44 mg of mutant FGF-21 peptide conjugate.

[0222] In some embodiments, administration results in normalization of triglyceride levels to 150 mg / dl or less.

[0223] In some embodiments, the administration results in at least a 10% decrease in non-HDL cholesterol levels from baseline, at least a 10% decrease in apoB levels from baseline, at least a 10% decrease in apoC3 levels from baseline, or a combination thereof.

[0224] In some embodiments, administration results in at least a 10% increase in HDL cholesterol levels from baseline, at least a 10% increase in adiponectin levels from baseline, or a combination thereof.

[0225] In some embodiments, the administration results in a decrease in the production of TG-rich lipoproteins.

[0226] In some embodiments, the administration results in improved clearance of TG-rich lipoproteins.

[0227] In some embodiments, the administration results in improved insulin sensitivity.

[0228] In some embodiments, a subject in need thereof has baseline hepatic steatosis.

[0229] In some embodiments, administration results in greater than a 30% reduction in liver fat.

[0230] In some embodiments, the method comprises administering the pharmaceutical composition to a subject in need thereof for 8 weeks or more.

[0231] In some embodiments, the subject in need thereof is a human.

[0232] In some embodiments, a subject in need thereof has fasting triglycerides (TG) of ≧500 mg / dL and ≦2000 mg / dL.

[0233] In some embodiments, the pharmaceutical composition comprises about 36 mg to about 44 mg of the mutant FGF-21 peptide conjugate. In some embodiments, administration results in a median decrease in hsCRP of at least 10%.

[0234] In some embodiments, the subject in need is undergoing background lipid-modifying therapy (LMT). In some embodiments, the LMT comprises a statin, prescription fish oil, a fibrate, or a combination thereof. In some embodiments, the subject in need is on background LMT and administration results in a reduction in non-HDL cholesterol levels of at least 10% from baseline. In some embodiments, the subject in need is on background LMT and administration results in a reduction in apoB cholesterol levels of at least 10% from baseline.

[0235] In some embodiments, the pharmaceutical composition is administered subcutaneously.

[0236] An embodiment of the present disclosure is a method of treating severe hypertriglyceridemia (SHTG) in a subject in need thereof, comprising administering to the subject in need thereof once every two weeks a pharmaceutical composition comprising about 36 mg to about 44 mg of a mutant fibroblast growth factor-21 (FGF-21) peptide conjugate and a pharmaceutically acceptable carrier, wherein the mutant FGF-21 peptide conjugate comprises: i) a mutant FGF-21 peptide comprising the amino acid sequence of SEQ ID NO:2; ii) a glycosyl moiety; and iii) a 20 kDa polyethylene glycol (PEG), wherein the mutant FGF-21 peptide is The glycosyl moiety is attached to a 20 kDa PEG by a covalent bond between the threonine at amino acid position 173 of NO:2 and a first site of the glycosyl moiety, and the glycosyl moiety is attached to a 20 kDa PEG by a covalent bond between a second site of the glycosyl moiety and the 20 kDa PEG, and administration of the pharmaceutical composition results in a reduction in triglyceride levels of at least 20% from baseline, and administration of the pharmaceutical composition results in one or more of the following: a median reduction of at least 10% from baseline in high-sensitivity C-reactive protein (hsCRP), at least 10% reduction in non-HDL cholesterol levels from baseline, at least 10% reduction in apoB, at least 10% reduction in apoC3, at least 10% increase in HDL cholesterol levels from baseline, at least 10% increase in adiponectin levels from baseline, and a reduction in liver fat of more than 30% from baseline.

[0237] In some embodiments, the glycosyl moiety of the FGF-21 peptide conjugate comprises at least one of an N-acetylgalactosamine (GalNAc) residue, a galactose (Gal) residue, a sialic acid (Sia) residue, a 5-amine analog of a Sia residue, a mannose (Man) residue, mannosamine, a glucose (Glc) residue, an N-acetylglucosamine (GlcNAc) residue, a fucose residue, a xylose residue, or a combination thereof.

[0238] In some embodiments, the glycosyl moiety of the FGF-21 peptide conjugate comprises at least one N-acetylgalactosamine (GalNAc) residue, at least one galactose (Gal) residue, at least one sialic acid (Sia) residue, or a combination thereof. In some embodiments, at least one Sia residue is a nine-carbon carboxylated sugar. In some embodiments, at least one Sia residue is N-acetyl-neuraminic acid (2-keto-5-acetamido-3,5-dideoxy-D-glycero-D-galactononulopyranos-1-onic acid) (Neu5Ac), N-glycolylneuraminic acid (Neu5Gc), 2-keto-3-deoxy-nonulosonic acid (KDN), or a 9-substituted sialic acid. In some embodiments, the 9-substituted sialic acid is 9-O-lactyl-Neu5Ac, 9-O-acetyl-Neu5Ac, 9-deoxy-9-fluoro-Neu5Ac, or 9-azido-9-deoxy-Neu5Ac.

[0239] In some embodiments, the glycosyl moiety of the FGF-21 peptide conjugate comprises the structure -GalNAc-Sia-.

[0240] In some embodiments, the 20 kDa PEG moiety is attached to the glycosyl moiety by a covalent bond to a linker, wherein the linker comprises at least one amino acid residue, in some embodiments, the at least one amino acid residue is glycine (Gly).

[0241] In some embodiments, the mutant FGF-21 peptide conjugate comprises the structure -GalNAc-Sia-Gly-PEG(20 kDa).

[0242] In some embodiments, the 20 kDa PEG of the FGF-21 peptide conjugate is a linear or branched PEG. In some embodiments, the 20 kDa PEG is a 20 kDa methoxy-PEG.

[0243] In some embodiments, the mutant FGF-21 peptide conjugate comprises the structure: [ka] Here, n is an integer selected from 450 to 460.

[0244] Without being bound by theory, the FGF-21 conjugates of the present disclosure are an attractive treatment for severe hypertriglyceridemia with the potential to simultaneously address multiple comorbidities, including cardiac, glycemic, and liver risks.

[0245] Specific examples of methods and kits are described herein for illustrative purposes. These are examples only. The techniques provided herein may be applied to systems other than the exemplary systems described above. Many changes, modifications, additions, omissions, and substitutions are possible in the practice of the present invention. The present invention includes variations of the described embodiments that will be apparent to those skilled in the art, including variations obtained by: replacing features, elements, and / or acts with equivalent features, elements, and / or acts; mixing and matching features, elements, and / or acts from different embodiments; combining features, elements, and / or acts from the embodiments described herein with features, elements, and / or acts of other technologies; and / or omitting to combine features, elements, and / or acts from the described embodiments.

[0246] The embodiments of the present invention described above are intended to be illustrative only, and those skilled in the art will understand that various modifications of detail may be made to these embodiments, all of which are within the scope of the present invention.

[0247] All publications mentioned herein are incorporated herein by reference in their entirety. Although the foregoing invention has been described in some detail for purposes of clarity and understanding, those skilled in the art will appreciate from a reading of this specification that various changes in form and detail may be made therein without departing from the true scope of the invention as set forth in the appended claims. [Example]

[0248] Example 1: Randomized Phase 2 Study of Pegozafermin in Severe Hypertriglyceridemia <Summary of a randomized phase 2 study of pegosafermin in severe hypertriglyceridemia> <Background> Pegozafermin is a long-acting glycopegylated recombinant analog of human fibroblast growth factor 21 in development for severe hypertriglyceridemia and nonalcoholic steatohepatitis.

[0249] <Method> ENTRIGUE was an 8-week, phase 2, double-blind, randomized, five-arm study of pegosafermin at four different doses versus placebo in patients with triglycerides ≥ 500 mg / dL and triglycerides ≤ 2000 mg / dL. The primary endpoint was percent change in triglycerides from baseline. Prespecified secondary endpoints included other lipids and substudies of hepatic fat fraction by magnetic resonance imaging, as well as markers of insulin sensitivity and inflammation.

[0250] <Result> A total of 85 patients were randomized to treatment (18 placebo, 67 pegosafermin at four different doses; 55% of patients received background lipid-lowering therapy; mean baseline triglycerides, 733 mg / dL). There was a significant reduction in median triglycerides for pooled pegosafermin doses versus placebo (57.3% vs. 11.9%, a difference of 45.4%, 95% confidence interval (CI): 30.3%, 57.1%; p<0.001), with reductions ranging from 36.4% to 63.4% across all four treatment groups versus placebo. Results were consistent regardless of whether patients were receiving background lipid-lowering therapy. Mean apoB and non-HDL-C changed by -10.5% and -18.3% with the pooled dose compared with 1.1% and -0.6% with placebo (p=0.019 and p=0.007, respectively). Liver fat (n=23) was significantly reduced in the pooled treatment groups versus placebo (34.8%, p=0.012). There were no serious adverse events related to study drug.

[0251] <Conclusion> Pegozafermin reduced triglycerides, non-HDL-C, apoB, and hepatic fat fraction. If these results are confirmed in a phase 3 trial, pegosafermin may be a promising treatment for severe hypertriglyceridemia.

[0252] Severe hypertriglyceridemia (SHTG; ≥ 500 mg / dL) increases the risk of both acute pancreatitis and cardiovascular disease 1~10 Although lifestyle modification strategies are often recommended as first-line treatment, triglyceride (TG) levels often remain elevated, requiring pharmacological treatment in almost all patients. 11~13 Current treatments for severe hypertriglyceridemia rarely lower TG to desirable levels, highlighting the need for new therapies. Furthermore, SHTG is commonly associated with obesity, metabolic syndrome, insulin resistance, type 2 diabetes mellitus (T2DM), and nonalcoholic fatty liver disease (NAFLD).12,14~16 , an ideal treatment would not only reduce TG levels but also provide benefits for other metabolic comorbidities.

[0253] Fibroblast growth factor 21 (FGF21) is an endogenous stress hormone that regulates lipid and glucose metabolism and energy expenditure. FGF21 is thought to lower TG by decreasing de novo lipogenesis in the liver, inhibiting the release of free fatty acids from adipose tissue, and increasing free fatty acid oxidation in muscle and liver. 17~20 Pegozafermin is a glycopegylated recombinant analog of human FGF21 designed to have a longer half-life than native FGF21 while recapitulating the receptor activity profile of the native hormone. It is being developed for SHTG and non-alcoholic steatohepatitis (NASH). Pegozafermin has previously demonstrated efficacy on serum lipids (TG, LDL, non-HDL, HDL), insulin resistance, HbA1c, body weight, and liver fat, albeit in the NASH patient population. 21,22 Previous clinical trials with pegosafermin and other FGF21 analogs have consistently demonstrated lipid improvements in both healthy volunteers and patients with NASH or diabetes, but FGF21 analogs have not been evaluated in SHTG. 21,23~27 The ENTRIGUE trial was designed to investigate pegosafermin as a novel therapeutic agent for the treatment of SHTG.

[0254] <Method> <Test Design> The ENTRIGUE study was a randomized, double-blind, placebo-controlled, dose-ranging, Phase 2 study designed to evaluate the efficacy, safety, and tolerability of pegosafermin administered subcutaneously once weekly (QW) or every two weeks (Q2W) in participants with SHTG. Participants with screening fasting triglycerides ≥ 500 mg / dL (5.6 mmol / L) and ≤ 2000 mg / dL (22.6 mmol / L) were eligible for enrollment, regardless of background lipid-modifying therapy with statins, prescribed omega-3 fatty acids, and fibrates. Participants were enrolled in one of two cohorts: 1) the main study cohort (no concurrent fibrate therapy allowed); or 2) the fibrate cohort.

[0255] The primary study cohort was randomized 1:1:1:1:1 to one of four doses of pegosafermin (9 mg QW, 18 mg QW, 27 mg QW, or 36 mg Q2W) or placebo, and the fibrate cohort was randomized 1:1 to either pegosafermin 27 mg QW or placebo QW for 8 weeks (Figure 4). All participants were stratified by TG levels (<750 mg / dL or ≥750 mg / dL [8.5 mmol / L]) and further stratified by whether or not they were receiving background therapy in the primary cohort. A magnetic resonance imaging proton density fat fraction (MRI-PDFF) substudy was conducted at a site capable of performing MRI-PDFF imaging. Participants in the fibrate cohort were required to have an MRI-PDFF ≥6.0% at enrollment. After completing the 8-week treatment period, participants underwent a 4-week safety follow-up period.

[0256] Participants were required to fast for 12–14 h and abstain from alcohol for 48 h before each lipid assessment throughout the study. After a stabilization period (4 weeks if on stable, approved lipid-modifying therapy; up to 6 weeks if discontinuing ineligible lipid-modifying therapy), an approximately 2-week qualification period consisted of two fasting TG assessments, separated by at least 1 week. If the mean TG levels from these two laboratory assessments were not within the inclusion range, an additional third assessment was performed, separated by at least 1 week from the previous assessment. The mean TG value from the last two assessments served as TG eligibility for the study and was the basis for participant TG stratification at randomization. Exclusion criteria included poorly controlled or recent diagnosis of hypertension, poorly controlled or recent diagnosis of type 2 diabetes within 6 months of screening, and a BMI >45 kg / m. 2 or cardiovascular or cerebrovascular disease. Additional eligibility and complete exclusion criteria are outlined in the study protocol.

[0257] The study was conducted at 50 clinical sites in the United States, Hungary, Poland, and the Czech Republic from September 2020 to June 2022. The study was approved by the local ethics committee or institutional review board. Participants provided written informed consent. All authors had access to the study data, participated in manuscript preparation, and are responsible for the data and analysis.

[0258] <Research results> The study objectives and endpoints were similar for the main study and the fibrate cohort. Because the number of patients enrolled in the fibrate cohort was small (n=6), data from both cohorts were presented combined. The primary efficacy endpoint was the percent change in serum triglycerides from baseline to week 8. Secondary efficacy endpoints included changes in specific serum lipids and lipoproteins, metabolic markers, and liver fat content assessed by MRI-PDFF. Safety endpoints included overall safety and tolerability assessments, markers of liver function, and immunogenicity.

[0259] Baseline TG levels were defined as the mean of the randomization-day assessment collected pre-dose and the two previous lipid-qualifying assessments collected during the TG qualifying period. TG values at week 8 were defined as the mean of TG values at week 7 and week 8. If TG values were missing at week 7 or week 8, the non-missing result was used as the TG value at week 8. Responder analyses of TG reduction at various threshold levels were performed, and the proportion of participants who achieved TG normalization (<150 mg / dL) was also analyzed.

[0260] <Statistical analysis> The study was designed to have at least 86% power to detect a 45% difference in TG between the pegosafermin and placebo groups, assuming a 50% reduction in TG in the pegosafermin group and a 5% reduction in TG in the placebo group. Pooled pegosafermin across all dose groups and individual pegosafermin dose groups were compared with placebo. All analyses were performed at a two-sided alpha level of 0.05 without adjusting for multiplicity, and confidence intervals (CIs) were two-sided (95%). Summary descriptive statistics were used to present demographic and baseline characteristics, safety endpoints, and pharmacodynamic parameters.

[0261] The patient breakdown and population analysis set are shown in Figure 5 and Table 3.

[0262] [Table 1]

[0263] The primary efficacy analysis was performed using the nonparametric van Elteren test stratified by baseline TG levels and background lipid therapy, and treatment differences were tested using pooled data based on the entire analysis set. Position shift estimates and Hodges-Lehmann two-sided 95% CIs are shown. For comparisons of individual pegosafermin treatment groups with placebo, an unstratified Wilcoxon rank-sum test was used due to small sample sizes. When the proportion of participants in a subgroup was less than 33% of the total cohort, only descriptive analyses were performed.

[0264] Secondary efficacy endpoints were analyzed using mixed-model repeated measures. When mixed-model assumptions were significantly violated, nonparametric methods were used for analysis. The proportion of participants with TG <500 mg / dL at week 8 was analyzed using stratified Cochran Mantel Haenszel (CMH) methods, using patients with both baseline and week 8 TG results. Unstratified chi-square tests were performed for comparisons between placebo and individual pegosafermin dose groups. No adjustments for multiple testing were made in this phase 2 study. Statistical analyses were performed using SAS®, version 9.4 or later. A complete description of the study endpoints and prespecified analyses is provided in the study protocol and statistical analysis plan.

[0265] <Result> <Patient characteristics> A total of 489 patients were screened, and 85 patients (17.4%) were randomized and treated (18 placebo; 67 pegosafermin at four different doses). The distribution among patients treated with pegosafermin was as follows: 9 mg QW, n=16; 18 mg QW, n=17; 27 mg QW, n=18; and 36 mg Q2W, n=16. Patient baseline characteristics, shown in Table 1, were fairly evenly distributed between groups, with a mean age of 53.7 years, 75.3% male, and a mean BMI of 33.1 kg / m. 250.6% had T2DM, 55.3% were receiving lipid-lowering therapy (e.g., statins, prescription omega-3 fatty acids, fibrates, bempedoic acid, or ezetimibe), and the mean baseline triglyceride level was 732.5 mg / dL. Other baseline lipids were typical for this population: LDL-C 89.1 mg / dL, HDL-C 28.4 mg / dL, and non-HDL-C 211.5 mg / dL. At clinical sites with MRI capability, a subset of patients (n = 24) underwent proton density fat fraction (PDFF) assessment to measure hepatic steatosis. All patients assessed by MRI-PDFF had evidence of hepatic steatosis (>5% hepatic fat) at baseline, with an overall mean of 20.1% (Table 1).

[0266] [Table 2A] [Table 2B]

[0267] <Efficacy endpoints> <Effect on triglyceride levels (primary endpoint)> Pegozafermin significantly reduced TG levels after 8 weeks of treatment across all treatment groups, with placebo-corrected reductions ranging from -24.6% to -51.5%. Pooled pegosafermin data showed a median percent change in TG levels of -45.4% (-57.3% vs. -11.9% placebo; 95% CI: -57.1%, -30.3%; p<0.001) (Figure 1A). The magnitude of TG reduction seen in this study was clinically significant, as 79.7% of patients treated with pegosafermin achieved target TG levels of <500 mg / dL compared with 29.4% of patients on placebo (95% CI: 29.4%, 74.7%; p<0.001) (Figure 1B). Furthermore, 60.9% of all patients treated with pegosafermin had a ≥50% reduction from baseline (95% CI: 36.7%, 69.5%; p<0.001), whereas at the highest QW dose (27 mg), 75.0% of patients demonstrated a ≥50% reduction in TG levels from baseline (95% CI: 45.1%, 93.1%; p<0.001), and 31.3% were able to normalize TG levels to <150 mg / dL (95% CI: 8.5%, 54.0%; p=0.012) (Figure 1B). TG reductions were comparable across all prespecified groups (Figure 1C) and consistent regardless of background lipid-lowering therapy or T2DM status (Figures 1C-1F).

[0268] Impact on overall lipid profile Pegozafermin treatment resulted in clinically meaningful improvements in non-HDL-C and apoB, with pooled mean percent reductions in LS for pegosafermin of -18.3% (95% CI: -30.7%, -5.1%; p=0.007) versus -0.6% for placebo, and -10.5% (95% CI: -21.5%, -2.0%; p=0.019) versus -1.1% for placebo, respectively (Figures 2A-2B). Pegozafermin treatment also resulted in a significant reduction in apoC3 (median percent change -41.9% versus -8.9% for placebo; 95% CI: -44.7%, -18.0%; p<0.001) (Figure 2C). Although small changes in LDL-C were detected in the pooled pegosafermin group (Figure 2D), the mean percent change from baseline in HDL-C levels in patients receiving 27 mg of pegosafermin weekly was significantly increased (44.5% vs. 9.7% for placebo; 95% CI: 14.5%, 55.1%; p=0.001) (Figure 2E). Further lipid data are listed in Table 4.

[0269] [Table 3A] [Table 3B] [Table 3C]

[0270] Liver and metabolic effects Patients treated with pegosafermin for 8 weeks had a significant reduction in hepatic steatosis compared to placebo (LS mean percent change -42.2% vs. -8.3%; 95% CI: -60.9%, -8.7%; p = 0.012) (Figure 3A). Representative MRI-PDFF images are shown in Figure 3B, and all individual treatment responses and images are shown in Figures 6A-6B. Many patients treated with pegosafermin achieved significant clinical thresholds, including a ≥30% reduction, a ≥50% reduction, or normalization (defined as <5%) of hepatic fat, achieving response rates of 88%, 41%, and 24%, respectively, compared to 0% for placebo, for all measures (Figure 3C). Patients receiving the 27 mg weekly dose also showed improvements in body weight and markers of ALT, AST, and hsCRP (Table 5).

[0271] Adiponectin increased significantly across all pegosafermin doses, with placebo-corrected values ranging from 50.6% to 87.6%, with a pooled mean percent change across pegosafermin doses of 62.8% (69.5% vs. 5.7% for placebo; 95% CI: 30.3%, 95.3%, p<0.001) (Figure 3D). Furthermore, in patients not receiving concomitant insulin, pegosafermin significantly reduced median insulin levels in both the 18 mg and 27 mg weekly dose groups (-21.5% [p=0.031] and -41.8% [p=0.006], respectively) compared with a 9.3% increase with placebo (Figure 3E). In patients randomized to the 27 mg weekly dose, additional metabolic benefits observed after 8 weeks of treatment included improvements in fasting plasma glucose and HbA1c (Table 5).

[0272] [Table 4A] [Table 4B]

[0273] <Safety> Treatment-emergent adverse events (TEAEs) were reported in 41 / 67 (61.2%) patients treated with pegosafermin versus 9 / 18 (50%) in placebo (Table 2). The most common TEAEs were related to gastrointestinal disorders and injection site reactions, all of which were mild to moderate in severity and mostly transient in duration. In the pooled pegosafermin groups, nausea, diarrhea, and injection site reactions occurred in 13.4%, 10.4%, and 9%, respectively, compared with 0%, 5.6%, and 0% for placebo. TEAE rates were higher for nausea (27.8%) and diarrhea (22.2%) with the 27 mg weekly dose. No Grade 3 or higher TEAEs were reported. One severe TEAE of hypertension was reported in the 27 mg QW group, but it was deemed unrelated to treatment and the study was discontinued. There were four additional emergency treatment interruptions in the 27 mg treatment group. Two were judged to be unrelated and two were judged to be related (Table 2). No deaths or transaminase elevation adverse events were reported.

[0274] [Table 5]

[0275] <Consideration> This placebo-controlled randomized trial demonstrated that treatment with the FGF21 analog pegozafermin resulted in significant reductions in TG in patients with SHTG, achieving TG levels ≤500 mg / dL in 80% of patients. Furthermore, non-HDL cholesterol, apoB, and apoC3 levels were significantly reduced, suggesting that pegozafermin reduces the production and improves clearance of TG-rich lipoproteins. While LDL cholesterol levels remained stable, HDL cholesterol increased numerically at all doses, particularly at the 27 mg dose. Despite the short 8-week study period, the 27 mg QW dose of pegozafermin improved various measures of insulin sensitivity, including adiponectin. Previous data from NASH patient populations suggest that improvements continue with longer treatment durations with the 27 mg QW dose. Notably, an absolute reduction in HbA1c of 0.9 was achieved at week 20 in NASH patients with baseline HbA1c levels ≥6.5%. 22 .

[0276] Another important finding of this study was the prevalence of hepatic fat in this severely hypertriglyceridemia population. 100% of subjects with MRI-PDFF data had baseline hepatic steatosis, defined by >5% hepatic fat. This raises the question of whether SHTG patients should be routinely screened for hepatic fat—a question that will need to be tested in future randomized trials. To our knowledge, these are the first data demonstrating significant reductions in hepatic fat with treatment targeting TG-rich lipoproteins in severe hypertriglyceridemia. Safety and tolerability remained consistent with previous data, with mild-to-moderate gastrointestinal disturbances being the most common TEAE. 21,22 There were no serious TEAEs related to the study drug.

[0277] Patients with severe hypertriglyceridemia often have metabolic comorbidities related to dyslipidemia and insulin resistance, such as obesity, metabolic syndrome, T2DM, and nonalcoholic fatty liver disease. Data from patients with residual dyslipidemia receiving lipid-modifying therapy in the United States showed that only 36.5% of patients achieved target or near-normal levels of TG, LDL-C, and HDL-C. 28 In this phase 2 study, we demonstrated that the FGF21 pathway may improve lipid and insulin sensitivity markers, potentially impacting metabolic health. Furthermore, we demonstrated that pegosafermin can reverse hepatic fat accumulation in a relatively short period of 8 weeks.

[0278] Limitations of this study include the lack of power to assess clinical events such as pancreatitis, liver failure, and cardiovascular endpoints. Further safety and tolerability data from longer drug exposure at the intended dose are needed.

[0279] In conclusion, the FGF21 analog pegozafermin significantly reduces TGC, non-HDL cholesterol, apoB, apoC3, and liver fat in patients with SHTG, and may positively impact other aspects of metabolic dysregulation. If these findings are confirmed in an adequately powered Phase 3 trial, pegozafermin may be useful for treating SHTG and simultaneously addressing several other cardiometabolic risk factors.

[0280] <References> 1. Wang GJ, Gao CF, Wei D, Wang C, Ding SQ. Acute pancreatitis: etiology and common pathogenesis. World J Gastroenterol 2009;15(12):1427‐30. DOI: 10.3748 / wjg.15.1427. 2. Anderson F, Thomson SR, Clarke DL, Buccimazza I. Dyslipidaemic pancreatitis clinical assessment and analysis of disease severity and outcomes. Pancreatology 2009;9(3):252‐7. DOI: 10.1159 / 000212091. 3. Yuan G, Al‐Shali KZ, Hegele RA. Hypertriglyceridemia: its etiology, effects and treatment. CMAJ 2007;176(8):1113‐20. DOI: 10.1503 / cmaj.060963. 4. Ganda OP, Bhatt DL, Mason RP, Miller M, Boden WE. Unmet need for adjunctive dyslipidemia therapy in hypertriglyceridemia management. J Am Coll Cardiol 2018;72(3):330‐343. DOI: 10.1016 / j.jacc.2018.04.061. 5. Toth PP, Granowitz C, Hull M, Liassou D, Anderson A, Philip S. High triglycerides are associated with increased cardiovascular events, medical costs, and resource use: a real‐world administrative claims analysis of statin‐treated patients with high residual cardiovascular risk. J Am Heart Assoc 2018;7(15):e008740. DOI: 10.1161 / JAHA.118.008740. 6. Klempfner R, Erez A, Sagit BZ, et al. Elevated triglyceride level is independently associated with increased all‐cause mortality in patients with established coronary heart disease: twenty‐two‐year follow‐up of the Bezafibrate Infarction Prevention Study and Registry. Circ Cardiovasc Qual Outcomes 2016;9(2):100‐8. DOI: 10.1161 / CIRCOUTCOMES.115.002104. 7. Nichols GA, Philip S, Reynolds K, Granowitz CB, Fazio S. Increased cardiovascular risk in hypertriglyceridemic patients with statin‐controlled LDL cholesterol. J Clin Endocrinol Metab 2018;103(8):3019‐3027. DOI: 10.1210 / jc.2018‐00470. 8. Libby P. Triglycerides on the rise: should we swap seats on the seesaw? Eur Heart J 2015;36(13):774‐6. DOI: 10.1093 / eurheartj / ehu500. 9. Bhatt DL, Steg PG, Miller M, et al. Cardiovascular Risk Reduction with Icosapent Ethyl for Hypertriglyceridemia. N Engl J Med 2019;380(1):11‐22. DOI: 10.1056 / NEJMoa1812792. 10. Nichols GA, Philip S, Reynolds K, Granowitz CB, Fazio S. Increased residual cardiovascular risk in patients with diabetes and high versus normal triglycerides despite statin‐controlled LDL cholesterol. Diabetes Obes Metab 2019;21(2):366‐371. DOI: 10.1111 / dom.13537. 11. Virani SS, Morris PB, Agarwala A, et al. 2021 ACC expert consensus decision pathway on the management of ASCVD risk reduction in patients with persistent hypertriglyceridemia: a report of the American College of Cardiology Solution Set Oversight Committee. J Am Coll Cardiol 2021;78(9):960‐993. DOI: 10.1016 / j.jacc.2021.06.011. 12. American College of Cardiology. Hypertriglyceridemia management according to the 2018 AHA / ACC guideline. August 2022 (https: / / www.acc.org / latest‐in‐cardiology / articles / 2019 / 01 / 11 / 07 / 39 / hypertriglyceridemia‐management‐according‐to‐the‐2018‐aha‐acc‐guideline). 13. Rosenson RS, Eckel RH. Hypertriglyceridemia in adults: management. August 2022 (https: / / www.uptodate.com / contents / hypertriglyceridemia‐in‐adults‐management). 14. Xing J, Guan X, Zhang Q, Chen S, Wu S, Sun X. Triglycerides mediate body mass index and nonalcoholic fatty liver disease: a population‐based study. Obes Facts 2021;14(2):190‐196. DOI: 10.1159 / 000514848. 15. Rashid N, Sharma PP, Scott RD, Lin KJ, Toth PP. Severe hypertriglyceridemia and factors associated with acute pancreatitis in an integrated health care system. J Clin Lipidol 2016;10(4):880‐890. DOI: 10.1016 / j.jacl.2016.02.019. 16. Pejic RN, Lee DT. Hypertriglyceridemia. J Am Board Fam Med 2006;19(3):310‐6. DOI: 10.3122 / jabfm.19.3.310. 17. Lin X, Liu YB, Hu H. Metabolic role of fibroblast growth factor 21 in liver, adipose and nervous system tissues. Biomedical reports 2017;6(5):495‐502. DOI: 10.3892 / br.2017.890. 18. Tillman EJ, Rolph T. FGF21: an emerging therapeutic target for non‐alcoholic steatohepatitis and related metabolic diseases. Front Endocrinol (Lausanne) 2020;11:601290. DOI: 10.3389 / fendo.2020.601290. 19. Kliewer SA, Mangelsdorf DJ. A dozen years of discovery: insights into the physiology and pharmacology of FGF21. Cell Metab 2019;29(2):246‐253. DOI: 10.1016 / j.cmet.2019.01.004. 20. Stojsavljevic‐Shapeski S, Duvnjak M, Virovic‐Jukic L, Hrabar D, Smircic Duvnjak L. New drugs on the block‐emerging treatments for nonalcoholic steatohepatitis. J Clin Transl Hepatol 2021;9(1):51‐59. DOI: 10.14218 / JCTH.2020.00057. 21. Frias JP, Lawitz EJ, Ortiz‐LaSanta G, et al. BIO89‐100 demonstrated robust reductions in liver fat and liver fat volume (LFV) by MRI‐PDFF, favorable tolerability and potential for weekly (QW) or every 2 weeks (Q2W) dosing in a phase 1b / 2a placebo‐controlled, double‐blind, multiple ascending dose study in NASH. J Endocr Soc 2021;5(Supplement_1):A5‐A6. DOI: 10.1210 / jendso / bvab048.010. 22. Alkhouri N, Loomba R, Frias JP, et al. Pegozafermin led to significant metabolic benefits, in addition to robust beneficial effects on the liver, in an open‐label cohort of a phase 1b / 2a study in subjects with non‐alcoholic steatohepatitis (NASH). J Hepatol 2022;77:S732. DOI: 10.1016 / S0168‐8278(22)01785‐8. 23. Gaich G, Chien JY, Fu H, et al. The effects of LY2405319, an FGF21 analog, in obese human subjects with type 2 diabetes. Cell Metab 2013;18(3):333‐40. DOI: 10.1016 / j.cmet.2013.08.005. 24. Talukdar S, Zhou Y, Li D, et al. A long‐acting FGF21 molecule, PF‐05231023, decreases body weight and improves lipid profile in non‐human primates and type 2 diabetic subjects. Cell Metab 2016;23(3):427‐40. DOI: 10.1016 / j.cmet.2016.02.001. 25. Charles ED, Neuschwander‐Tetri BA, Pablo Frias J, et al. Pegbelfermin (BMS‐986036), pEGylated FGF21, in patients with obesity and type 2 diabetes: results from a randomized phase 2 study. Obesity (Silver Spring) 2019;27(1):41‐49. DOI: 10.1002 / oby.22344. 26. Kaufman A, Abuqayyas L, Denney WS, Tillman EJ, Rolph T. AKR‐001, an Fc‐FGF21 analog, showed sustained pharmacodynamic effects on insulin sensitivity and lipid metabolism in type 2 diabetes patients. Cell Rep Med 2020;1(4):100057. DOI: 10.1016 / j.xcrm.2020.100057. 27. Harrison SA, Ruane PJ, Freilich BL, et al. Efruxifermin in non-alcoholic steatohepatitis: a randomized, double-blind, placebo-controlled, phase 2a trial. Nat Med 2021;27(7):1262-1271. DOI: 10.1038 / s41591-021-01425-3. 28. Wong ND, Chuang J, Wong K, Pham A, Neff D, Marrett E. Residual dyslipidemia among United States adults treated with lipid modifying therapy (data from National Health and Nutrition Examination Survey 2009‐2010). Am J Cardiol 2013;112(3):373‐9. DOI: 10.1016 / j.amjcard.2013.03.041.

[0281] Example 2: Pegozafermin provides beneficial lipid effects in patients with severe hypertriglyceridemia (SHTG) regardless of prior lipid-modifying therapy: Analysis of the Phase 2 ENTRIGUE trial <Background> Fibroblast growth factor 21 (FGF21) is an endogenous stress hormone that regulates glucose and lipid metabolism and energy expenditure. Pegozafermin (PGZ), a glyco-PEGylated analog of human FGF21 developed for severe hypertriglyceridemia (SHTG), has been shown to significantly improve lipid profiles in patients with SHTG. Most patients with SHTG are treated with lipid-modifying therapy (LMT). In this analysis, we evaluated the lipid effects of PGZ based on the subjects' background LMT status.

[0282] <Method> ENTRIGUE was a phase 2, double-blind, randomized, five-arm study. Patients with fasting triglycerides (TG) ≥ 500 mg / dL and ≤ 2000 mg / dL were randomized to receive PGZ at four doses, once weekly (9 mg, 18 mg, 27 mg) or once every two weeks (36 mg), compared with placebo for 8 weeks. Subjects could be on background LMT, including statins, prescription fish oil, fibrates, or others. The primary endpoint was percent change in TG from baseline.

[0283] <Result> Of the 85 subjects randomized to placebo (n=18) or PGZ (n=67), 55% were on background LMT: 45% were on statins (55% of which were high-intensity); 14% were on prescription fish oil; and 7% were on fibrates. PGZ significantly reduced triglycerides in patients with background LMT, with a placebo-adjusted median reduction of 42.6% (95% CI -56.29, -22.99, p=0.001). Overall, 79.7% of patients treated with PGZ had their triglycerides reduced to <500 mg / dL compared with 29.4% of those treated with placebo (85.3% for PGZ vs. 45.5% for placebo in patients with LMT). Treatment with PGZ significantly reduced non-HDL cholesterol and ApoB, with placebo-adjusted mean reductions of 17.9% (p=0.007) and 11.8% (p=0.019), respectively. These reductions were more pronounced in patients undergoing background LMT, with placebo-adjusted reductions in non-HDL-C and ApoB of 21.7% and 16.8%, respectively. LDL-C did not change significantly in the overall population (LS mean difference 1.7%, p=0.87). However, PGZ reduced LDL-C in subjects undergoing background LMT (LS mean reduction 9.0%).

[0284] <Conclusion> PGZ significantly reduced TG and atherogenic lipids in patients with SHTG. This analysis demonstrated that PGZ reduced TG when added to background LMT. A phase 3 program is planned to confirm these findings.

[0285] 7A-7B show the median change in triglycerides from baseline at week 8. Pegozafermin demonstrates a significant reduction in triglycerides over and above background treatment.

[0286] Figure 8 shows the median change in triglycerides from baseline at week 8. Pegozafermin demonstrates a significant reduction in triglycerides in addition to statins, prescription fish oil, and fibrates.

[0287] 9A-9B show that treatment with pegosafermin resulted in a reduction in triglycerides among subjects receiving background high-intensity statins.

[0288] FIG. 10 shows that patients treated with pegozafermin reach initial treatment goals regardless of background therapy.

[0289] FIG. 11 shows that pegozafermin treatment resulted in an improvement in non-HDL cholesterol regardless of background treatment.

[0290] FIG. 12 shows that pegozafermin treatment resulted in an improvement in apolipoprotein B, regardless of background treatment.

[0291] FIG. 13 shows that there was no significant change in LDL cholesterol regardless of background treatment.

[0292] FIG. 14 shows that pegozafermin treatment resulted in improvements in HDL cholesterol, regardless of background treatment.

[0293] Example 3: FGF21 analogue pegozafermin in severe hypertriglyceridemia: a randomized phase 2 clinical trial <Summary> Pegozafermin, a long-acting glycopegylated analog of human fibroblast growth factor 21, is being developed for the treatment of severe hypertriglyceridemia and nonalcoholic steatohepatitis. Here, we report the results of a phase 2, double-blind, randomized, five-arm study in patients with severe hypertriglyceridemia (triglycerides ≥ 500 mg / dL and ≤ 2000 mg / dL) administered pegosafermin at four different doses (n = 67, 52 men) for 8 weeks compared with placebo (n = 18, 12 men). Treated patients demonstrated a significant reduction in pooled median triglycerides in the pegosafermin group compared with placebo (57.3% vs. 11.9%, difference vs. placebo: -43.7%, 95% confidence interval [Cl]: -57.1%, -30.3%; p < 0.001), meeting the study's primary endpoint. Median triglyceride reductions ranged from 36.4% to 63.4% across all treatment groups and were consistent regardless of background lipid-lowering therapy. Secondary endpoint results included significant reductions in mean ApoB and non-HDL-C concentrations (-10.5% and -18.3% for the pooled doses compared with 1.1% and -0.6% for placebo (95% CI: -21.5%, -2.0%; p=0.019 and 95% CI: -30.7%, -5.1%; p=0.007, respectively) and significant reductions in liver fat fraction for the pooled treatments (n=17) versus placebo (n=6) as assessed in a magnetic resonance imaging substudy (pooled pegosafermin -42.2%, placebo -8.3%; 95% CI: -60.9%, -8.7%; p=0.012). All p-values are based on comparisons with placebo. No serious adverse events related to study drug were observed.

[0294] <Introduction> Severe hypertriglyceridemia (SHTG; ≥ 500 mg / dL) increases the risk of both acute pancreatitis and cardiovascular disease 1~10 Although lifestyle modification strategies are often recommended as first-line treatment, triglyceride (TG) levels often remain elevated, requiring pharmacological treatment in almost all patients. 11~13Current treatments for SHTG rarely lower TG to desirable levels, highlighting the need for new therapies. Furthermore, SHTG is commonly associated with obesity, metabolic syndrome, insulin resistance, type 2 diabetes mellitus (T2DM), and nonalcoholic fatty liver disease (NAFLD). 12,14~16 , an ideal treatment would not only reduce TG levels but also provide benefits for other metabolic comorbidities.

[0295] Fibroblast growth factor 21 (FGF21) is an endogenous stress hormone that regulates lipid and glucose metabolism and energy expenditure. Preclinical data suggest that in the liver, FGF21 reduces fat through increased adenosine monophosphate-activated protein kinase (AMPK) signaling, stimulates fatty acid oxidation to reduce de novo lipogenesis (DNL), alleviates new triglyceride accumulation, and promotes the secretion of triglycerides in the form of very low-density lipoproteins (VLDL) to reduce existing fat stores. In adipose tissue, FGF21 activates brown adipose tissue by inducing the expression of uncoupling protein 1, browning white adipose tissue and thereby improving insulin sensitivity and accelerating triglyceride-rich lipoprotein turnover (e.g., VLDL metabolism). 17~22 Importantly, FGF21 may increase the expression of low-density lipoprotein receptor (LDLR) and accelerate the uptake of generated VLDL remnants via the ApoE-LDLR pathway. 22 .

[0296] Pegozafermin is a glycopegylated recombinant analog of human FGF21 designed to have a longer half-life than native FGF21 while recapitulating the receptor activity profile of the native hormone. It is being developed for the treatment of SHTG and nonalcoholic steatohepatitis (NASH). Pegozafermin has previously shown beneficial effects on serum lipids (triglycerides [LDL], low-density lipoprotein cholesterol [LDL-C], non-high-density lipoprotein cholesterol [non-HDL-C], and high-density lipoprotein cholesterol [HDL-C]), insulin resistance, HbA1c, body weight, and liver fat in patients with NASH. 23,24 Previous clinical trials with pegosafermin and other FGF21 analogs have consistently demonstrated lipid improvements in both healthy volunteers and patients with NASH or diabetes, but FGF21 analogs have not been evaluated in SHTG. 23,25~29 To our knowledge, the ENTRIGUE trial was the first clinical trial to investigate an FGF21 analogue as a novel therapeutic agent for the treatment of SHTG.

[0297] <Method> <Test Design> The ENTRIGUE study was a randomized, double-blind, placebo-controlled, dose-ranging, Phase 2 study designed to evaluate the efficacy, safety, and tolerability of pegosafermin administered subcutaneously once weekly (QW) or every two weeks (Q2W) in participants with SHTG. Participants with screening fasting TG ≥ 500 mg / dL (5.6 mmol / L) and ≤ 2000 mg / dL (22.6 mmol / L) were eligible for enrollment, regardless of background lipid-modifying therapy with statins, prescribed omega-3 fatty acids, and fibrates (fibrate expansion cohort only). Participant sex, as determined by self-report, was not considered in the study design.

[0298] Participants were enrolled in one of two cohorts: 1) the main study cohort (concurrent fibrate therapy was not permitted); or 2) the fibrate expansion cohort. Because fibrates are commonly used to treat SHTG, a fibrate expansion cohort was initiated as a protocol amendment (v2.0) after study initiation to evaluate pegosafermin in subjects receiving stable fibrate therapy. The main study cohort was randomized 1:1:1:1:1 to one of four doses of pegosafermin (9 mg QW, 18 mg QW, 27 mg QW, or 36 mg Q2W) or placebo, and the fibrate cohort was randomized 1:1 to either pegosafermin 27 mg QW or placebo QW for 8 weeks (Figure 4). All participants were stratified by TG levels (<750 mg / dL or ≥750 mg / dL [8.5 mmol / L]) and, in the main cohort, further stratified by whether they were receiving background therapy. Because participants in the fibrate expansion cohort were required to have an MRI-PDFF ≥6.0% at enrollment, an MRI-PDFF substudy was initiated at sites capable of performing MRI-PDFF imaging. Twenty-four subjects (6 from the fibrate expansion cohort) underwent baseline MRI-PDFF measurements, of which 23 completed follow-up scans at the end of the study period. After completing the 8-week treatment period, all participants underwent a 4-week safety follow-up period.

[0299] Participants were required to fast for 12–14 h and abstain from alcohol for 48 h before each lipid assessment throughout the study. After a stabilization period (4 weeks if on stable, approved lipid-modifying therapy; up to 6 weeks if discontinuing ineligible lipid-modifying therapy), an approximately 2-week qualification period consisted of two fasting TG assessments, separated by at least 1 week. If the mean TG levels from these two laboratory assessments were not within the inclusion range, an additional third assessment was performed, separated by at least 1 week from the previous assessment. The mean TG value from the last two assessments served as TG eligibility for the study and was the basis for participant TG stratification at randomization. Exclusion criteria included poorly controlled or recent diagnosis of hypertension, poorly controlled or recent diagnosis of type 2 diabetes within 6 months of screening, HbA1c ≥ 9.5%, and BMI > 45 kg / m. 2 , or cardiovascular or cerebrovascular disease.

[0300] <Research results> The study objectives and endpoints were similar for the main study and the fibrate cohort. Because the number of patients enrolled in the fibrate cohort was small (n=6), data from both cohorts were presented combined. The primary efficacy endpoint was the percent change in serum triglycerides from baseline to week 8. Secondary efficacy endpoints included changes in specific serum lipids and lipoproteins, metabolic markers, and liver fat content assessed by MRI-PDFF. Safety endpoints included overall safety and tolerability assessments, markers of liver function, and immunogenicity.

[0301] Baseline TG levels were defined as the average of the randomization assessment collected pre-dose and the two preceding lipid assessments collected during the TG qualification period. TG values at week 8 were defined as the average of TG values at week 7 and week 8. If TG values were missing at week 7 or week 8, the non-missing result was used as the TG value at week 8. Responder analyses of TG reduction at various threshold levels were performed, and the proportion of participants who achieved TG normalization (<150 mg / dL) was also analyzed.

[0302] <Statistical analysis> The study was designed to have at least 86% power to detect a 45% difference in TG between the pegosafermin and placebo groups, assuming a 50% reduction in TG in the pegosafermin group and a 5% reduction in TG in the placebo group. Pooled pegosafermin across all dose groups and individual pegosafermin dose groups were compared with placebo. All analyses were performed at a two-sided alpha level of 0.05 without adjusting for multiplicity, and confidence intervals (CIs) were two-sided (95%). Summary descriptive statistics were used to present demographic and baseline characteristics, safety endpoints, and pharmacodynamic parameters.

[0303] Efficacy analyses were performed on the complete analysis set, including patients with at least one postbaseline TG level. Normality tests were performed, and nonparametric tests were used when normality was significantly impaired. Prespecified QQ plots suggested that the distribution of TG data was highly skewed, violating the normality assumption required for the MMRM method. Therefore, the primary efficacy analysis was performed using the nonparametric van Elteren test stratified by baseline TG levels and background lipid therapy, and treatment differences were tested using pooled data. Position shift estimates and Hodges-Lehmann two-sided 95% CIs are shown. For comparisons of individual pegosafermin treatment groups with placebo, an unstratified Wilcoxon rank-sum test was used due to small sample sizes. When the number of participants within a subgroup was too low for meaningful comparisons (n < 6), only descriptive analyses were performed. The placebo-corrected change was defined as the difference between the change from baseline in the pegosafermin treatment group and the change from baseline in the placebo group. Each week 8 value was defined as the mean of the week 7 value, week 8 value, and the early termination value that fell within the analysis window.

[0304] Secondary efficacy endpoints were analyzed using mixed-model repeated measures. When mixed-model assumptions were significantly violated, nonparametric methods were used for analysis. The proportion of participants with TG <500 mg / dL at week 8 was analyzed using stratified Cochran Mantel Haenszel (CMH) methods using patients with both baseline and week 8 TG results. Unstratified chi-square tests were used for comparisons between placebo and individual pegosafermin treatment groups. Statistical analysis was performed using SAS®, version 9.4 or later.

[0305] <Result> <Patient characteristics> 489 patients were screened, and 85 patients (17.4%) were randomized and treated. The distribution among patients treated with pegosafermin was as follows: placebo, n=18; 9 mg QW, n=16; 18 mg QW, n=17; 27 mg QW, n=18; and 36 mg Q2W, n=16. Post-baseline TG levels were available for 82 patients in the full analysis set. Patient baseline characteristics, shown in the table below, were reasonably balanced across groups, with a mean age of 53.7 years, 75.3% male, and a mean BMI of 33.1 kg / m. 2 50.6% had T2DM, 55.3% were receiving background lipid-lowering therapy (including statins, prescription omega-3 fatty acids, fibrates [fibrate cohort], bempedoic acid, and ezetimibe), and had a median baseline triglyceride level of 622.0 mg / dL. Other baseline lipids were at mean levels typical of this population: LDL-C 89.1 mg / dL, HDL-C 28.4 mg / dL, and non-HDL-C 211.5 mg / dL. At clinical sites with MRI capability, a subset of patients (n=24) underwent baseline proton density fat fraction (PDFF) assessment to measure hepatic steatosis. All patients assessed by MRI-PDFF had evidence of hepatic steatosis (>5% hepatic fat) at baseline, with an overall mean of 20.1% (see table below).

[0306] [Table 6A] [Table 6B]

[0307] Patient outcomes and results of the population analysis are shown in Figure 15 and Table 3.

[0308] <Efficacy endpoints> <Effect on triglyceride levels (primary endpoint)> Pegozafermin significantly reduced TG levels across all treatment groups after 8 weeks of treatment, with placebo-corrected changes from baseline ranging from -29.0% to -52.9%. Pooled pegosafermin data showed a placebo-corrected median percent change of -43.7% (-57.3% vs. placebo -11.9%; 95% CI -57.1%, -30.3%; p<0.001) (Figure 16A). A total of 79.7% of patients treated with pegosafermin achieved target TG levels of <500 mg / dL compared with 29.4% of patients receiving placebo (placebo-corrected 52.1%, 95% CI: 29.4%, 74.7%; p<0.001) (Figure 16B). Furthermore, 60.9% of all patients treated with pegosafermin had a ≥50% reduction from baseline compared with 5.9% of patients receiving placebo (53.1% placebo-corrected, 95% CI: 36.7%, 69.5%; p<0.001). Meanwhile, at the highest QW dose (27 mg), 75.0% of patients achieved a ≥50% reduction in TGs from baseline compared with 0% of placebo patients (69.1% placebo-corrected, 95% CI: 45.1%, 93.1%; p<0.001), and 31.3% were able to normalize TGs to <150 mg / dL (31.3% placebo-corrected, 95% CI: 8.5%, 54.0%; p=0.012) (Figure 16B). TG reduction was comparable across all pre-specified groups (Figure 6B) and remained consistent regardless of background lipid-lowering treatment or T2DM status (Figures 16C-16F).

[0309] Impact on overall lipid profile Pegozafermin treatment resulted in improvements in non-HDL-C and apolipoprotein B (ApoB), with least squares (LS) mean changes from baseline of -18.3% in the pegosafermin group and -0.6% in the placebo group (placebo-corrected -17.9%, 95% CI: -30.7%, -5.1%, p=0.007) and -10.5% in the pegosafermin group and 1.1% in the placebo group (placebo-corrected -11.8%, 95% CI: -21.5%, -2.0%, p=0.019), respectively (Figures 17A-17B). Treatment with pegozafermin also resulted in a significant reduction in ApoC3 (median percent change -41.9% vs. placebo -8.9% [placebo-corrected -32.0%, 95% CI: -44.7%, -18.0%; p<0.001]) (Figure 17C). Although small changes in LDL-C were detected in the pooled pegozafermin group (Figure 17D), the LS mean percent change from baseline in HDL-C levels in subjects receiving pegozafermin 27 mg weekly was significantly increased (44.5% vs. 9.7% in the placebo group [placebo-corrected 34.8%, 95% CI: 14.5%, 55.1%; p=0.001]) (Figure 17E). Weekly treatment with 27 mg of pegosafermin reduced ApoB48 by 73%, suggesting improved clearance of plasma chylomicrons and their remnants in addition to a reduction in ApoB100 particles. Further lipid data are presented below.

[0310] [Table 7A] [Table 7B]

[0311] Liver and metabolic effects Patients treated with pegosafermin for 8 weeks had a significant reduction in hepatic steatosis compared to placebo (LS mean percent change -42.2% vs. -8.3%; 95% CI: -60.9%, -8.7%; p = 0.012) (Figure 18A). Representative MRI-PDFF images are shown in Figure 18B, and all individual treatment images and responses are shown in Figures 6A and 6B. Many patients treated with pegosafermin achieved significant clinical thresholds, including a ≥30% reduction, a ≥50% reduction, or normalization (defined as <5%) of hepatic fat, achieving response rates of 88%, 41%, and 24%, respectively, compared to 0% for placebo, for all measures (Figure 18C). Patients receiving the 27 mg weekly dose also saw improvements in inflammatory markers: alanine aminotransferase, aspartate aminotransferase, and high-sensitivity C-reactive protein (see table below).

[0312] [Table 8]

[0313] <Safety> Treatment-emergent adverse events (TEAEs) were reported in 41 / 67 (61.2%) patients treated with pegosafermin versus 9 / 18 (50%) with placebo (see table below).

[0314] [Table 9A] [Table 9B]

[0315] The most common TEAEs were related to gastrointestinal disturbances and injection site reactions, all of which were mild to moderate in severity and mostly transient in duration. In the pooled pegosafermin groups, nausea, diarrhea, and injection site reactions occurred in 13.4%, 10.4%, and 9%, respectively, compared with 0%, 5.6%, and 0% for placebo. The TEAE rates were higher for nausea (27.8%) and diarrhea (22.2%) at the 27 mg weekly dose. No Grade 3 or higher TEAEs were reported. At week 8, there were no clinically significant differences in blood pressure between the placebo and pegosafermin treatment groups. The mean changes in systolic blood pressure for placebo and pegosafermin were -4.1 mmHg and 0.7 mmHg at week 4 and 0.3 mmHg and 1.7 mmHg at week 8, respectively. However, in the 27 mg QW group, one serious TEAE of hypertension was reported in a patient with newly diagnosed hypertension before enrollment, which was determined to be unrelated to treatment and led to study discontinuation. There were three additional treatment emergency interruptions in the 27 mg treatment group: two patients with TEAEs deemed related by the investigator (one with nausea and vomiting and one with abdominal cramps) and one patient with nausea and abdominal pain assessed as unrelated to pegosafermin. No deaths, systemic hypersensitivity reactions, or liver transaminase elevation adverse events were reported.

[0316] <Consideration> This placebo-controlled, randomized trial demonstrated that treatment with the FGF21 analog pegozafermin significantly reduced TG in patients with SHTG. Significant reductions were also observed in atherogenic lipoproteins, including non-HDL-C and ApoB, and ApoC3, a key regulator of lipoprotein lipase, suggesting that pegozafermin reduces the production and improves the clearance of TG-rich lipoproteins. While LDL-C levels remained relatively stable, there was a numerical increase in HDL cholesterol across all doses, most pronounced at the 27 mg dose. The 27 mg dose was also the most effective in reducing TG and lipoproteins. The biweekly administration regimen likely had a smaller effect across various outcome parameters due to the drug's pharmacokinetics and variability in TG levels.

[0317] Eligibility criteria allowed for enrollment of subjects on a stabilizing regimen of approved lipid-modifying therapy (LMT), such as statins, prescription fish oil, and / or fibrates. Approximately 55% of subjects were enrolled in background LMT, with the majority taking statins (45% [25% high-intensity statins]), followed by prescription fish oil (14%) and fibrates (7%). Initially, fibrates were excluded due to potential crosstalk between FGF21 and the peroxisome proliferator-activated receptor alpha (PPARα) pathway. 30 However, this study was ultimately amended to include a fibrate cohort, with the additional criterion that these subjects had to have at least 6% liver fat at baseline. Because enrollment in this group proved difficult (n = 6), subjects taking fibrates were likely underrepresented in the final study population compared with clinical practice. Nevertheless, with the exception of this potential caveat regarding fibrates, the overall use of LMT in the study population appears to largely reflect the actual treatment patterns of SHTG patients. Christian et al. reported that baseline medication utilization (up to 6 months prior to the index date) was approximately 31% for statins and approximately 14% for triglyceride-lowering drugs.31 In the same study, additional medication use after the index date increased slightly to 38% and 35% for statins and TG-lowering medications, respectively, leaving a significant number of patients untreated. Similarly, a study by Toth et al. reported that 30-50% of TG-treatment-naive patients had not initiated medication within 4 months of their index date (among patients prescribed medication, approximately 50% received statins, 30% received fibrates, and 8% received omega-3 fatty acids). 32 More recently, data from the Rochester Epidemiology Project showed that only 46% of patients with isolated hypertriglyceridemia (TG ≥ 500 mg / dL) underwent LMT within 18 months of detection of elevated TG levels. 33 .

[0318] Note that in this study, the effects of pegosafermin were consistent regardless of the presence or absence of background LMT, suggesting that pegosafermin may significantly improve many important lipid parameters when used as an adjunct to diet and exercise or as add-on therapy in patients unable to achieve TG <500 mg / dL. Furthermore, the effects of pegosafermin were comparable regardless of LMT drug class. This is an important finding because data from patients with residual dyslipidemia undergoing LMT in the United States showed that only 36.5% achieved target or near-normal levels of TG, LDL-C, and HDL-C. 34 .

[0319] Although LDL-C remains the primary target for CV risk reduction, there is great interest in finding new therapies that can address residual CV risk after LDL treatment. Most clinical trials utilizing triglyceride-lowering therapies (i.e., fibrates, niacin, and omega-3 fatty acids, with the exception of icosapent ethyl) have not demonstrated an add-on reduction in CV events in patients receiving statin therapy. 4For example, the REDUCE-IT trial (icosapent ethyl [IPE] 4 g / day) found a 25% reduction (p<0.001) in a composite of CV events in high-risk patients, but only part of the benefit was predicted by TG reduction, suggesting that the results were due to broader pleiotropic effects of IPE. The PR-MINENT trial found no benefit on CV outcomes despite TG reduction with pemafibrate. 35 The aforementioned study was conducted in patients with TG <500 mg / dL, so extrapolation to the SHTG population requires caution. PROMINENT reported that pemafibrate showed a small reduction in TG (-26.2%) and VLDL-C (-25.8%), but produced placebo-corrected increases in LDL-C, ApoB, and non-HDL-C (10 mg / dL, 5 mg / dL, and 3 mg / dL, respectively), with no difference in CV outcomes. 35 Similar to pemafibrate, a recently published paper on evinacumab (an angiogenesis inhibitor-like 3 [ANGPTL3]) also observed increases in LDL-C in SHTG patients across three cohorts, regardless of the presence or absence of lipoprotein lipase pathway mutations, although it should be noted that both non-HDL-C and ApoB were decreased in this study. 36 It is not unexpected that LDL-C can sometimes increase dramatically in patients treated for SHTG, especially when treated with agents such as fenofibrate or EPA / DHA. This is known as the "beta shift" phenomenon, and LDL-C levels may rise due to increased lipolysis of VLDL via lipoprotein lipase. 37 Although there was a relatively small increase in LDL-C that was not different from placebo in this study, we speculate that the 45% of patients receiving background statin therapy may have influenced the observed LDL-C response. Indeed, in a post-hoc analysis of patients receiving lipid-modifying therapy (including but not limited to statin therapy), the placebo-adjusted LS mean difference in LDL-C was -9.0%, compared with a LS mean difference of 1.7% in the pegosafermin group. 38Because patients enrolled in this study had a mean baseline LDL-C of <90 mg / dL, which is relatively well controlled for this population, when considering the overall atherosclerotic burden, the effects associated with minimal changes in LDL-C may be offset by the large reductions in non-HDL-C, ApoB, and triglyceride-rich lipoprotein cholesterol [TRL-C].

[0320] Newer RNA-based therapies (antisense oligonucleotides [ASO] and RNA interference [RNAi]) are also being developed to significantly lower TGs in both familial chylomicronemia syndrome (FCS) and severe hypertriglyceridemia. Early data suggest that traditional ASO approaches have safety concerns. For example, volanesorsen (an ASO APOC3 inhibitor) is approved in the EU for the treatment of FCS. 39 However, it has not been approved by the FDA due to concerns about bleeding and thrombocytopenia. Additionally, Pfizer and Ionis discontinued their clinical programs for vupanorsen (an ASO targeting ANGPTL3) due to its modest efficacy in reducing non-HDL-C and TGs and its association with dose-dependent increases in liver fat and liver enzymes. 40 The development of second-generation ASOs, such as olezarsen [an APOC3 inhibitor], is a major advancement, reflected in the FDA fast track designation for FCS patients. siRNA agents also show promise, but these agents also have some safety signals. Data from the SHASTA-2 trial evaluating ARO-APOC3 for SHTG suggest that this agent may be associated with elevated LDL-C, whereas data from the ARCHES-2 trial evaluating ARO-ANGPTL3 for mixed dyslipidemia showed a reduction in LDL-C. 41,42It is unclear whether the differential effects on LDL-C are related to different baseline triglyceride levels in the two populations or whether they depend on differences in gene targets. Interestingly, both trials reported increases in HbA1c in the treatment groups, especially in patients with baseline diabetes.

[0321] In the 8-week study reported herein, pegosafermin significantly reduced TG, non-HDL-C, ApoB, and liver fat, increased HDL-C with minimal changes in LDL-C, and improved liver transaminases, while maintaining a favorable safety and tolerability profile. Taken together, these data suggest that pegosafermin provides overall metabolic benefits with no identified safety concerns to date.

[0322] Although patients with SHTG are at increased risk of CV events, the main clinical risk in patients with TG ≥ 500 mg / dL is acute pancreatitis due to saturation or impairment of lipoprotein lipase-mediated lipolysis, resulting in the accumulation of TG-rich particles that are hydrolyzed by pancreatic lipase, releasing free fatty acids and subsequently causing pro-inflammatory signaling in adjacent pancreatic tissue. 43 Preclinical data suggest that FGF21 may have a role in modulating inflammation and injury induced by experimental pancreatitis. 44 Furthermore, FGF21 appears to promote β-cell survival and protect isolated rat pancreatic islets and insulin-producing INS cells from glucose-lipotoxicity and cytokine-induced apoptosis. 44 .

[0323] Valdivielso et al. demonstrated that elevated chylomicron levels are necessary to induce acute pancreatitis in the setting of elevated serum triglycerides. 45The subjects in this study had significantly elevated ApoB48, a specific marker of chylomicron particles, at baseline (median range 2.60-4.90 mg / dL) compared with healthy subjects (median 0.51 mg / dL), hyperlipidemic subjects (median 0.7 mg / dL), and obese subjects (median 0.82 mg / dL), indicating an increased risk of developing acute pancreatitis. 46 Pegozafermin significantly reduced ApoB48 (73% reduction with 27 mg once weekly), suggesting its ability to improve clearance of chylomicrons and chylomicron remnants. Taskinen et al. recently demonstrated that patients with loss-of-function mutations in APOC3, which increase lipoprotein lipase activity, have lower plasma concentrations of VLDL, IDL, and ApoB48 particles. 47 Because production rates of CM-ApoB48 and VLDL ApoB100 were unaffected, enhanced clearance of residuals may be the primary mechanism for the observed reduction.

[0324] Pegozafermin demonstrated a robust 50% reduction in ApoC3 at the 27 mg dose, suggesting that increased lipoprotein lipase activity may contribute to the observed reduction in ApoB48. Indeed, post-hoc analyses evaluating the correlation between percent change in TG and ApoC3 at week 8 in the pooled pegozafermin groups demonstrated a reasonable correlation between the two [Pearson r (linear correlation) = 0.87; Spearman r = 0.80, p-value < 0.001], indicating that greater reductions in TG were associated with greater reductions in ApoC3.

[0325] Current guidelines from the National Cholesterol Education Program Adult Treatment Panel III recommend lowering TG levels to <500 mg / dL to prevent acute pancreatitis, with a secondary focus on CV risk reduction. Data from a large retrospective claims study have shown that SHTG patients with TG levels <400 mg / dL during follow-up have a lower incidence of clinical events, and patients with TG levels <300 mg / dL during follow-up have significantly lower incidence rate ratios for pancreatitis, overall cardiovascular events, acute myocardial infarction (AMI), heart failure (HF), reperfusion, and acute coronary syndrome. However, the greatest clinical benefit (more robust incidence rate ratios overall and additional significance for ischemic stroke) was seen when follow-up levels were lowered to <200 mg / dL. 31 In the current study, 80% (pooled data) of patients receiving pegosafermin were able to suppress TGs to <500 mg / dL, and 44% and 31% of subjects receiving the 27 mg weekly dose achieved TG levels <200 mg / dL and <150 mg / dL, respectively, suggesting that pegosafermin may have a favorable effect on the risk of acute pancreatitis and cardiovascular events.

[0326] SHTG patients often have metabolic comorbidities related to dyslipidemia and insulin resistance, such as obesity, metabolic syndrome, T2DM, and nonalcoholic fatty liver disease, further increasing their risk of cardiovascular morbidity and mortality. The dramatic increase in obesity and T2DM over the past few decades has exacerbated the development of NAFLD, making it a rising health problem in the United States and globally. NAFLD is now the most common form of chronic liver disease in the United States and is often considered the hepatic manifestation of metabolic syndrome, a patient population frequently affected by atherogenic dyslipidemia. A key finding of this study was the prevalence of hepatic fat in this SHTG population. One hundred percent of patients who underwent MRI-PDFF screening had baseline hepatic steatosis, defined as liver fat >5% (range 6.2–39.2%). Interestingly, baseline MRI-PDFF values did not correlate with baseline TG values, but all patients tested who had baseline fasting TG levels >500 mg / dL had hepatic steatosis >5%.

[0327] Pegozafermin therapy demonstrated a significant reduction in hepatic fat accumulation, reaching the primary reduction goals of ≥30% and ≥50% in 88% and 41% of subjects, respectively. These are important thresholds, and the literature has established that a relative reduction in MRI-PDFF of ≥30% is associated with a histologic response (classified as a responder), while a relative reduction in MRI-PDFF of ≥50% elicits a significantly higher histologic response (defined as a super-responder). 48 In addition to the strong association between fatty liver and histologic findings, the presence of fatty liver is also associated with more severe acute pancreatitis, which may lead to local complications, persistent organ failure, and increased mortality, regardless of the underlying etiology. 49,50 More recently, Wu et al. reported that hyperlipidemic pancreatitis had the highest incidence of NAFLD (65%), and the severity of AP, systemic inflammatory response syndrome, and incidence of organ failure were higher in patients with NAFLD compared with non-NAFLD patients. 51 .

[0328] Pegozafermin treatment normalized liver fat to ≤5% in 24% of subjects in just 8 weeks. To our knowledge, these are the first data reporting a significant reduction in quantified liver fat by targeting TG-rich lipoproteins in SHTG, suggesting potential benefit in reducing the risk of severe acute pancreatitis. The mechanism by which pegosafermin reduces liver fat has not yet been fully elucidated. Based on preclinical data in hepatocytes, FGF21 is thought to affect hepatic steatosis by regulating AMPK phosphorylation to control lipid accumulation, decreasing sterol regulatory element-binding transcription factor 1 (SREBF1) to inhibit lipid synthesis, increasing PPARα mRNA and PPARα nuclear translocation to affect fatty acid oxidation, and promoting lipid transport and VLDL secretion. 21 Furthermore, FGF21 appears to increase hepatic expression of the LDLR, which not only functions to remove VLDL and LDL from the circulation but also to promote the post-translational degradation of ApoB and subsequently reduce secretion of VLDL particles. 21 In adipose tissue, FGF21 promotes TRL turnover by activating BAT and browning WAT. 22,55 Furthermore, FGF21 has been shown to suppress adipose tissue lipolysis, increase adiponectin levels, and reduce insulin resistance, which may also affect hepatic steatosis. 18,53 Overall, the safety and tolerability profile of pegosafermin was consistent with previous data, with mild to moderate gastrointestinal disturbances being the most common TEAE. 23,24 There were no serious TEAEs related to the study drug. One limitation of this study is the lack of power to assess clinical events such as pancreatitis, liver failure, and cardiovascular endpoints. Another limitation is that the majority of subjects were Caucasian males, which may limit the generalizability of the data. Although we recognize that fibrate use is likely underestimated in this study, the overall use pattern is very similar to other real-world data reported in patients with SHTG. Further safety and tolerability data from longer drug exposure at target doses are needed.

[0329] In conclusion, the FGF21 analog pegozafermin significantly reduces atherogenic lipoproteins, ApoC3, and liver fat in patients with SHTG, potentially positively impacting other aspects of metabolic dysregulation. Indeed, these "metabolic patients" are likely to benefit most from a treatment that can function as a metabolic regulator across multiple comorbidities. If these findings are confirmed in an appropriately powered Phase 3 trial, pegozafermin may be useful for treating SHTG and simultaneously addressing several other cardiometabolic risk factors.

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Claims

1. A pharmaceutical composition for use in the treatment of severe hypertriglyceridemia (SHTG) in a subject, The pharmaceutical composition comprises 9 mg to 30 mg of a mutant fibroblast growth factor-21 (FGF-21) peptide conjugate and a pharmaceutically acceptable carrier. The aforementioned pharmaceutical composition is prepared for once-weekly administration. The mutant FGF-21 peptide conjugate is i) A mutant FGF-21 peptide containing the amino acid sequence of SEQ ID NO: 2, ii) Glycosyl moiety, iii) Contains 20 kDa polyethylene glycol (PEG), The mutant FGF-21 peptide is bound to the glycosyl moiety by a covalent bond between the threonine at amino acid position 173 of SEQ ID NO:2 and the first site of the glycosyl moiety. A pharmaceutical composition in which the glycosyl moiety is bonded to 20 kDaPEG by a covalent bond between the second site of the glycosyl moiety and 20 kDaPEG.

2. A pharmaceutical composition for reducing triglyceride levels by at least 20% from baseline in a subject having severe hypertriglyceridemia (SHTG), The pharmaceutical composition comprises 9 mg to 30 mg of a mutant fibroblast growth factor-21 (FGF-21) peptide conjugate and a pharmaceutically acceptable carrier. The aforementioned pharmaceutical composition is prepared for once-weekly administration. The mutant FGF-21 peptide conjugate is i) A mutant FGF-21 peptide containing the amino acid sequence of SEQ ID NO: 2, ii) Glycosyl moiety, iii) Contains 20 kDa polyethylene glycol (PEG), The mutant FGF-21 peptide is bound to the glycosyl moiety by a covalent bond between the threonine at amino acid position 173 of SEQ ID NO:2 and the first site of the glycosyl moiety. A pharmaceutical composition in which the glycosyl moiety is bonded to 20 kDa PEG by a covalent bond between the second site of the glycosyl moiety and 20 kDa PEG.

3. A pharmaceutical composition for use in the treatment of severe hypertriglyceridemia (SHTG) in a subject, The pharmaceutical composition comprises 31 mg to 44 mg of mutant fibroblast growth factor-21 (FGF-21) peptide conjugate and a pharmaceutically acceptable carrier. The aforementioned pharmaceutical composition is prepared for administration once every two weeks. The mutant FGF-21 peptide conjugate is i) A mutant FGF-21 peptide containing the amino acid sequence of SEQ ID NO: 2, ii) Glycosyl moiety, iii) Contains 20 kDa polyethylene glycol (PEG), The mutant FGF-21 peptide is bound to the glycosyl moiety by a covalent bond between the threonine at amino acid position 173 of SEQ ID NO:2 and the first site of the glycosyl moiety. A pharmaceutical composition in which the glycosyl moiety is bonded to 20 kDaPEG by a covalent bond between the second site of the glycosyl moiety and 20 kDaPEG.

4. A pharmaceutical composition for reducing triglyceride levels by at least 20% from baseline in a subject having severe hypertriglyceridemia (SHTG), The pharmaceutical composition comprises 31 mg to 44 mg of mutant fibroblast growth factor-21 (FGF-21) peptide conjugate and a pharmaceutically acceptable carrier. The aforementioned pharmaceutical composition is prepared for administration once every two weeks. The mutant FGF-21 peptide conjugate is i) A mutant FGF-21 peptide containing the amino acid sequence of SEQ ID NO: 2, ii) Glycosyl moiety, iii) Contains 20 kDa polyethylene glycol (PEG), The mutant FGF-21 peptide is bound to the glycosyl moiety by a covalent bond between the threonine at amino acid position 173 of SEQ ID NO:2 and the first site of the glycosyl moiety. A pharmaceutical composition in which the glycosyl moiety is bonded to 20 kDa PEG by a covalent bond between the second site of the glycosyl moiety and 20 kDa PEG.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein administration of the pharmaceutical composition to a target normalizes the triglyceride level to 150 mg / dl or less.

6. The pharmaceutical composition according to any one of claims 1 to 4, wherein administration to a subject of the pharmaceutical composition results in a reduction of at least 10% of non-HDL cholesterol levels from baseline, a reduction of at least 10% of apoB levels from baseline, a reduction of at least 10% of apoC3 levels from baseline, or a combination thereof.

7. The pharmaceutical composition according to any one of claims 1 to 4, wherein administration to a subject of the pharmaceutical composition results in an increase of at least 10% from baseline in HDL cholesterol levels, an increase of at least 10% from baseline in adiponectin levels, or a combination thereof.

8. The pharmaceutical composition according to any one of claims 1 to 4, wherein administration of the pharmaceutical composition to a target results in a decrease in the production of TG-rich lipoprotein.

9. The pharmaceutical composition according to any one of claims 1 to 4, wherein administration of the pharmaceutical composition to a target subject results in improved clearance of TG-rich lipoprotein.

10. The pharmaceutical composition according to any one of claims 1 to 4, wherein administration of the pharmaceutical composition to a target subject results in improvement of insulin sensitivity.

11. The pharmaceutical composition according to any one of claims 1 to 4, wherein the subject has baseline hepatic steatosis.

12. The pharmaceutical composition according to any one of claims 1 to 4, wherein administration of the pharmaceutical composition to a target results in a reduction of more than 30% of liver fat.

13. The pharmaceutical composition according to any one of claims 1 to 4, wherein the pharmaceutical composition is administered to a subject for eight weeks or more.

14. The pharmaceutical composition according to any one of claims 1 to 4, wherein the target is a human subject.

15. The pharmaceutical composition according to any one of claims 1 to 4, wherein the subject is fasting triglycerides (TG) of ≥ 500 mg / dL and ≤ 2000 mg / dL.

16. The pharmaceutical composition according to claim 1 or claim 2, comprising 9 mg of mutant FGF-21 peptide conjugate.

17. The pharmaceutical composition according to claim 1 or claim 2, wherein the pharmaceutical composition comprises 15 mg to 18 mg of mutant FGF-21 peptide conjugate.

18. The pharmaceutical composition according to claim 1 or claim 2, wherein the pharmaceutical composition comprises 27 mg to 30 mg of a mutant FGF-21 peptide conjugate.

19. The pharmaceutical composition according to claim 3 or claim 4, wherein the pharmaceutical composition comprises 36 mg to 44 mg of mutant FGF-21 peptide conjugate.

20. The pharmaceutical composition according to claim 18, wherein administration to a subject of the pharmaceutical composition results in at least a 10% reduction in an alanine transaminase (ALT) marker, at least a 10% reduction in an aspartate aminotransferase (AST) marker, at least a 10% median reduction in high-sensitivity C-reactive protein (hsCRP), or a combination thereof.

21. The pharmaceutical composition according to claim 18, wherein administration to a subject of the pharmaceutical composition results in a reduction of at least 10% of fasting plasma glucose, a reduction of at least 0.2% of HBA1c, or a combination thereof.

22. The pharmaceutical composition according to claim 19, wherein administration of the pharmaceutical composition to a subject results in a median reduction of at least 10% in hsCRP.

23. The subject is a pharmaceutical composition according to any one of claims 1 to 4, which is undergoing background lipid modification therapy (LMT).

24. The pharmaceutical composition according to claim 23, wherein LMT comprises a statin, a formula fish oil, a fibrate, or a combination thereof.

25. The pharmaceutical composition according to claim 1, wherein the subject has undergone background LMT, and administration of the pharmaceutical composition to the subject results in a reduction of at least 10% of the non-HDL cholesterol level from baseline.

26. The pharmaceutical composition according to claim 1 or 2, wherein the subject has undergone background LMT, and administration of the pharmaceutical composition to the subject results in a reduction of at least 10% from baseline apoB cholesterol levels.

27. ​​The pharmaceutical composition according to any one of claims 1 to 4, wherein administration of the pharmaceutical composition to a subject results in a median reduction of at least 30% of the triglyceride level from baseline.

28. The pharmaceutical composition according to any one of claims 1 to 4, wherein administration of the pharmaceutical composition to a subject results in a median reduction of at least 40% of the triglyceride level from baseline.

29. The pharmaceutical composition according to any one of claims 1 to 4, wherein the pharmaceutical composition is prepared for subcutaneous administration.

30. The pharmaceutical composition according to any one of claims 1 to 4, wherein the glycosyl portion of the mutant FGF-21 peptide conjugate comprises at least one of the following: N-acetylgalactosamine (GalNAc) residue, galactose (Gal) residue, sialic acid (Sia) residue, a 5-amine analog of the Sia residue, mannose (Man) residue, mannosamine, glucose (Glc) residue, N-acetylglucosamine (GlcNAc) residue, fucose residue, xylose residue, or a combination thereof.

31. The pharmaceutical composition according to any one of claims 1 to 4, wherein the glycosyl portion of the mutant FGF-21 peptide conjugate comprises at least one N-acetylgalactosamine (GalNAc) residue, at least one galactose (Gal) residue, at least one sialic acid (Sia) residue, or a combination thereof.

32. The pharmaceutical composition according to claim 31, wherein at least one SiO residue is a carboxylated sugar having 9 carbon atoms.

33. The pharmaceutical composition according to claim 32, wherein at least one Sia residue is N-acetylneuraminic acid (2-keto-5-acetamido-3,5-dideoxy-D-glycero-D-galactonunuropyranose-1-onic acid (Neu5Ac), N-glycolylneuraminic acid (Neu5Gc), 2-keto-3-deoxy-nonurosonic acid (KDN), or a 9-substituted sialic acid.

34. The pharmaceutical composition according to claim 33, wherein the 9-substituted sialic acid is 9-O-lactyl-Neu5Ac, 9-O-acetyl-Neu5Ac, 9-deoxy-9-fluoro-Neu5Ac, or 9-azido-9-deoxy-Neu5Ac.

35. The pharmaceutical composition according to any one of claims 1 to 4, wherein the glycosyl moiety of the mutant FGF-21 peptide conjugate comprises the structure -GalNAc-Sia-.

36. The pharmaceutical composition according to any one of claims 1 to 4, wherein the 20 kDaPEG portion of the mutant FGF-21 peptide conjugate is covalently bound to the glycosyl portion of the linker, and the linker comprises at least one amino acid residue.

37. The pharmaceutical composition according to claim 36, wherein at least one amino acid residue is glycine (Gly).

38. The pharmaceutical composition according to any one of claims 1 to 4, wherein the mutant FGF-21 peptide conjugate comprises the structure-GalNAc-Sia-Gly-PEG (20 kDa).

39. The mutant FGF-21 peptide conjugate contains the following structure: 【Transformation 6】 The pharmaceutical composition according to any one of claims 1 to 4, wherein n is an integer selected from 450 to 460.

40. The pharmaceutical composition according to any one of claims 1 to 4, wherein the 20 kDaPEG of the mutant FGF-21 peptide conjugate is a linear or branched PEG.

41. The pharmaceutical composition according to any one of claims 1 to 4, wherein the 20 kDa PEG of the mutant FGF-21 peptide conjugate is 20 kDa methoxy-PEG.

42. A pharmaceutical composition for use in the treatment of severe hypertriglyceridemia (SHTG) in a subject, The pharmaceutical composition comprises 27 mg to 30 mg of a mutant fibroblast growth factor-21 (FGF-21) peptide conjugate and a pharmaceutically acceptable carrier. The aforementioned pharmaceutical composition is prepared for once-weekly administration. The mutant FGF-21 peptide conjugate is i) A mutant FGF-21 peptide containing the amino acid sequence of SEQ ID NO: 2, ii) Glycosyl moiety, iii) Contains 20 kDa polyethylene glycol (PEG), The mutant FGF-21 peptide is bound to the glycosyl moiety by a covalent bond between the threonine at amino acid position 173 of SEQ ID NO:2 and the first site of the glycosyl moiety. The glycosyl moiety is bonded to 20kDaPEG by a covalent bond between the second site of the glycosyl moiety and 20kDaPEG. Administration of the aforementioned pharmaceutical composition to a subject results in at least a 20% reduction in triglyceride levels from baseline in the subject. The administration of the aforementioned pharmaceutical composition to the target is A decrease of at least 10% from baseline in the alanine transaminase (ALT) marker. A decrease of at least 10% from baseline in aspartate aminotransferase (AST) markers. A median reduction of at least 10% in high-sensitivity C-reactive protein (hsCRP) from baseline. A decrease of at least 10% from baseline in fasting plasma glucose, A decrease of at least 0.3% in HBA1c from baseline. A reduction of at least 10% in non-HDL cholesterol levels from baseline. A decrease of at least 10% in apoB levels from baseline. A decrease of at least 10% in apoC3 levels from baseline. An increase of at least 10% in HDL cholesterol levels from baseline. An increase of at least 10% in adiponectin levels from baseline, and A reduction of more than 30% in liver fat from baseline. A pharmaceutical composition that provides one or more of the following effects.

43. A pharmaceutical composition for use in the treatment of severe hypertriglyceridemia (SHTG) in a subject, The pharmaceutical composition comprises 36 mg to 44 mg of mutant fibroblast growth factor-21 (FGF-21) peptide conjugate and a pharmaceutically acceptable carrier. The aforementioned pharmaceutical composition is prepared for administration once every two weeks. The mutant FGF-21 peptide conjugate is i) A mutant FGF-21 peptide containing the amino acid sequence of SEQ ID NO: 2, ii) Glycosyl moiety, iii) Contains 20 kDa polyethylene glycol (PEG), The mutant FGF-21 peptide is bound to the glycosyl moiety by a covalent bond between the threonine at amino acid position 173 of SEQ ID NO:2 and the first site of the glycosyl moiety. The glycosyl moiety is bonded to 20kDaPEG by a covalent bond between the second site of the glycosyl moiety and 20kDaPEG. Administration of the aforementioned pharmaceutical composition to a subject results in at least a 20% reduction in triglyceride levels from baseline in the subject. The administration of the aforementioned pharmaceutical composition to the target is A median reduction of at least 10% in high-sensitivity C-reactive protein (hsCRP) from baseline. A reduction of at least 10% in non-HDL cholesterol levels from baseline. A decrease of at least 10% in apoB levels from baseline. A decrease of at least 10% in apoC3 levels from baseline. An increase of at least 10% in HDL cholesterol levels from baseline. An increase of at least 10% in adiponectin levels from baseline, and A reduction of more than 30% in liver fat from baseline. A pharmaceutical composition that provides one or more of the following effects.

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