Methods for treating NASH using mutant FGF-21 peptide conjugates

Abstract

Therapeutic regimens and uses of mutant fibroblast growth factor-21 (FGF-21) peptide conjugates in the treatment of non-alcoholic steatohepatitis are provided.

Description

[Technical Field]

[0001] <Related Applications> This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 373,352, filed August 24, 2022, U.S. Provisional Patent Application No. 63 / 373,694, filed August 26, 2022, U.S. Provisional Patent Application No. 63 / 382,058, filed November 2, 2022, U.S. Provisional Patent Application No. 63 / 482,078, filed January 30, 2023, U.S. Provisional Patent Application No. 63 / 494,011, filed April 4, 2023, and U.S. Provisional Patent Application No. 63 / 510,041, 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 a file named 180234-011706.xml, created on August 20, 2023, and having a size of 40,011 bytes, the contents of which are incorporated herein by reference.

[0003] <Technical field> Therapeutic regimens and uses of mutant fibroblast growth factor-21 (FGF-21) peptide conjugates comprising a polyethylene glycol (PEG) moiety attached to the mutant FGF-21 peptide via a glycosyl moiety are provided. [Background technology]

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

[0005] Methods for treating non-alcoholic steatohepatitis (NASH) in a subject in need thereof are provided. In some embodiments, the methods comprise administering to a subject in need thereof once weekly a pharmaceutical composition comprising about 15 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 comprises the amino acid sequence of SEQ ID NO:3. and a 20 kDa PEG is attached to the glycosyl moiety 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 the 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 at least one of the following: reduction in liver fat; improvement in liver fibrosis score; resolution of NASH; improvement in NAFLD activity score by ≥ 2 points; improvement in VCTE score; improvement in FAST score; improvement in FIB-4 score; reduction in liver size assessed by magnetic resonance imaging-proton density fat fraction; reduction in levels of one or more biomarkers including Pro-C3, alanine transaminase (ALT), enhanced liver fibrosis (ELF) panel, CK-18, inflammatory marker high-sensitivity C-reactive protein (hs-CRP), hemoglobin A1c (HbA1c), non-HDL-c, LDL-c, and triglycerides; and increase in levels of HDL-c and / or adiponectin.

[0006] In some embodiments, the subject is a human.

[0007] In some embodiments, the pharmaceutical composition is administered subcutaneously.

[0008] In some embodiments, the glycosyl moiety 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, a mannosamine, a glucose (Glc) residue, an N-acetylglucosamine (GlcNAc) residue, a fucose residue, a xylose residue, or a combination thereof. In some embodiments, the glycosyl moiety 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 9-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-galactonunopyranose-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. In some embodiments, the glycosyl moiety comprises the structure -GalNAc-Sia-.

[0009] In some embodiments, the 20 kDa PEG moiety is covalently attached to the glycosyl moiety via 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). 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 structure: [ka] Here, n is an integer selected from 450 to 460.

[0010] 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.

[0011] In some embodiments, the mutant FGF-21 peptide conjugates exhibit equal or greater potency than wild-type FGF-21 when tested in vitro in KLB-FGFR1, KLB-FGFR2, and KLB-FGFR3 expressing cells.

[0012] In some embodiments, the method comprises administering to a subject in need thereof once weekly a pharmaceutical composition comprising about 25 to about 30 mg of a mutant FGF-21 peptide conjugate.

[0013] In some embodiments, the method comprises administering to a subject in need thereof once weekly a pharmaceutical composition comprising about 30 mg of a mutant FGF-21 peptide conjugate.

[0014] Provided is a method for treating non-alcoholic steatohepatitis (NASH) in a subject in need thereof, the method comprising administering to the subject in need thereof once every two weeks a pharmaceutical composition comprising about 18 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 comprises the amino acid sequence of SEQ ID NO:3. and a 20 kDa PEG is attached to the glycosyl moiety 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 the 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 at least one of the following: reduction in liver fat; improvement in liver fibrosis score; resolution of NASH; improvement in NAFLD activity score by ≥ 2 points; improvement in VCTE score; improvement in FAST score; improvement in FIB-4 score; reduction in liver size as assessed by magnetic resonance imaging-proton density fat fraction; reduction in levels of one or more biomarkers including Pro-C3, alanine transaminase (ALT), enhanced liver fibrosis (ELF) panel, CK-18, inflammatory marker high-sensitivity C-reactive protein (hs-CRP), hemoglobin A1c (HbA1c), triglycerides, non-HDL-c, LDL-c; and increase in levels of HDL-c and / or adiponectin.

[0015] In some embodiments, the subject is a human.

[0016] In some embodiments, the pharmaceutical composition is administered subcutaneously.

[0017] In some embodiments, the glycosyl moiety 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, a mannosamine, a glucose (Glc) residue, an N-acetylglucosamine (GlcNAc) residue, a fucose residue, a xylose residue, or a combination thereof. In some embodiments, the glycosyl moiety 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 9-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-galactonunulopyranos-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. In some embodiments, the glycosyl moiety comprises the structure -GalNAc-Sia-.

[0018] In some embodiments, the 20 kDa PEG moiety is covalently attached to the glycosyl moiety via 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). 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 structure: [ka] Here, n is an integer selected from 450 to 460.

[0019] In some embodiments, the 20 kDa PEG is a linear or branched PEG, hi some embodiments, the 20 kDa PEG is a 20 kDa methoxy-PEG.

[0020] In some embodiments, the mutant FGF-21 peptide conjugates exhibit equal or greater potency than wild-type FGF-21 when tested in vitro in KLB-FGFR1, KLB-FGFR2, and KLB-FGFR3 expressing cells.

[0021] In some embodiments, the method comprises administering to a subject in need thereof once weekly a pharmaceutical composition comprising about 40 to about 50 mg of a mutant FGF-21 peptide conjugate.

[0022] In some embodiments, the method comprises administering to a subject in need thereof once every two weeks a pharmaceutical composition comprising about 44 mg of a mutant FGF-21 peptide conjugate. [Brief explanation of the drawings]

[0023] [Figure 1] FIG. 1 depicts a single ascending dose (SAD) study design according to some embodiments.

[0024] [Figure 2] FIG. 2 is a table illustrating a baseline demographic design according to some embodiments.

[0025] [Figure 3] FIG. 3 is a table illustrating baseline laboratory parameters according to some embodiments.

[0026] [Figure 4] FIG. 4 is a table illustrating a safety summary according to some embodiments.

[0027] [Figure 5]5A and 5B show the pharmacokinetics of compounds described herein as a single dose, according to some embodiments. The PK profile is generally dose-proportional with a T range of approximately 53 to 100 hours.

[0028] [Figure 6] 6A and 6B are graphs showing the mean percent change (%) in triglycerides from baseline at days 8 and 15, according to some embodiments.

[0029] [Figure 7] FIG. 7 is a graph showing the mean percent change (%) from baseline in triglycerides over time, according to some embodiments.

[0030] [Figure 8] 8A and 8B are graphs showing the mean percent change (%) in total cholesterol from baseline at days 8 and 15, according to some embodiments.

[0031] [Figure 9] 9A and 9B are graphs showing the mean percent change (%) in LDL cholesterol from baseline at days 8 and 15, according to some embodiments.

[0032] [Figure 10] 10A and 10B are graphs showing the mean percent change (%) in HDL cholesterol from baseline at days 8 and 15, according to some embodiments.

[0033] [Figure 11] FIG. 11 is a table showing a comparison of the study population to other FGF-21SAD studies.

[0034] [Figure 12] FIG. 12 is a table showing a comparison of Phase 1 trials to other FGF-21 products.

[0035] [Figure 13] FIG. 13 is a table showing a comparison of Phase 1 trials to other FGF-21 products.

[0036] [Figure 14] FIG. 14 is a table showing a comparison of Phase 1 trials for thyroid hormone receptor beta agonists and FXR.

[0037] [Figure 15] FIG. 15 shows a standard procedure for a pERK functional assay according to some embodiments.

[0038] [Figure 16] Figures 16A and 16C show the results of a pERK functional assay in cells expressing only KLB using FGF-21. Figures 16B and 16D show the results of a pERK functional assay in cells expressing only KLB using BIO89-100, according to some embodiments. In KLB-expressing cells, FGF-21 and BIO89-100 were not active in ERK phosphorylation (pERK).

[0039] [Figure 17] Figures 17A and 17C show the results of a pERK functional assay in KLB / FGFR1-expressing cells using FGF-21. Figures 17B and 17D show the results of a pERK functional assay in KLB / FGFR1-expressing cells using BIO89-100, according to some embodiments. Both FGF-21 and BIO89-100 activate KLB-FGFR1 cells. BIO89-100 showed a 15- to 20-fold increase in potency sensitivity relative to FGF-21 in the pERK assay.

[0040] [Figure 18]Figures 18A and 18C show the results of a pERK functional assay in KLB / FGFR2-expressing cells using FGF-21. Figures 18B and 18D show the results of a pERK functional assay in KLB / FGFR2-expressing cells using BIO89-100, according to some embodiments. Both FGF-21 and BIO89-100 activate KLB-FGFR2 cells. BIO89-100 showed a 3- to 5-fold increase in potency sensitivity relative to FGF-21 in the pERK assay.

[0041] [Figure 19] Figures 19A and 19C show the results of a pERK function assay in KLB / FGFR3-expressing cells using FGF-21. Figures 19B and 19D show the results of a pERK function assay in KLB / FGFR3-expressing cells using BIO89-100 according to some embodiments. In KLB / FGFR3-expressing cells, BIO89-100 showed efficacy comparable to FGF-21.

[0042] [Figure 20] Figures 20A and 20C show the results of a pERK functional assay in KLB / FGFR4-expressing cells using FGF-21. Figures 20B and 20D show the results of a pERK functional assay in KLB / FGFR4-expressing cells using BIO89-100, according to some embodiments. In KLB / FGFR4-expressing cells, FGF-21 and BIO89-100 were barely active, increasing pERK by less than two-fold even at the highest concentration of 3000 nM.

[0043] [Figure 21] FIG. 21 is a summary of FGF-21 versus BIO89-100 according to some embodiments, showing that BIO89-100 is more potent than FGF-21 but has similar activity.

[0044] [Figure 22]Figures 22A and 22C show the results of a pERK functional assay in cells expressing only KLB using FGF-19. Figures 22B and 22D show the results of a pERK functional assay in cells expressing only KLB using FGF-21, according to some embodiments. In KLB-expressing cells, FGF-19 and FGF-21 were inactive (approximately a 2-fold increase in pERK at 3000 nM).

[0045] [Figure 23] Figures 23A and 23C show the results of a pERK functional assay in KLB / FGFR1-expressing cells using FGF-19, and Figures 23B and 23D show the results of a pERK functional assay in KLB / FGFR1-expressing cells using FGF-21, according to some embodiments.

[0046] [Figure 24] Figures 24A and 24C show the results of a pERK functional assay in KLB / FGFR2-expressing cells using FGF-19, and Figures 24B and 24D show the results of a pERK functional assay in KLB / FGFR2-expressing cells using FGF-21, according to some embodiments.

[0047] [Figure 25] Figures 25A and 25C show the results of a pERK functional assay in KLB / FGFR3-expressing cells using FGF-19, and Figures 25B and 25D show the results of a pERK functional assay in KLB / FGFR3-expressing cells using FGF-21, according to some embodiments.

[0048] [Figure 26] Figures 26A and 26C show the results of a pERK function assay in KLB / FGFR4-expressing cells using FGF-19. Figures 26B and 26D show the results of a pERK function assay in KLB / FGFR4-expressing cells using FGF-21, according to some embodiments. FGF-21 had no activity, but FGF-19 had activity and efficacy.

[0049] [Figure 27] Figures 27A and 27D show the results of pERK functional assays in cells expressing only KLB using EGF, Figures 27B and 27E show the results of pERK functional assays in cells expressing only KLB using FGF-21, and Figures 27C and 27F show the results of pERK functional assays in cells expressing only KLB using FGF-23.

[0050] [Figure 28] Figures 28A and 28D show the results of a pERK function assay in KLB / FGFR1-expressing cells using EGF, Figures 28B and 28E show the results of a pERK function assay in KLB / FGFR1-expressing cells using FGF-21, and Figures 28C and 28F show the results of a pERK function assay in KLB / FGFR1-expressing cells using FGF-23.

[0051] [Figure 29] Figures 29A and 29D show the results of pERK functional assay in KLB / FGFR2-expressing cells using EGF, Figures 29B and 29E show the results of pERK functional assay in KLB / FGFR2-expressing cells using FGF-21, and Figures 29C and 29F show the results of pERK functional assay in KLB / FGFR2-expressing cells using FGF-23.

[0052] [Figure 30] Figures 30A and 30D show the results of a pERK functional assay in KLB / FGFR3-expressing cells using EGF, Figures 30B and 30E show the results of a pERK functional assay in KLB / FGFR3-expressing cells using FGF-21, and Figures 30C and 30F show the results of a pERK functional assay in KLB / FGFR3-expressing cells using FGF-23.

[0053] [Figure 31]Figures 31A and 31D show the results of pERK functional assay in KLB / FGFR4-expressing cells using EGF, Figures 31B and 31E show the results of pERK functional assay in KLB / FGFR4-expressing cells using FGF-21, and Figures 31C and 31F show the results of pERK functional assay in KLB / FGFR4-expressing cells using FGF-23.

[0054] [Figure 32] Figures 32A-32C provide a summary of pERK functional assays in KLB-only, KLB / FGFR1, KLB / FGFR2, KLB / FGFR3, and KLB / FGFR4-expressing cells.

[0055] [Figure 33] Figures 33A-33F show a comparison of the efficacy of FGF-21 and BIO89-100.

[0056] [Figure 34] Figures 34A-34F show the efficacy of FGF-21 in FGFR-expressing cells (dose-response curves from three independent experiments).

[0057] [Figure 35] Figures 35A-35G show the efficacy of FGF-21 in FGFR-expressing cells (average of three independent experiments).

[0058] [Figure 36] Figures 36A-36F show the efficacy of BIO89-100 in FGFR-expressing cells (dose-response curves from two independent experiments).

[0059] [Figure 37] Figures 37A-37G show the efficacy of BIO89-100 in FGFR-expressing cells (average of two independent experiments).

[0060] [Figure 38A] Figure 38A shows the Phase 1b / 2a NASH study design.

[0061] [Figure 38B] FIG. 38B is a table illustrating baseline characteristics according to some embodiments.

[0062] [Figure 39] Figure 39A is a graph showing the mean reduction in liver fat versus baseline. Figure 39B is a table showing the relative reduction in liver fat in different cohorts. Figures 39A and 39B show that BIO89-100 demonstrated robust liver fat reduction with a high responder rate.

[0063] [Figure 40] Figure 40A is a graph showing mean percent change versus baseline. Figure 40B is a graph showing ALT-absolute change versus baseline.

[0064] [Figure 41] Figure 41A is a graph showing a ≧2 pt improvement in NAS. *Accompanied by a ≧2 pt improvement in ballooning or inflammation. Figure 41B is a graph showing responder rates by NAS component.

[0065] [Figure 42] Figure 42 is a graph of the change in key histological efficacy endpoints.

[0066] [Figure 43] Figure 43A is a graph showing mean relative change from baseline in VCTE, FAST score, FIB-4 score, and Pro-C3. Bar values ​​are absolute change relative to baseline. **p<0.01; ***p<0.001. Figure 43B is a graph showing responders by clinically relevant thresholds.

[0067] [Figure 44]Figure 44A is a graph showing absolute change in HbA1c. Figure 44B is a graph showing change in adiponectin. Figure 44C is a graph showing change in body weight. P values ​​for change from baseline based on MMRM analysis; all data are from Cohort 7. **p<0.01; ***p<0.001.

[0068] [Figure 45] Figure 45 is a graph showing the percent change from baseline in TG, LDL-C, non-HDL-C, and HDL-C at week 20. *p<0.05; ***p<0.001.

[0069] [Figure 46] Figure 46A is a graph showing improvement in liver health. Figures 46B and 46C are graphs showing improvement in comorbidities associated with NASH.

[0070] [Figure 47] Figure 47 is a table showing the Phase 2b NASH clinical trial design.

[0071] [Figure 48] Figures 48A-48B show arithmetic mean pegosafermin serum concentration-time profiles across dosing regimens. Shown as steady-state values ​​on day 29 for the QW (Figure 48A) and Q2W (Figure 48B) regimens. The concentration on day 57 (i.e., 336 hours after the day 43 dose instead of the day 29 dose) was used as the equivalent trough value for the Q2W regimen (Figure 48B).

[0072] [Figure 49] Figure 49. Correlation of percent change from baseline in alanine aminotransferase (ALT) levels at week 13 with percent change in MRI-PDFF at week 13.

[0073] [Figure 50] Figure 50 is a responder analysis of hepatic steatosis assessed by MRI-PDFF (pharmacodynamic MRI population).

[0074] [Figure 51] Figure 51 is a summary of the protocol corrections.

[0075] [Figure 52] Figure 52 is additional supporting text for the method.

[0076] [Figure 53] Figure 53 shows the dosing and evaluation schedule (Cohorts 1-4).

[0077] [Figure 54] Figure 54 is the dosing and assessment schedule (Cohorts 5 and 6).

[0078] [Figure 55] Figure 55 is the population analysis set.

[0079] [Figure 56] Figure 56 shows changes in liver fat fraction (pharmacodynamics population - MRI population).

[0080] [Figure 57] Figure 57: Liver volume change (pharmacodynamic population).

[0081] [Figure 58] Figure 58: Changes in alanine aminotransferase (ALT) levels (pharmacodynamic population).

[0082] [Figure 59] Figure 59 is the change in alanine aminotransferase (ALT) levels in patients with baseline ALT levels >45 U / L (pharmacodynamic population).

[0083] [Figure 60] Figure 60: Change in aspartate aminotransferase (AST) levels (pharmacodynamic population).

[0084] [Figure 61] Figure 61 shows changes in N-terminal propeptide of type III collagen (PRO-C3) levels (pharmacodynamic population).

[0085] [Figure 62] Figure 62: Change in triglyceride levels (pharmacodynamic population).

[0086] [Figure 63] Figure 63: Change in low density lipoprotein cholesterol (LDL-C) levels (pharmacodynamic population).

[0087] [Figure 64] Figure 64: Change in High Density Lipoprotein Cholesterol (HDL-C) Levels (Pharmacodynamic Population).

[0088] [Figure 65] Figure 65. Changes in non-high density lipoprotein cholesterol (non-HDL-C) levels (pharmacodynamic population).

[0089] [Figure 66] Figure 66: Change in Homeostasis Model Assessment of Insulin Resistance (HOMA-IR) levels (pharmacodynamic population).

[0090] [Figure 67] Figure 67 shows the change in glucose levels (pharmacodynamic population).

[0091] [Figure 68] Figure 68 shows the change in glycated hemoglobin (HbA1C) levels (pharmacodynamic population).

[0092] [Figure 69] Figure 69 is the change in body weight (pharmacodynamic population).

[0093] [Figure 70] Figure 70: Changes in adiponectin levels (pharmacodynamic population).

[0094] [Figure 71] Figure 71: Changes in free fatty acid (FFA) levels (pharmacodynamic population).

[0095] [Figure 72] Figure 72: Changes in adipose tissue insulin resistance index (Adipo-IR) levels (pharmacodynamic population).

[0096] [Figure 73] Figure 73 shows the randomization of participants by center (randomization group).

[0097] [Figure 74] Figure 74 shows the demographics and baseline characteristics of the BC-NASH and PNASH subpopulations (randomized population).

[0098] [Figure 75] Figure 75 shows the demographics and baseline characteristics (Pharmacodynamic MRI population) of participants who had a 30% or greater relative reduction in liver fat fraction versus placebo (MRI-PDFF responders) versus those who did not (MRI-PDFF non-responders).

[0099] [Figure 76] Figure 76 shows the research design.

[0100] [Figure 77] Figure 77 shows the primary analysis (central reader). Pegozafermin significantly improved the NAFLD Activity Score (NAS) and all components of the NAS.

[0101] [Figure 78] Figure 78 is the primary analysis (central reader). PGZ demonstrated clinically meaningful changes to the key histologic efficacy endpoint.

[0102] [Figure 79]Figure 79. Number of visible tumor nodules in STAM™ male mice treated with vehicle, pegosafermin, or sorafenib.

[0103] [Figure 80] Figure 80 shows the Phase 2b study design. 1. Improvement of liver fibrosis by one or more stages and no worsening of steatohepatitis, defined as no increase in NAS for ballooning, inflammation, and steatosis. 2. Resolution of steatohepatitis is defined as no fatty liver disease, isolated or simple steatosis without steatohepatitis, an NAS score of 0-1 for inflammation, an NAS score of 0 for ballooning, and any value for steatosis. *Some placebo patients were randomized to receive pegosafermin in the extension phase.

[0104] [Figure 81] Figure 81 shows the design of a biopsy reading method.

[0105] [Figure 82] Figure 82 is the patient treatment and analysis set.

[0106] [Figure 83] Figure 83 shows baseline characteristics across treatment groups.

[0107] [Figure 84] Figure 84 demonstrates statistical significance for fibrosis improvement with weekly and biweekly dosing of pegosafermin. *Relative risks shown are calculated by dividing the drug response by the placebo response. Relative risks calculated using statistical methods show similar results.

[0108] [Figure 85] Figure 85 shows confirmation of the robustness of the primary efficacy outcome (fibrosis improvement without worsening of NASH at week 24) by additional pre-specified analyses.

[0109] [Figure 86]Figure 86 is a descriptive analysis of cirrhosis (F4) patients from a clinical trial. 1. Improvement of liver fibrosis by one or more stages and no worsening of steatohepatitis, defined as no increase in NAS for ballooning, inflammation, and steatosis.

[0110] [Figure 87] Figure 87 shows the NASH cure data. *The relative risks shown are calculated by dividing the drug response by the placebo response. Relative risks calculated using statistical methods show similar results.

[0111] [Figure 88] Figure 88 shows confirmation of the robustness of the primary efficacy outcome (NASH resolution without worsening fibrosis at week 24) by additional pre-specified analyses.

[0112] [Figure 89] Figure 89 is a graph showing ≧2 point NAS improvement and no worsening of fibrosis at week 24. 1 Full analysis set. Analysis by Cochran-Mantel-Haenzel (CMH) test stratified by T2DM status (yes vs. no) and fibrosis stage (F2 vs. F3). ***p<0.001 vs. placebo.

[0113] [Figure 90] Figure 90A: Mean relative reduction in liver fat vs. baseline at week 24. 1 Analysis by mixed model repeated measures (MMRM). ***p<0.001 vs. placebo. Figure 90B: Proportion of patients achieving a ≥ 50% reduction in liver fat at week 24. 2 Analysis by Cochran-Mantel-Haenszel (CMH) study stratified by T2DM status (yes vs. no) and fibrosis stage (F2 vs. F3). ***p<0.001 vs. placebo.

[0114] [Figure 91]Figure 91A is the mean relative decrease in ALT vs. baseline at week 24. Figure 91B is the mean relative decrease in AST vs. baseline at week 24. Analysis by mixed model repeated measures (MMRM). ***p<0.001 vs. placebo.

[0115] [Figure 92] Figure 92A: cT1 responders at week 24. Figure 92B: Absolute change from baseline in VCTE at week 24. Figure 92C: Percent change in PRO-C3 from baseline at week 24. Full analysis set for Fibroscan and PRO-C3, and MRI-PDFF analysis set for cT1, analysis by MMRM for cT1 and PRO-C3, ANCOVA for VCTE. Patients are designated as cT1 responders with a decrease of ≥ 80 ms compared to baseline. cT1 was performed at available sites. *p<0.05, **p<0.01, ***p<0.001 vs. placebo.

[0116] [Figure 93] Figures 93A-93B are HbA1c change from baseline at week 24. Full analysis set for either the overall population or FAS with baseline HbA1c >= 6.5%. Analysis by MMRM. *p<0.05, ***p<0.001 vs. placebo.

[0117] [Figure 94] Figures 94A-94C show percent change in serum lipids from baseline at week 24 (Figure 94A: triglycerides; Figure 94B: non-HDL cholesterol; Figure 94C: HDL cholesterol). Full analysis set with Elteren test for triglycerides (reported as median) and MMRM. Subjects missing triglycerides at week 24 are excluded from non-parametric analysis. Non-HDL-cholesterol and HDL-cholesterol (reported as LS mean) have change from baseline (absolute or %) as dependent variable. p<0.05, **p<0.01, ***p<0.001 vs. placebo.

[0118] [Figure 95] Figure 95 shows treatment-related TEAEs.

[0119] [Figure 96] Figure 96A is a graph showing improvement of fibrosis by one stage or more without worsening of NASH. Figure 96B is a graph showing resolution of NASH without worsening of fibrosis.

[0120] [Figure 97] Figure 97 is a graph showing the change in patient key markers (ELF score, VCTE, ALT, HbA1c, MRI-PDFF) relative to background GLP1.

[0121] Among these disclosed benefits and improvements, other objects and advantages of the present disclosure will become apparent from the following description taken in conjunction with the accompanying drawings. While detailed embodiments of the present disclosure are disclosed herein, it should be understood that the disclosed embodiments are merely exemplary of the present disclosure, which may be embodied in various forms. Moreover, each of the examples given in connection with various embodiments of the present disclosure is intended to be illustrative, not limiting. DETAILED DESCRIPTION OF THE INVENTION

[0122] Nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) are chronic diseases in which fat accumulates in the liver, causing liver damage and inflammation. To date, there are no specific treatments for these disorders.

[0123] <Definition> For clarity and readability, the following definitions are provided. The technical features described in these definitions can be understood in all embodiments and in each embodiment of the present disclosure. Additional definitions and explanations may be specifically provided in 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 terminology and laboratory procedures used in this disclosure, in cell culture, molecular genetics, organic chemistry, and nucleic acid chemistry and hybridization, are those widely known and commonly used in the art. Standard techniques are used for nucleic acid and peptide synthesis. The 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 are provided throughout this specification.

[0124] <Enzyme> Enzymes are catalytically active biological molecules 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.

[0125] <Protein> A protein typically comprises one or more peptides or polypeptides. Proteins typically fold 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 the order of its amino acid sequence.

[0126] <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. Typically, in the art to produce a recombinant protein, i.e., a protein or peptide is "recombinantly produced." Typically, heterologous systems used in the art to produce recombinant proteins are bacteria (e.g., Escherichia coli), yeast (e.g., Saccharomyces cerevisiae), or certain mammalian cell culture strains.

[0127] <Expression host> An expression host refers to an organism used to produce a recombinant protein. Common expression hosts are bacteria such as E. coli, yeasts such as Saccharomyces cerevisiae or Pichia pastoris, or mammalian cells, such as human cells.

[0128] <RNA、mRNA> RNA is the common abbreviation for ribonucleic acid. It is a nucleic acid molecule, 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 specific sequence of monomers is called an RNA sequence.

[0129] <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 deoxyadenosine monophosphate, deoxythymidine monophosphate, deoxyguanosine monophosphate, and deoxycytidine monophosphate monomers, which themselves 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 (i.e., 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, nucleotides from the first strand hybridize with nucleotides from the second strand, for example, by A / T base pairing and G / C base pairing.

[0130] <Nucleic acid molecule sequence / Nucleic acid sequence> The sequence of a nucleic acid molecule is generally understood to be the specific and distinct order, ie, succession of its nucleotides.

[0131] <Amino acid molecule sequence / 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.

[0132] <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 are identical. That is, it typically represents the percentage of nucleotides at sequence positions that match identical nucleotides in a reference sequence, such as a natural or wild-type sequence. The degree of identity is determined by comparing sequences that are the same 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 the present disclosure, sequence identity specifically relates to the percentage of nucleotides / amino acids in two or more sequences of the same length that have the same positions. Gaps are typically considered non-identical positions, regardless of their actual position in the alignment.

[0133] Newly introduced amino acids "Newly introduced amino acid" refers to an amino acid 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 C-terminal amino acid sequence adjacent to the proline residue.

[0134] <Functional group> This term is understood according to the general understanding of those skilled in the art and refers to chemical moieties present on molecules, particularly peptides, or on amino acids of peptides or glycosyl residues attached to peptides, which can participate in covalent or non-covalent bonds to other chemical molecules, allowing for the attachment of glycosyl residues or PEG.

[0135] <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 called 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, which depends on the expression host used, does not usually alter the state of the protein considered to have its native or native / wild-type sequence.

[0136] Mutated This term should be understood in accordance with the general understanding of those skilled in the art. An amino acid sequence is called a "mutant" when it contains at least one added, deleted, or replaced amino acid, i.e., an amino acid mutation, compared to its native or naturally occurring amino acid sequence. A mutant protein is also called a variant. 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 such that the mutant FGF-21 peptide contains a newly introduced O-linked glycosylation site. The amino acid exchange is typically indicated as S172T, which means, for example, that the amino acid serine at position 172 in the amino acid sequence of SEQ ID NO: 1 is replaced with the amino acid threonine.

[0137] <Pharmaceutically effective amount> A pharmaceutically effective amount in this disclosure is typically understood to be an amount sufficient to induce a medicinal effect.

[0138] <Treatment / Processing> The term "therapy" refers to "treating" a disease or condition or the "treatment" of a disease or condition, suppressing a disease (delaying or stopping its progression), alleviating the symptoms or side effects of a disease (including pain relief), and reducing a disease (causing regression of the disease).

[0139] <Therapeutically effective amount or effective amount> It is an amount of a compound sufficient to treat a disease or condition, suppress a disease or condition, alleviate the symptoms or side effects of a disease, and / or cause regression of a disease or condition.

[0140] <Half-life> As used herein, the term "half-life" is defined as the time required for the plasma concentration of a drug, i.e., a mutant FGF-21 peptide and / or its conjugate, to decrease by half in a subject upon administration of the mutant FGF-21 peptide and / or conjugate.

[0141] <O-linked glycosylation> "O-linked glycosylation" occurs at serine or threonine residues (Tanner et al., Biochim. Biophys. Acta. 906:81-91 (1987), and Hounsell et al, Glycoconj. J. 13:19-26 (1996)). In some embodiments, the O-linked glycosylation site is an amino acid motif within the amino acid sequence of the peptide that is recognized by a glycosyltransferase as a point of attachment for a glycosyl residue, and includes the proline-threonine (PT) amino acid motif that is not present in the native / wild-type amino acid sequence. Specifically, the threonine residue is adjacent to proline and is newly introduced on the C-terminal side of the proline residue. Next, the glycosyl moiety is attached to the -OH group of the threonine residue by a glycosyltransferase.

[0142] <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 described herein.

[0143] <Adjacent> Adjacent refers to an amino acid that is immediately adjacent to another amino acid in an amino acid sequence, either at the N-terminal or C-terminal end of each amino acid. In the present disclosure, a newly introduced threonine residue is adjacent to a proline residue on the C-terminal side of the proline residue. In the present disclosure, for example, a newly introduced threonine residue is adjacent to a proline residue on the C-terminal side of the proline residue.

[0144] <Glycosyl moiety> The glycosyl moiety is a moiety consisting of one or more identical or different glycosyl residues that link the 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 mono-, di-, tri-, or oligoglycosyl. The glycosyl moiety can include one or more sialic acid residues, one or more N-acetylgalactosamine (GalNAc) residues, one or more galactose (Gal) residues, and others. The glycosyl moiety can also be modified with, for example, PEG or methoxy-PEG (m-PEG), an alkyl derivative of PEG.

[0145] <Glycoconjugation> The term "glycoconjugation" as used herein refers to the enzymatically mediated conjugation of a PEG-modified glycosyl moiety to a glycosyl residue of an amino acid or (poly)peptide, such as the mutant FGF-21 of the present disclosure. A taxonomic 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 branching core moiety and multiple linear polymer chains connected to the branching 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 branching moiety can also be derived from some amino acids, such as lysine. Branched PEGs can be represented by the general formula R(-PEG-OX)m, where 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 "glycosyl-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.

[0146] <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.

[0147] <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-acetylneuraminic 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. The third sialic acid family member 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).

[0148] <Pharmaceutically acceptable excipients> 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.

[0149] <Medicine containers> A "pharmaceutical container" is a container suitable for carrying a pharmaceutical composition and is typically made of an inert material and is sterile.

[0150] <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, such as 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, intraarteriolar, 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.

[0151] <Diabetes and diabetes-related diseases> "Diabetes" is commonly known and characterized as a disease often referred to as diabetes mellitus. This term describes a group of metabolic disorders in which a person's glucose (blood sugar) levels are high because of insufficient insulin production, an inability of the body's cells to respond properly to insulin, or both. Patients with hyperglycemia typically have polyuria (frequent urination) and increased thirst (polydipsia) and hunger (polyphagia). "Diabetes-related diseases" are diseases characterized by the same symptoms, such as obesity, polyuria, polydipsia, and polyphagia.

[0152] <Type 2 diabetes> "Type 2 diabetes" is the most common form of diabetes / diabetes mellitus. Type 2 diabetes most commonly develops in adults and is more likely to occur in obese, sedentary individuals. Unlike type 1 diabetes, which cannot currently be prevented, many of the risk factors for type 2 diabetes can be modified. The International Diabetes Foundation lists four symptoms that indicate the need for diabetes testing: a) frequent urination, b) weight loss, c) lack of energy, and d) excessive thirst. Insulin resistance, which is usually a precursor to type 2 diabetes, is a condition in which more insulin than normal is required for glucose to enter cells. Insulin resistance in the liver results in increased glucose production, while resistance in peripheral tissues means glucose uptake is impaired.

[0153] <Non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH)> It is a condition in which fat accumulates in the liver, followed by liver damage and inflammation.

[0154] <Metabolic syndrome> This is a defined cluster of risk factors (biochemical and physiological changes) associated with the development of type 2 diabetes and cardiovascular disease.

[0155] Abbreviations used herein include: PEG, poly(ethylene glycol); PPG, poly(propylene glycol); Ara, arabinosyl; Fru, fructosyl; Fuc, fucosyl; Gal, galactosyl; GalNAc, N-acetylgalactosaminyl; Glc, glucosyl; GlcNAc, N-acetylglucosaminyl; Man, mannosyl; ManAc, mannosaminyl acetate; Xyl, xylosyl; NeuAc, sialyl or N-acetylneuraminyl; Sia, sialyl or N-acetylneuraminyl; and derivatives and analogs thereof.

[0156] Natural FGF-21 has a relatively short half-life in vitro, and the reported circulating half-life ranges from 0.5 to 4 hours in rodents and non-human primates, limiting its clinical applicability. The half-life of recombinant human FGF-21 is 1 to 2 hours. To improve the pharmacokinetic properties of FGF-21, various half-life extension strategies have been developed.

[0157] See also WO2019 / 043457, the entire content of which is incorporated herein.

[0158] <PEGylation> In glycopegylation, the 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 common final structure is protein-glycosyl moiety-optional linker-PEG. In some embodiments, it is protein-protein (N-, C- or internal) amino acid-1 or more glycosyl residues-optional linker (e.g., amino acid linker)-linear or branched PEG moieties of various lengths, and the glycosyl moiety may contain 1 or more glycosyl residues. The 1 or more glycosyl residues that make up at least part of the structure can be any glycosyl residue that can link the protein to the PEG moiety. In the art, various methods for glycopegylating proteins are known and will be described in detail below.

[0159] In some embodiments, the fibroblast growth factor-21 (FGF-21) peptide conjugate i) a mutant FGF-21 peptide and ii) polyethylene glycol (PEG) of 20 kDa, The mutant FGF-21 peptide of i) comprises at least one threonine (T) residue adjacent to at least one proline (P) residue C-terminal to the at least one proline residue, thereby forming at least one O-linked glycosylation site not present in the corresponding native FGF-21, the native FGF-21 having at least 95% amino acid sequence identity with SEQ ID NO:1, and the 20 kDa PEG of ii) is covalently attached to the mutant FGF-21 peptide via at least one glycosyl moiety at at least one threonine residue.

[0160] In some embodiments, the mutant FGF-21 peptide conjugate comprises a mutant FGF-21 peptide comprising the amino acid sequence PT. In some embodiments, 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 the proline and threonine are based on the amino acid sequence set forth in SEQ ID NO:1. In some 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, particularly consisting 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 some 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.

[0161] In some embodiments, the mutant FGF-21 peptide comprises the amino acid sequence P172T, where the positions of the proline and threonine are based on the amino acid sequence shown in SEQ ID NO:1.

[0162] In some embodiments, 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 set forth in SEQ ID NO:1, and in particular, the mutant FGF-21 peptide comprises the amino acid sequence set forth in SEQ ID NO:2.

[0163] In some embodiments, the mutant FGF-21 peptide comprises the mutation Q157T, where the position of the amino acid Q is based on the amino acid sequence set forth in SEQ ID NO:1, and in particular, the mutant FGF-21 peptide comprises the amino acid sequence set forth in SEQ ID NO:4.

[0164] In some embodiments, the mutant FGF-21 peptide comprises the mutation D6T, where the position of amino acid D is based on the amino acid sequence set forth in SEQ ID NO:1, and in particular, the mutant FGF-21 peptide comprises the amino acid sequence set forth in SEQ ID NO:5.

[0165] In some embodiments, the mutant FGF-21 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-28. In some embodiments, the mutant FGF-21 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-5. In some embodiments, the mutant FGF-21 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-4. In some embodiments, the mutant FGF-21 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2.

[0166] In some 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 some embodiments, at least one glycosyl moiety comprises the structure -GalNAc-Sia-.

[0167] In some embodiments, the mutant FGF-21 peptide conjugate comprises a 20 kDa PEG moiety, wherein the PEG moiety is linked to at least one glycosyl moiety via an amino acid residue, particularly 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: [ka] Here, n is an integer selected from 450 to 460.

[0168] In some embodiments, the mutant FGF-21 peptide conjugate comprises a 20 kDa PEG, which is a linear PEG or a branched PEG, and in more particular embodiments, a linear PEG. In some embodiments, the 20 kDa PEG is a 20 kDa methoxy-PEG.

[0169] In some embodiments, pharmaceutical compositions are provided comprising at least one mutant FGF-21 peptide conjugate described herein 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 1 mg / mL to 45 mg / mL, from 10 mg / mL to 40 mg / mL, or, for example, 26±4 mg / mL. The buffering agent may be Tris buffer. The buffering agent may be present at a concentration of 1 mM to 100 mM, 2 mM to 75 mM, 5 mM to 50 mM, 10 mM to 25 mM, or, for example, 16±2 mM. The pH may be in the range of 6.0 to 8.5, 6.5 to 8.0, 6.75 to 8.0, or, for example, 7.5±0.3. The pharmaceutical composition may further comprise an inorganic salt, such as, but not limited to, NaCl. The pharmaceutical composition may comprise a salt present at a concentration of 30 mM to 200 mM, 40 mM to 150 mM, 50 mM to 100 mM, or, for example, 56±2 mM. The pharmaceutical composition may further comprise a tonicity adjuster. Suitable tonicity adjusters include, but are not limited to, glycerol, amino acids, sodium chloride, proteins, sugars, and sugar alcohols. In some embodiments, the tonicity adjuster is a sugar, for example, the tonicity adjuster is sucrose. The tonicity adjuster is present at a concentration of 50 mM to 200 mM, 100 mM to 175 mM, 135 mM to 160 mM, or, for example, 150±2 mM. The pharmaceutical composition may further comprise a surfactant, particularly a non-ionic surfactant. The surfactant or non-ionic surfactant may be a polysorbate-based non-ionic surfactant, such as polysorbate 20 or polysorbate 80. The surfactant or non-ionic surfactant may be present in a concentration of 0.01 mg / mL to 1 mg / mL, 0.05 to 0.5 mg / mL, or, for example, 0.2±0.02 mg / mL.

[0170] In some embodiments, the pharmaceutical composition comprises 0.1 mg / mL to 50 mg / mL of a mutant FGF-21 peptide conjugate, 1 mM to 100 mM of a buffering agent, e.g., Tris buffer, 30 mM to 200 mM of a salt, e.g., NaCl, 50 mM to 200 mM of a tonicity adjusting agent, e.g., sucrose, and 0.01 mg / mL to 1 mg / mL of a surfactant or non-ionic surfactant, e.g., polysorbate 20, and has a pH of 6.0 to 8.5.

[0171] Also included in the present disclosure are pharmaceutical containers containing the mutant FGF-21 peptide conjugates described herein and / or pharmaceutical compositions comprising said mutant FGF-21 peptide conjugates. Suitable pharmaceutical containers include, but are not limited to, syringes, vials, infusion bottles, ampoules, carpules, syringes with needle protection systems, or carpules in pen injectors.

[0172] <FGF-21コンジュゲート> 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.

[0173] In another exemplary embodiment, a mutant FGF-21 peptide conjugate can comprise a mutant FGF-21 peptide and a glycosyl group linked to a mutant amino acid residue of the mutant FGF-21 peptide.

[0174] In an exemplary embodiment, 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.

[0175] <Modified sugars> In an exemplary embodiment, a mutant FGF-21 peptide is reacted with a modified sugar to form a peptide conjugate. The modified sugar includes a "sugar donor moiety" and a "sugar transfer moiety." A sugar donor moiety is any portion of a modified sugar that is attached to a peptide via either the glycosyl moiety or the amino acid moiety as a conjugate described herein. The sugar donor moiety includes an atom that is chemically altered during the conversion of the modified sugar to the glycosyl linking group of the mutant FGF-21 peptide conjugate.

[0176] For the modified sugars described herein, the saccharyl moiety can be a saccharide, deoxysaccharide, aminosaccharide, or N-acylsaccharide. The terms "saccharide" and its equivalents, "saccharyl," "sugar," and "glycosyl," refer to monomers, dimers, oligomers, and polymers. The sugar moiety can also be functionalized with a modifying group. The modifying group is typically conjugated to the saccharyl moiety via conjugation with an amine, sulfhydryl, or hydroxyl, e.g., primary hydroxyl, moiety of the sugar. In an exemplary embodiment, the modifying group is attached through an amine moiety of the sugar, e.g., an amide, urethane, or urea formed through reaction of the amine with a reactive derivative of the modifying group.

[0177] Any saccharyl moiety can be utilized as the sugar donor portion of the modified sugar. The saccharyl moiety can be a known sugar, such as mannose, galactose, or glucose, or a species with the stereochemistry of a known sugar. The general formula for these modified sugars is shown in the following formula: [ka]

[0178] Other saccharyl moieties useful in the methods described herein include, but are not limited to, fucose and sialic acid, as well as amino sugars such as glucosamine, galactosamine, mannosamine, and 5-amine analogs of sialic acid. The saccharyl moiety may be a naturally occurring structure or may be modified to provide a site for attaching a modifying group. For example, in one embodiment, the modified sugar provides a sialic acid derivative in which the 9-hydroxy moiety is replaced with an amine. The amine is readily derivatized with an activated analog of the selected modifying group. Examples of modified sugars useful in the methods described herein are described in PCT Patent Application No. PCT / US05 / 002522, which is incorporated herein by reference in its entirety.

[0179] In a further exemplary embodiment, the invention utilizes modified sugars in which the 6-hydroxyl position has been converted to the corresponding amine moiety, which contains a linker-modification group cassette, such as those described above. Examples of glycosyl groups that can be used as the core of these modified sugars include Gal, GalNAc, Glc, GlcNAc, Fuc, Xyl, Man, etc. An exemplary modified sugar according to this embodiment has the following structure: [ka] In the formula, R 11 ~R 14 R is a member independently selected from H, OH, C(O)CH3, NH, and NHC(O)CH3. 10 is linked to, for example, another glycosyl residue (-O-glycosyl). 14 is OR 1 , NHR 1 or NH-LR 1 R 1 and NH-LR 1 is as described herein.

[0180] In yet another exemplary embodiment, the glycosyl group used as the core of the modified sugar in which the 6-hydroxyl position is converted to the corresponding amine moiety comprises Gal and / or GalNAc.

[0181] Glycosyl linking groups In an exemplary embodiment, a mutant FGF-21 peptide conjugate is provided that includes a modified sugar and a mutant FGF peptide described herein. In this embodiment, the sugar donor portion of the modified sugar (e.g., the saccharyl moiety and the modifying group) serves as a "glycosyl linking group." Note that "glycosyl linking group" may also refer to the glycosyl moiety present between the peptide and the modifying group.

[0182] In the exemplary embodiments below, the present disclosure is illustrated with reference to the use of selected derivatives of furanoses and pyranoses. Those skilled in the art will appreciate that the structures and compositions described are generally applicable to the entire genus that includes glycosyl linking groups and modified sugars. Thus, glycosyl linking groups can include virtually any monosaccharide or oligosaccharide.

[0183] In an exemplary embodiment, the methods described herein can utilize a glycosyl linking group having the formula: 1 is a modifying group, for example a polymeric modifying group. [ka] Exemplary bonds are formed, for example, between the NH moiety of the glycosyl moiety and a chemical group of complementary reactivity on the modifying group. For example, R 1 If contains a carboxylic acid moiety, this moiety is activated and coupled to the NH2 moiety of the glycosyl residue to form the structure NHC(O)R 1 J is preferably an "intact" glycosyl moiety that is not degraded upon exposure to conditions that cleave pyranose or furanose structures, e.g., oxidizing conditions, e.g., sodium periodate conditions.

[0184] Examples of linkers include alkyl and heteroalkyl moieties. Linkers include linking groups, such as acyl-based linking groups, such as -C(O)NH-, -OC(O)NH-, and the like. Linking groups are used to connect components of a conjugate, such as between a glycosyl moiety and a linker (L) or between a linker and a modifying group (R 1 ) is a bond formed between the carboxylic acid moiety of the glycosyl group and the amine group. Other exemplary linking groups are ethers, thioethers, and amines. For example, in one embodiment, the linker is an amino acid residue, such as a glycine residue. The carboxylic acid moiety of the glycine is converted to the corresponding amide by reaction with the amine of the glycosyl residue, and the amine of the glycine is converted to the corresponding amide or urethane by reaction with the activated carboxylic acid or carbonate of the modifying group.

[0185] NH-LR 1 A typical species has the formula: -NH{C(O)(CH2) a NH} s {C(O)(CH2) b (OCH2CH2) c O(CH2) d NH} t R 1 where the indices s and t are independently 0 or 1. The indices a, b, and d are independently integers from 0 to 20, and c is an integer from 1 to 2500. Other similar linkers are based on species in which the -NH moiety is replaced with other chemical groups, such as -S, -O, or -CH2. As understood in the art, one or more of the portions of the brackets corresponding to the indices s and t can be replaced with a substituted or unsubstituted alkyl or heteroalkyl moiety.

[0186] In some embodiments, the compounds described herein are comprised of NH-L-R', where NH-L-R' is NHC(O)(CH) a NHC(O)(CH2) b (OCH2CH2) c O(CH2) d NHR 1 , NHC(O)(CH2) b (OCH2CH2) c O(CH2) d NHR 1 , NHC(O)O(CH2) b (OCH2CH2) c O(CH2) d NHR 1 , NH(CH2) a NHC(O)(CH2) b (OCH2CH2) c O(CH2) d NHR 1 , NHC(O)(CH2) a NHR 1 , NH(CH2) a NHR 1 , and NHR 1 In these formulas, the indices a, b, and d are independently selected from integers of 0 to 20, preferably 1 to 5. The index c is an integer of 1 to about 2500.

[0187] In exemplary embodiments, c is selected such that the PEG moiety is about 1 kD, 5 kD, 10 kD, 15 kD, 20 kD, 25 kD, 30 kD, 35 kD, 40 kD, 45 kD, 50 kD, 55 kD, 60 kD, or 65 kD.

[0188] In some embodiments, c is selected such that the PEG moiety is in the range of 15-25 kD, 16-25 kD, 17-25 kD, 18-25 kD, 19-25 kD, 20-25 kD, 21-25 kD, 22-25 kD, 23-25 ​​kD, 24-25 kD, 15-20 kD, 16-20 kD, 17-20 kD, 18-20 kD, 19-20 kD, 20-30 kD, 21-30 kD, 22-30 kD, 23-30 kD, 24-30 kD, 25-30 kD, 26-30 kD, 27-30 kD, 28-30 kD, or 29-30 kD. In some embodiments, c is selected such that the PEG moiety is 20 kD, 22 kD, 23 kD, 24 kD, 25 kD, 26 kD, 27 kD, 28 kD, 29 kD, or 30 kD.

[0189] For clarity, the glycosyl linking groups in the remainder of this section are based on sialyl moieties, however, one of skill in the art will recognize that other glycosyl moieties, such as mannosyl, galactosyl, glucosyl, or fucosyl, can be used in place of the sialyl moiety.

[0190] In exemplary embodiments, the glycosyl linking group is an intact glycosyl linking group, and the glycosyl moiety or moieties forming the linking group are not degraded by chemical processes (e.g., sodium metaperiodate) or enzymatic processes (e.g., oxidases). Selected conjugates of the invention include modifying groups attached to the amine moiety of an aminosaccharide, e.g., mannosamine, glucosamine, galactosamine, sialic acid, and the like. In exemplary embodiments, the disclosure provides peptide conjugates comprising an intact glycosyl linking group having a formula selected from the following formulas: [ka]

[0191] In Formula I, R 2 is H, CH2OR 7 , COOR 7 OR 7 where R 7 represents H, substituted or unsubstituted alkyl, or substituted or unsubstituted heteroalkyl. 7 When R is a carboxylic acid or carboxylate, both forms are represented by the designation of the single structure COO- or COOH. In Formulas I and II, the symbol R 3 , R 4 , R 5 , R 6 and R 6 ' are independently H, substituted or unsubstituted alkyl, OR 8 , NHC(O)R 9 The index d is 0 or 1. R 8 and R 9 R is independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, sialic acid, or polysialic acid. 3 , R 4 , R 5 , R 6 or R 6 At least one of R' contains a modifying group. The modifying group can be a polymeric modifying moiety, such as PEG, linked via a bond or linking group. In an exemplary embodiment, R 6 and R 6 The R's, together with the carbon to which they are attached, are components of the pyruvyl side chain of sialic acid. In a further exemplary embodiment, the pyruvyl side chain is functionalized with a polymeric modifying group. In another exemplary embodiment, R 6 and R 6 ' are components of the side chain of sialic acid, along with the carbon to which they are attached, and the polymer modifying group is R 5 is a component of.

[0192] Examples of modifying group-intact glycosyl linking group cassettes according to this motif are based on sialic acid structures, such as cassettes having the formula: [ka]

[0193] In the above formula, R 1 and L are as described above. 1 Further details regarding the structure of the group are provided below.

[0194] In a further exemplary embodiment, a conjugate is formed between a peptide and a modified sugar, with the modifying group attached via a linker at the 6-carbon position of the modified sugar. Thus, an exemplary glycosyl linking group according to this embodiment has the formula: [ka] In the above formula, the radicals are as described above. Glycosyl linking groups include, but are not limited to, glucose, glucosamine, N-acetyl-glucosamine, galactose, galactosamine, N-acetylgalactosamine, mannose, mannosamine, and N-acetyl-mannosamine.

[0195] In some embodiments, the present disclosure provides a mutant FGF-21 peptide conjugate comprising the following glycosyl linking group: [ka] In the formula, D is —OH and R 1 -L-HN-. G is a member selected from H and R 1 R is a member selected from -L- and -C(O)(C1-C6)alkyl. 1 is a moiety comprising a linear or branched poly(ethylene glycol) residue. L is a linker, e.g., a chemical bond ("zero order"), substituted or unsubstituted alkyl, and substituted or unsubstituted heteroalkyl. In an exemplary embodiment, when D is OH, G is and when G is -C(O)(C1-C6)alkyl, D is R 1 -L-NH-.

[0196] In some embodiments, the peptide conjugate comprises a glycosyl linking group having the formula: [ka]

[0197] In some embodiments, the glycosyl linking group has the formula: [ka] where the index t is 0 or 1.

[0198] In some embodiments, the glycosyl linking group has the formula: [ka] where the index t is 0 or 1.

[0199] In some embodiments, the glycosyl linking group has the formula: [ka] In the formula, p represents an integer of 1 to 10, and a is either 0 or 1.

[0200] In some embodiments, the glycoPEGylated peptide conjugate is selected from the formulae set forth below: [ka]

[0201] In the above formula, the index t is an integer between 0 and 1, and the index p is an integer between 1 and 10. 15 ' is H, OH (e.g., Gal-OH), a sialyl moiety, a sialyl linking group (i.e., a sialyl linking group-polymeric modifying group (Sia-LR) 1 ), or a polymer-modified sialyl moiety (e.g., Sia-Sia-LR 1 ) ("Sia-Siap")) represents a sialyl moiety to which is attached. Exemplary polymer-modified sugar moieties have structures according to Formulae I and II. Exemplary peptide conjugates of the present disclosure include R 15 The oxygen with an open valence of Formulas I and II is, in some embodiments, attached to a carbon of a Gal or GalNAc moiety via a glycosidic bond. In a further exemplary embodiment, the oxygen is attached to a carbon at the 3-position of a galactose residue. In an exemplary embodiment, the modified sialic acid is α2,3-linked to the galactose residue. In another exemplary embodiment, the sialic acid is α2,6-linked to the galactose residue.

[0202] In an exemplary embodiment, the sialyl linking group is a polymeric sialyl moiety (e.g., Sia-Sia-LR 1 )("Sia-Sia p " is a sialyl moiety attached to the glycosyl linking group, wherein the glycosyl linking group is linked to the galactosyl moiety via the sialyl moiety. [ka] Exemplary species according to this motif include Sia-LR, using enzymes that form Sia-Sia bonds, e.g., CST-11, ST8Sia-II, ST8Sia-III, and ST8Sia-IV. 1 is prepared by conjugating to the terminal sialic acid of the glycan.

[0203] In another exemplary embodiment, the glycan of the peptide conjugate has a formula selected from the following group and combinations thereof: [ka]

[0204] In each of the above formulas, R 15 Further, exemplary mutant FGF-21 peptide conjugates described herein include R' having a structure according to Formula I or II. 15 The glycan comprises at least one glycan having a moiety.

[0205] In another exemplary embodiment, the glycosyl linking group comprises at least one glycosyl linking group having the formula: [ka] In the formula, R 15 is the sialyl linking group, and the index p is an integer selected from 1 to 10.

[0206] In an exemplary embodiment, the glycosyl linking moiety has the formula: [ka] In the formula, b is an integer of 0 to 1. The index s is an integer of 1 to 10, and the index f is an integer of 1 to 2,500.

[0207] 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 5 kDa. In another exemplary embodiment, the PEG moiety has a molecular weight of 10 kDa. In another exemplary embodiment, the PEG moiety has a molecular weight of 40 kDa.

[0208] In an exemplary embodiment, 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.

[0209] In an exemplary embodiment, 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.

[0210] In some embodiments, the mutant FGF-21 peptide is PEGylated according to the methods described herein. In some embodiments, the mutant FGF-21 peptide comprises a mutant S 172 T and R 176 The amino acid sequence of SEQ ID NO:1 includes the amino acid sequence of SEQ ID NO:2, and the positions of the amino acids S and R are based on the amino acid sequence set forth in SEQ ID NO:1. In some embodiments, the variant FGF-21 peptide comprises the amino acid sequence set forth in SEQ ID NO:2. As described in detail herein, the at least one glycosyl moiety attached to the threonine residue and linking the newly introduced threonine residue to a PEG moiety can be virtually any possible glycosyl moiety. The only restriction is that it must be capable of being attached to the threonine and of being attached to PEG or m-PEG, e.g., via a linker, e.g., an amino acid residue, or glycine. In some embodiments, the at least one glycosyl moiety comprises N-acetylgalactosamine (GalNAc), galactose (Gal), and / or sialic acid (Sia). In some embodiments, the at least one glycosyl moiety comprises the structure -GalNAc-Sia-, i.e., two glycosyl moieties, GalNAc and Sia, and the PEG residue can be attached to GalNAc or Sia, particularly Sia. The glycosyl moiety that is not attached to a PEG moiety can be attached to the newly introduced threonine residue.

[0211] 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, particularly a small amino acid such as alanine or glycine, more particularly 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.

[0212] In some embodiments, a 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, which 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.

[0213] In some embodiments, a 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, which 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.

[0214] In some embodiments, a 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, which is further attached via a glycine residue to a 20 kDa, 25 kDa, or 30 kDa PEG.

[0215] In some embodiments, a 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, which is further attached to a 20 kDa or 30 kDa PEG via a glycine residue.

[0216] In some embodiments, a 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, which is further attached to a 20 kDa PEG via a glycine residue.

[0217] In some embodiments, the mutant FGF-21 peptide conjugate comprises the following structure: [ka] Here, n is an integer selected from 450 to 460.

[0218] In some embodiments, the 20 kDa PEG can be linear or branched. In some embodiments, the 20 kDa PEG is linear 20 kDa PEG. In some embodiments, the 20 kDa PEG is 20 kDa methoxy-PEG (mPEG, m-PEG). PEGs and mPEGs of various 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.

[0219] <Mutant FGF-21 peptides and their conjugates> As described herein, mutants of fibroblast growth factor-21 (FGF-21) have been generated with surprising properties, including mutants with consistently long half-lives. The mutants are peptide conjugates comprising: 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, wherein the mutant FGF-21 peptide is capable of forming at least one O-linked glycosylation that is not present in a corresponding native FGF-21 having at least 95% of its amino acid sequence identical to SEQ ID NO:1; ii) a 20-30 kDa polyethylene glycol (PEG) covalently attached via at least one glycosyl moiety to at least one threonine residue of said mutant FGF-21 peptide; Includes.

[0220] 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 disclosure, option (ii) is an exemplary embodiment. As described herein, multiple threonine residues can be introduced adjacent to the C-terminus of the existing proline residue. Thus, the mutant FGF-21 of the present disclosure can contain both additionally introduced threonine residues and threonine residues introduced in place of the native amino acid.

[0221] 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.

[0222] General 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).

[0223] 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.

[0224] In some embodiments, the variant FGF-21 peptide comprises the amino acid sequence PT, a threonine residue C-terminally adjacent to a proline residue, which sequence is not present in the native FGF-21 amino acid sequence.

[0225] The mutant FGF-21 peptide optionally 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 150 T (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 variant FGF-21 peptide comprises at least one amino acid sequence selected from the group consisting of P, T (e.g., SEQ ID NO:28), and combinations thereof, wherein the positions of proline and threonine are based on the native amino acid sequence of FGF-21 shown in SEQ ID NO:1. 172 T, P 156 T, P 5 T and combinations thereof, for example, P 172 T, P 156 T and combinations thereof. More specifically, the mutant FGF-21 peptide comprises at least one amino acid sequence selected from the group consisting of a sequence motif P based on the amino acid sequence set forth in SEQ ID NO:1. 172 It contains T, and the positions of proline and threonine are based on the amino acid sequence shown in SEQ ID NO:1.

[0226] 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 PEG attachment, particularly attachment of glycosyl-PEG moieties. This was unexpected, as literature reports that β-Klotho binding of FGF-21 requires an intact C-terminus.

[0227] In some embodiments, the mutant FGF-21 peptide comprises a mutant S 172 T and R 176 A, and the positions of amino acids S and R are based on the amino acid sequence shown in SEQ ID NO: 1. In particular, the mutant FGF-21 peptide comprises the amino acid sequence shown in SEQ ID NO: 2. 176 A was found to be beneficial to the overall stability of the protein after introducing an O-linked glycosylation site at threonine 173. This mutation removed the relatively bulky arginine side chain and replaced 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.

[0228] In an alternative embodiment, the mutant FGF-21 peptide has the mutation Q 157 The variant FGF-21 peptide comprises the amino acid set forth in SEQ ID NO:4, i.e., D6T, and the position of the amino acid Q is based on the amino acid sequence set forth in SEQ ID NO:1. In particular, the variant FGF-21 peptide comprises the amino acid set forth in SEQ ID NO:4, i.e., D6T, and the position of the amino acid D is based on the amino acid sequence set forth in SEQ ID NO:1. In particular, the variant FGF-21 peptide comprises the amino acid sequence set forth in SEQ ID NO:5.

[0229] 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, more particularly an amino acid sequence selected from the group consisting of SEQ ID NOs:2-5, even more particularly an amino acid sequence selected from the group consisting of SEQ ID NOs:2-4, and most particularly an amino acid sequence set forth in SEQ ID NO:2.

[0230] <Pharmaceutical Compositions and Treatment Methods> In some embodiments, a pharmaceutical composition comprises a mutant FGF-21 peptide conjugate and a pharmaceutically acceptable carrier, such as water or a physiologically compatible buffer. The pharmaceutical composition typically comprises a therapeutically effective or pharmaceutically active amount of the mutant FGF-21 peptide conjugate as the active agent.

[0231] In some embodiments, the pharmaceutical compositions are suitable for use in various drug delivery systems. Formulations suitable for use in the present disclosure are described in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, PA, 17th ed. (1985). The pharmaceutical compositions are used for prophylactic and / or therapeutic treatment by parenteral, intranasal, topical, oral, or local administration, such as by subcutaneous injection, aerosol inhalation, or transdermal absorption. Generally, pharmaceutical compositions are administered parenterally, for example, subcutaneously or intravenously.

[0232] In some embodiments, the present disclosure provides compositions for parenteral administration, comprising 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 also include pharmaceutically acceptable auxiliary substances necessary to approximate physiological conditions, such as pH adjusters, buffers, tonicity adjusters, wetting agents, detergents, etc.

[0233] The pharmaceutical compositions of the present invention may be sterilized by conventional sterilization techniques or sterile filtered. The resulting aqueous solutions may be packaged for immediate use or lyophilized. The lyophilized formulation is mixed with a sterile aqueous carrier prior to administration. Compositions containing FGF peptide conjugates can be administered for prophylactic and / or therapeutic purposes, particularly for the treatment of diabetes or diabetes-related conditions, such as type 2 diabetes, NASH, and metabolic syndrome. In therapeutic applications, the compositions are administered to subjects already suffering from diabetes or a diabetes-related condition in an amount sufficient to cure or at least partially prevent the symptoms of the disease and its complications. An amount sufficient to accomplish this is defined as a "therapeutically effective amount," which typically depends on the patient's health and weight. When tested in various animal models of NASH and type 2 diabetes, effective doses range from 0.1 mg / kg to 6 mg / kg.

[0234] In some embodiments, the present disclosure provides a method of treating a disease and / or a disorder or symptom thereof, comprising administering to a subject (e.g., a mammal, such as a human) a therapeutically effective amount of a compound (such as a mutant FGF-21 peptide conjugate described herein) or a pharmaceutical composition comprising said compound. Thus, one embodiment is a method of treating a subject suffering from diabetes or a diabetes-related disorder (e.g., type 2 diabetes, NAFLD, NASH, or metabolic syndrome), or a symptom thereof. The method comprises administering to the mammal a compound described herein or a composition comprising said compound in an amount sufficient to treat the disease or disorder or a symptom thereof, under conditions such that the disease or disorder is treated. In some embodiments, the method comprises subcutaneously administering to the mammal a compound described herein or a composition comprising same in an amount sufficient to treat the disease or disorder or a symptom thereof, under conditions such that the disease or disorder is treated.

[0235] In some embodiments, a mutant FGF-21 peptide conjugate (e.g., 89Bio-100, also known as pegozafermin) is administered to a human subject in a weekly treatment dosing regimen of about 3 mg to about 30 mg. In some embodiments, a mutant FGF-21 peptide conjugate (e.g., 89Bio-100) is administered to a human subject in a biweekly treatment dosing regimen of about 18 mg to about 44 mg. In some embodiments, a mutant FGF-21 peptide conjugate (e.g., 89Bio-100) is administered in a single-dose treatment dosing regimen of 0.45 mg, 1.2 mg, 3 mg, 9.1 mg, 15 mg, 18.2 mg, 39 mg, or 78 mg, or placebo in a 6:2 ratio (7:3 ratio for the 9.1 mg dose). See also Examples 1 and 4 below.

[0236] In some embodiments, the therapeutic dosage regimen may be in the range of about 3 mg to about 44 mg, about 3 mg to about 36 mg, about 3 mg to about 30 mg, about 3 mg to about 27 mg, about 3 mg to about 18 mg, about 3 mg to about 15 mg, about 9 mg to about 44 mg, about 9 mg to about 36 mg, about 9 mg to about 30 mg, about 9 mg to about 27 mg, about 9 mg to about 18 mg, about 9 mg to about 15 mg, about 15 mg to about 44 mg, about 15 mg to about 36 mg, about 15 mg to about 30 mg, about Including the range of 15 mg to about 27 mg, the range of about 15 mg to about 18 mg, the range of about 18 mg to about 44 mg, the range of about 18 mg to about 36 mg, the range of about 18 mg to about 30 mg, the range of about 18 mg to about 27 mg, the range of about 18 mg to about 30 mg, the range of about 18 mg to about 27 mg, the range of about 3 mg to about 9 mg, the range of about 9 mg to about 15 mg, the range of about 9 mg to about 18 mg, the range of about 18 mg to about 27 mg, the range of about 27 mg to about 30 mg, the range of about 18 mg to about 27 mg, the range of about 3 mg to about 18 mg, and the range of about 18 mg to about 36 mg. In some embodiments, the therapeutic dosage 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 the ranges indicated. In some embodiments, the therapeutic dosage 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 indicated. In some embodiments, the therapeutic dosage 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 the ranges indicated. In some embodiments, the therapeutic dosing regimen includes a range of about 10 mg to about 20 mg, or a range of about 15 mg to about 20 mg, and any integer within the range indicated. In some embodiments, the therapeutic dosing regimen includes 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 that is 10% above or below the specifically specified amount. For example, about 10 mg refers to a range of 9.0 to 11 mg. In some embodiments, the therapeutic dosing regimen includes a dose of 9.1 mg, about 18.2 mg, or about 39 mg.

[0237] The therapeutic dosing regimen can be administered to a human in need thereof to treat at least one of diabetes (e.g., type 2 diabetes), NAFLD, NASH, or metabolic syndrome. In some embodiments, the therapeutic dosing regimen is administered to a human in need thereof to reduce triglyceride levels. In some embodiments, the therapeutic dosing regimen is administered to a human to reduce triglyceride levels (see, e.g., Example 1).

[0238] The therapeutic dosing regimen may be administered to a human in need thereof to treat NASH. In some embodiments, the therapeutic dosing regimen is administered to a human in need thereof to reduce liver fat. In some embodiments, the therapeutic dosing regimen is administered to a human in need thereof to lower ALT. In some embodiments, the therapeutic dosing regimen is administered to a human in need thereof to reduce NAFLD activity score. In some embodiments, the therapeutic dosing regimen is administered to a human in need thereof to reduce or ameliorate fibrosis. In some embodiments, the therapeutic dosing regimen is administered to a human in need thereof to improve HbA1c levels. In some embodiments, the therapeutic dosing regimen results in an increase in adiponectin levels. In some embodiments, the therapeutic dosing regimen is administered to a human in need thereof to reduce weight. In some embodiments, the therapeutic dosing regimen is administered to a human in need thereof to improve lipid parameters. In some embodiments, the therapeutic dosing regimen is administered to a human in need thereof to reduce triglyceride levels. In some embodiments, the therapeutic dosing regimen is administered to a human to reduce triglyceride levels (see, e.g., Example 5).

[0239] The above-described therapeutic dosing regimen may also be administered to a human in need thereof to reduce cravings for sweet foods and beverages (see, eg, Example 2).

[0240] In some embodiments, a pharmaceutical composition is provided comprising any one or at least one of the mutant FGF-21 peptide conjugates described herein and a pharmaceutically acceptable carrier. The mutant FGF-21 peptide conjugate may be present in the pharmaceutical composition at a concentration ranging from 0.1 mg / mL to 50 mg / mL, 1 mg / mL to 45 mg / mL, or 10 mg / mL to 40 mg / mL, e.g., 26±4 mg / mL. In some embodiments, the pharmaceutical composition further comprises a buffer, e.g., a Tris buffer. In some embodiments, the buffer is present at a concentration of 1 mM to 100 mM, 2 mM to 75 mM, 5 mM to 50 mM, or 10 mM to 25 mM, e.g., 16±2 mM. In some embodiments, the pH is in the range of 6.0 to 8.5, 6.5 to 8.0, or 6.75 to 8.0, e.g., 7.5±0.3. In some embodiments, the pharmaceutical composition further comprises a salt, e.g., an inorganic salt, e.g., NaCl. In some embodiments, the salt is present at a concentration of 30 mM to 200 mM, 40 mM to 150 mM, 50 mM to 100 mM, e.g., 56±2 mM. The pharmaceutical composition may further comprise a tonicity adjuster. Such tonicity adjusters include, but are not limited to, glycerol, amino acids, sodium chloride, proteins, sugars, and sugar alcohols. In some embodiments, the tonicity adjuster is a sugar, e.g., the tonicity adjuster is sucrose. In some embodiments, the tonicity adjuster is present at a concentration of 50 mM to 200 mM, more preferably 100 mM to 175 mM, 135 mM to 160 mM, e.g., 150±2 mM. In some embodiments, the pharmaceutical composition further comprises a surfactant. In some embodiments, the surfactant is a non-ionic surfactant. In some embodiments, the surfactant or non-ionic surfactant is a polysorbate-based non-ionic surfactant, such as polysorbate 20 or polysorbate 80. In some embodiments, the surfactant or non-ionic surfactant is present at a concentration of 0.01 mg / mL to 1 mg / mL, 0.05 to 0.5 mg / mL, e.g., 0.2±0.02 mg / mL.

[0241] In some embodiments, the pharmaceutical composition comprises 0.1 mg / mL to 50 mg / mL of a mutant FGF-21 peptide conjugate, 1 mM to 100 mM of a buffer, such as Tris buffer, 30 mM to 200 mM of a salt, such as NaCl, 50 mM to 200 mM of a tonicity adjuster, such as sucrose, and 0.01 mg / mL to 1 mg / mL of a surfactant or non-ionic surfactant, such as polysorbate 20, and has a pH of 6.0 to 8.5.

[0242] 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. 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, 32, 34, 36, 38, 40, 42 mg / mL.

[0243] In some embodiments, the liquid pharmaceutical composition comprises 10 to 48 mg / mL of FGF-21 peptide conjugate, for example, about 10 mg / mL, 12 mg / mL, 14 mg / mL, 15 mg / mL, 16 mg / mL, 18 mg / mL, 20 mg / mL, 22 mg / mL, 24 mg / mL, 26 mg / mL, 28 mg / mL, 30 mg / mL, 32 mg / mL, 34 mg / mL, 36 mg / mL, 38 mg / mL, 40 mg / mL, 42 mg / mL, 44 mg / mL, 66 mg / mL, or 48 mg / mL of FGF-21 peptide conjugate.

[0244] In some embodiments, the liquid pharmaceutical composition comprises, or alternatively 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 alternatively consists of, about 10 mg / mL to about 48 mg / mL of a mutant blast 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 about 250 mOsmol / kg to about 510 mOsmol / kg. In some embodiments, the liquid formulation comprises or consists of 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, and further comprises 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 (pH 7-8), and a pharmaceutically acceptable carrier.In some embodiments, the liquid formulation comprises or consists of 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 has the amino acid sequence of SEQ ID NO:3. The formulation further comprises: a 20 kDa PEG; a threonine at amino acid position 173 of NO:2; a glycosyl moiety attached to the glycosyl moiety via a covalent bond between the threonine at amino acid position 173 of NO:2 and a first site of the glycosyl moiety; the glycosyl moiety attached to the 20 kDa PEG via a covalent bond between the second site of 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); a 20 mM buffer solution (pH 7-8); and a pharmaceutically acceptable carrier. In some embodiments, the formulation has an osmolality of about 250 mOsmol / kg to about 550 mOsmol / kg. 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 comprising a variant 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 variant 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; and about 50 mM to about 500 mM arginine, about 50 mM to about 250 mM alanine, about 50 mM to about 250 mM proline, and about 50 mM to about 500 mM PEG. , 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 5% (v / v) PEG400, or a combination thereof, about 0.01 to about 0.1% (w / v) polysorbate 80 (PS-80) or polysorbate 20 (PS-20), about 20 mM buffer solution (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 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; and The formulation further comprises about 50 mM to about 250 mM glycine, about 50 mM to about 250 mM MgCl, about 1% to about 5% (v / v) glycerol, about 1% to 5% (v / v) PEG 400, or a combination thereof, 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 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, or about 0.6 to about 1, e.g., about 0.6, 0.7, 0.8, 0.9, or 0.1. In some embodiments, the molar ratio of mutant FGF-21 to arginine is about 0.006 to about 0.008, 0.006 to about 0.009, 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, 0.007, 0.008, 0.009.

[0245] In some embodiments, the liquid formulation has an osmolality of about 250 mOsmol / kg to about 550 mOsmol / kg.

[0246] In some embodiments, the liquid pharmaceutical composition comprises 20 mg / mL PEG-FGF21, 150 mM arginine, and 0.02% (w / v) PS-80 in 20 mM Tris, at a pH of 7.5. In some embodiments, the liquid pharmaceutical formulation comprises 20 mg / mL PEG-FGF21, 150 mM arginine, and 0.02% (w / v) PS-80 in 20 mM phosphate, at a pH of 7.5. In some embodiments, the composition has an osmolality of about 250 mOsm / kg to 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, 275 mM arginine, and 0.02% (w / v) PS-80 in 20 mM Tris, at a pH of 7 to 8. In some embodiments, the composition has an osmolality of about 505 mOsm / kg. In some embodiments, the liquid pharmaceutical formulation comprises 18 to 44 mg / mL PEG-FGF21, 200 to 350 mM arginine, and 0.02% (w / v) PS-80 in 20 mM Tris, at a pH of 7.0 to 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 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 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 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 HCl, 20 mM Tris, and 0.02% (w / v) PS80, and 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, and 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, and 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, and 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, and 0.02% (w / v) PS80, has 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, and 0.02% (w / v) PS80, has 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, and 0.02% (w / v) PS80, has a pH of 7.1, and an osmolality of about 485 mOsm / kg.

[0247] 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.

[0248] In some embodiments, the liquid pharmaceutical composition further comprises one or more active agents. In some embodiments, PEG-FGF21 is formulated with one or more active agents. In some embodiments, the one or more active agents may include a peptide, a small molecule, or a combination thereof. In some embodiments, the one or more active agents may include a hormone. For example, the one or more active agents include oxyntomodulin, insulin, leptin, glucagon, a eroxisome proliferator-activated receptor (PPAR) agonist, an FXR (farnesoid X receptor) agonist, a thyroid hormone receptor beta (TRβ) agonist, a sodium glucose cotransporter 2 (SGLT2) inhibitor, an analog thereof, or a combination thereof. As used herein, an "analog" is a molecule having a modification, including one or more amino acid substitutions, deletions, inversions, or additions, when compared to the wild-type peptide sequence.

[0249] 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 may range 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.

[0250] The liquid pharmaceutical composition may further comprise a tonicity adjuster. Suitable tonicity adjusters include glycerol, amino acids, sodium chloride, proteins, or sugars and sugar alcohols. For example, the adjuster comprises arginine, such as arginine HCl or arginine sulfate. The tonicity adjuster is present at a concentration of 50 mM to 500 mM. For example, the adjuster (e.g., arginine HCl) comprises 150 mM to 500 mM, 150 to 275 mM, or 245 to 275 mM arginine. In some embodiments, the adjuster comprises arginine, for example, arginine HCl or arginine sulfate, is present at a concentration of 31.6 mg / ml (150 mM) to 54.8 mg / ml (260 mM).

[0251] The liquid pharmaceutical composition may further comprise a non-ionic surfactant. The non-ionic surfactant may be a polysorbate-based non-ionic surfactant, such as polysorbate 20 or polysorbate 80. In some embodiments, the surfactant is 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).

[0252] 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.

[0253] 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 buffering agent, such as Tris buffer, 150 mM to 500 mM of tonic arginine, and 0.02% to 1% (w / v) of a polysorbate-based nonionic surfactant, such as polysorbate 80, and has a pH of 7.0 to 8.0.

[0254] In some embodiments, the liquid formulation comprises 0.02% (w / v) PS80 (0.2 mg / ml). In some embodiments, the buffer is Tris or phosphate buffer. In some embodiments, the liquid formulation comprises 20 mM Tris buffer. In some embodiments, the liquid formulation comprises 15 mg / ml variant FGF-21. In some embodiments, the liquid formulation comprises 28 mg / ml variant FGF-21. In some embodiments, the liquid formulation comprises 30 mg / ml variant FGF-21. In some embodiments, the liquid formulation comprises 36 mg / ml variant FGF-21. In some embodiments, the liquid formulation comprises 44 mg / ml 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.

[0255] 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, and 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, and 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.

[0256] In some embodiments, the pharmaceutical composition is a liquid pharmaceutical composition comprising: (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 (b) a mutant FGF-21 peptide conjugate, wherein the FGF-21 peptide is attached to a glycosyl moiety 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; (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) 5 to 25 mM buffer (pH 7 to 8); and (e) a pharmaceutically acceptable carrier.

[0257] In some embodiments, the pharmaceutical composition is a liquid pharmaceutical composition comprising: (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 (b) a mutant FGF-21 peptide conjugate, wherein the FGF-21 peptide is linked to a glycosyl moiety 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 linked to a 20 kDa PEG by a covalent bond between a second site of the glycosyl moiety and the 20 kDa 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) 5 to 25 mM buffer (pH 7 to 8); and (e) a pharmaceutically acceptable carrier.

[0258] 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 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; (b) 150 mM to 500 mM arginine, 50 mM to 25 ... (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.

[0259] 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.

[0260] In some embodiments, the buffer is a Tris or phosphate buffer. In some embodiments, the liquid formulation comprises 20 mM Tris buffer. In some embodiments, the pH of the liquid formulation is 7.0 to 7.5.

[0261] In some embodiments, the liquid pharmaceutical composition comprises 20-44 mg / ml of the mutant FGF-21 peptide conjugate.

[0262] 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.

[0263] In some embodiments, the liquid pharmaceutical composition comprises about 28 mg / mL of a mutant FGF-21 peptide conjugate, about 260 mM arginine HCl, about 20 mM Tris, 0.02% (w / v) PS-80, and has a pH of about 7.1.

[0264] 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.

[0265] 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, 0.02% (w / v) PS-80, and has a pH of about 7.1.

[0266] 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, 0.02% (w / v) PS-80, and has a pH of about 7.1.

[0267] 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, 0.02% (w / v) PS-80, and has a pH of about 7.1.

[0268] In some embodiments, the liquid formulation has an osmolality of about 250 mOsmol / kg to about 550 mOsmol / kg.

[0269] In some embodiments, the liquid formulation is a liquid formulation described in US Pat. No. 11,596,669, the entirety of which is incorporated herein by reference.

[0270] In some embodiments, the present disclosure also includes 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, such as, but not limited to, a syringe, a vial, an infusion bottle, an ampoule, a carpule, a syringe with a needle protection system, or a carpule in an injector pen.

[0271] <Treatment method> In some embodiments, methods are disclosed for treating (e.g., controlling, alleviating, reversing, mitigating, or slowing the progression of) or preventing (e.g., delaying the onset of or reducing the risk of developing) diabetes and related diseases, particularly type 2 diabetes, NAFLD, non-alcoholic steatohepatitis (NASH), and / or metabolic syndrome in a subject in need thereof.

[0272] In some embodiments, methods are provided for treating diabetes and related diseases, particularly type 2 diabetes, NAFLD, nonalcoholic steatohepatitis (NASH), and / or metabolic syndrome. The methods comprise administering to a subject in need thereof a therapeutically effective amount of a mutant FGF-21 peptide conjugate described herein, or a pharmaceutical composition comprising at least one mutant FGF-21 peptide conjugate described herein. In some embodiments, the subject in need thereof is a human.

[0273] In some embodiments, methods for treating diabetes or a diabetes-related disorder are disclosed, the methods comprising administering to a subject in need thereof an amount of a mutant FGF-21 peptide conjugate described herein or obtainable by the methods described herein, or a pharmaceutical composition comprising the same. The diabetes or diabetes-related disorder may include at least one of type 2 diabetes, NAFLD, NASH, or metabolic syndrome. In some embodiments, methods for treating NASH are disclosed, the methods comprising administering to a subject in need thereof an amount of a mutant FGF-21 peptide conjugate described herein or obtainable by the methods described herein, or a pharmaceutical composition comprising the same. In some embodiments, methods for treating NAFLD are disclosed, the methods comprising administering to a subject in need thereof an amount of a mutant FGF-21 peptide conjugate described herein or obtainable by the methods described herein, or a pharmaceutical composition comprising the same. In some embodiments, a method for preventing the progression of NASH is disclosed, the method comprising administering to a subject in need thereof an amount of a mutant FGF-21 peptide conjugate described herein or obtainable by the methods described herein, or a pharmaceutical composition comprising the same. In some embodiments, a method for preventing the progression of NAFLD is disclosed, the method comprising administering to a subject in need thereof an amount of a mutant FGF-21 peptide conjugate described herein or obtainable by the methods described herein, or a pharmaceutical composition comprising the same. In some embodiments, the subject is a human subject. In some embodiments, the administration reduces HbA1C levels. A reduction in HbA1C levels indicates a sustained reduction in blood glucose levels over time.A variety of exemplary indicators known in the art are described herein, including, but not limited to, glucose, insulin, body weight, serum lipids (total cholesterol, LDL, triglycerides), liver enzymes (ALT, AST), liver weight, relative liver weight (% body weight), NAFLD activity score (NAS), fibrosis score (e.g., liver fibrosis), pro-inflammatory cytokines (e.g., IL1β, MCP-1), fibrosis biomarkers (αSMA, collagen 1α), and decreases in liver cholesterol, liver triglycerides, and liver fatty acids. An increase in at least one of adiponectin (e.g., high molecular weight (HMW)) or HDL is also an indicator of the clinical efficacy of the compounds and compositions described herein. In some embodiments, the therapeutic method of the method for preventing disease progression is evaluated by magnetic resonance imaging - proton density fat fraction that determines the size of the liver (e.g., reduction in the size of the liver).

[0274] The NAFLD fibrosis score is used to distinguish between patients with progressive fibrosis (F3 - F4) and non - progressive fibrosis (F0 - F2) among patients with non - alcoholic fatty liver disease. A fibrosis score of F0 - F1 (2 - 7 kPa) means that there is little or no scarring in the liver. A fibrosis score of F2 (7.5 - 10 kPa) indicates moderate scarring that spreads outside the liver. A fibrosis score of F3 (10 - 14 kPa) indicates severe scarring that expands and obstructs normal blood flow. A fibrosis score of F4 (14 kPa or higher) means late - stage scarring or cirrhosis where the scarring is permanent and the damage is irreversible.

[0275] <Methods for treating NASH> In some embodiments, methods for treating (e.g., controlling the progression of, alleviating, remitting, reducing, or delaying) non - alcoholic steatohepatitis (NASH) in a subject in need thereof, or for preventing (e.g., delaying the onset of, or reducing the risk of developing) it are disclosed.

[0276] In some embodiments, methods for treating nonalcoholic steatohepatitis (NASH) are provided, comprising administering to a subject in need thereof a therapeutically effective amount of a mutant FGF-21 peptide conjugate described herein or a pharmaceutical composition comprising at least one of the mutant FGF-21 peptide conjugates described herein. In some embodiments, the subject in need thereof is a human.

[0277] In some embodiments, administering comprises administering an effective dose of the composition that results in resolution of NASH (NAS score of 0-1 for inflammation, NAS score of 0 for ballooning).

[0278] In some embodiments, administering comprises administering an effective dose of the composition that results in at least one of the following: a reduction in the level of a marker of NASH (e.g., a serum marker), a reduction in symptoms associated with NASH, a reduction in liver fibrosis, or a reduction in NAS score.

[0279] In some embodiments, a method for treating NASH is disclosed, the method comprising administering to a subject in need thereof an amount of a mutant FGF-21 peptide conjugate described herein or obtainable by the methods described herein, or a pharmaceutical composition comprising the same. In some embodiments, a method for preventing the progression of NASH is disclosed, the method comprising administering to a subject in need thereof an amount of a mutant FGF-21 peptide conjugate described herein or obtainable by the methods described herein, or a pharmaceutical composition comprising the same. In some embodiments, the subject is a human. In some embodiments, the administration reduces HbA1C levels. A reduction in HbA1C levels indicates a sustained reduction in blood glucose levels over time. Various exemplary indicators known in the art are described herein, including, but not limited to, reductions in glucose, insulin, body weight, serum lipids (total cholesterol, LDL, triglycerides), liver enzymes (ALT, AST), liver weight, relative liver weight (% body weight), NAFLD activity score (NAS), fibrosis score (e.g., liver fibrosis), FIB-4 score, FAST score, proinflammatory cytokines (e.g., IL1β, MCP-1), fibrosis biomarkers (αSMA, collagen 1α), liver cholesterol, liver triglycerides, and liver fatty acids. Increases in at least one of adiponectin (e.g., high molecular weight (HMW)) or HDL are also indicators of clinical efficacy of the compounds and compositions described herein. In some embodiments, treatment of a method for preventing disease progression is assessed by magnetic resonance imaging (MRI)—proton density fat fraction—to determine liver size (e.g., reduction in liver size).

[0280] In some embodiments, indicators or endpoints used to determine the effectiveness of treatment include one or more of the following: NAS score NASH dissipation Improvement of fibrosis

[0281] In some embodiments, the biomarkers used to determine the effectiveness of treatment include one or more of the following: Fibrosis-4 (FIB-4) score FibroScan-AST (FAST) score Vibration-Controlled Transient Elastography (VCTE) for assessing fibrosis and steatosis triglycerides Non-high-density lipoprotein (non-HDL) cholesterol High-density lipoprotein (HDL-c) Low-density lipoprotein (LDL-c) Hemoglobin A1c (HbA1c) Homeostasis Model Assessment of Insulin Resistance (HOMA-IR) Liver function tests: alanine transaminase (ALT), aspartate transaminase (AST) Adiponectin N-terminal propeptide of type III collagen (Pro-C3) Free fatty acids and Adipo-IR (fasting free fatty acids x fasting insulin) Inflammatory marker high-sensitivity C-reactive protein (hs-CRP) Total cholesterol OGTT including C-peptide, glucose, and insulin IGF-1, total CK-18 Enhanced Liver Fibrosis (ELF) Panel

[0282] In some embodiments, the present disclosure includes any one of the mutant FGF-21 peptide conjugates described herein or a pharmaceutical composition comprising the same for use in a method for treating diabetes or a diabetes-related disorder. The diabetes or diabetes-related disorder may include at least one of type 2 diabetes, NAFLD, NASH, or metabolic syndrome. In some embodiments, the diabetes or diabetes-related disorder affects a human subject. In some embodiments, the use reduces HbA1C levels, where a reduction in HbA1C levels indicates a sustained reduction in blood glucose levels over an extended period of time. Various exemplary indicators known in the art are described herein, including, but not limited to, reductions in glucose, insulin, body weight, serum lipids (total cholesterol, LDL, triglycerides), liver enzymes (ALT, AST), liver weight, relative liver weight (% body weight), NAFLD activity score (NAS), fibrosis score (e.g., liver fibrosis), proinflammatory cytokines (e.g., IL1β, MCP-1), fibrosis biomarkers (αSMA, collagen 1α), liver cholesterol, liver triglycerides, and liver fatty acids. An increase in at least one of high molecular weight (HMW) adiponectin or HDL is also an indicator of the clinical effectiveness of the compounds and compositions described herein.

[0283] In some embodiments, the use of a mutant FGF-21 peptide conjugate described herein in the preparation of a medicament for use in a method for treating diabetes or a diabetes-related disorder is provided. The diabetes or diabetes-related disorder may include at least one of type 2 diabetes, NAFLD, NASH, or metabolic syndrome. In some embodiments, the diabetes or diabetes-related disorder affects a human subject. In some embodiments, the use reduces HbA1C levels, where a reduction in HbA1C levels indicates a sustained reduction in blood glucose levels over an extended period of time. Various exemplary indicators known in the art are described herein, including, but not limited to, reductions in glucose, insulin, body weight, serum lipids (total cholesterol, LDL, triglycerides), liver enzymes (ALT, AST), liver weight, relative liver weight (% body weight), NAFLD activity score (NAS), fibrosis score (e.g., liver fibrosis), proinflammatory cytokines (e.g., IL1β, MCP-1), fibrosis biomarkers (αSMA, collagen 1α), liver cholesterol, liver triglycerides, and liver fatty acids. An increase in at least one of adiponectin (e.g., high molecular weight (HMW)) or HDL is also an indicator of the clinical effectiveness of the compounds and compositions described herein.

[0284] In some embodiments, a mutant fibroblast growth factor-21 (FGF-21) peptide conjugate is provided, the peptide conjugate comprising: i) a mutant FGF-21 peptide comprising the amino acid sequence of SEQ ID NO:2; ii) a glycosyl moiety comprising the structure -GalNAc-Sia-, and iii) 30 kDa polyethylene glycol (PEG), The variant FGF-21 peptide is linked to a 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 linked to a 30 kDa PEG by a covalent bond between a second site on the glycosyl moiety and the 30 kDa PEG. In some embodiments, the 30 kDa PEG moiety is linked to the glycosyl moiety by a covalent bond to a linker, and the linker comprises at least one amino acid residue. Exemplary amino acids include polar but neutral amino acids (e.g., serine, threonine, cysteine, tyrosine, asparagine, and glutamine) and nonpolar amino acids with relatively simple side chains (e.g., glycine, alanine, valine, leucine). In some embodiments, the at least one amino acid residue is at least one glycine (Gly). In some embodiments, the mutant FGF-21 peptide conjugate comprises the structure -GalNAc-Sia-Gly-PEG(30 kDa). The mutant FGF-21 peptide conjugates described herein can comprise a 30 kDa PEG, which is a linear or branched PEG. In some embodiments, the 30 kDa PEG is a linear PEG. In some embodiments, the 30 kDa PEG is a 30 kDa methoxy-PEG. In some embodiments, single or multiple administrations of the composition may be carried out, with dose levels and patterns selected by the treating physician. In any event, the pharmaceutical composition should provide a quantity of the mutant FGF-21 peptide conjugate of the present disclosure sufficient to effectively treat the subject in need of such treatment.

[0285] In some embodiments, in the pharmaceutical composition, the mutant FGF-21 peptide conjugate is present at a concentration ranging from 0.1 mg / mL to 50 mg / mL.

[0286] All components of the pharmaceutical compositions and their specific concentrations were carefully selected after testing numerous different conditions, compounds, and their concentrations. Thus, the pharmaceutical compositions disclosed herein are not an arbitrary selection of compounds and compound concentrations, but rather a specific and rational selection of conditions found to be optimal for aqueous pharmaceutical compositions containing mutant FGF-21 peptide conjugates or mutant FGF-21 peptides according to the present disclosure for use as pharmaceuticals.

[0287] In some embodiments, the pharmaceutical composition includes a buffering agent such as a phosphate or Tris buffer, particularly a Tris buffer, e.g., tris(hydroxymethyl)aminomethane (THAM). Optionally, the buffering agent is present at a concentration of 1 mM to 100 mM, 2 mM to 75 mM, 5 mM to 50 mM, or 10 mM to 25 mM, e.g., 16±2 mM. Tris buffer was selected because it has been found to provide superior protein solubility compared to other buffer systems and is suitable for maintaining a pH of 7.5, which is believed to be optimal for long-term storage of PEGylated mutant FGF-21 peptide conjugates. Furthermore, the likelihood of Tris crystallization at low temperatures is lower than with phosphate-based buffers.

[0288] In some embodiments, the mutant FGF-21 peptide conjugate may precipitate at a pH below 6.0. Optionally, the pH of the pharmaceutical composition is in the range of 6.0 to 8.5, 6.5 to 8.0, 6.75 to 8.0, or 7.0 to 8.0, e.g., 7.5±0.3. It has been observed that a pH of 7 to 8 results in the least fragmentation in SDS-PAGE and the least aggregation in SEC. This pH has also been determined to be an appropriate setting with respect to viscosity. Because the pH of the solution may depend on the solution temperature, the pH should be adjusted and measured at 25±2°C. The pH is adjusted with HCl. The pharmaceutical composition may further comprise a salt, particularly an inorganic salt, e.g., NaCl. Optionally, the salt is present at a concentration of 30 mM to 200 mM, 40 mM to 150 mM, 50 mM to 100 mM, e.g., 56±2 mM. The presence of a salt (e.g., NaCl) is beneficial in reducing the increased viscosity in PEG-containing samples. For the same reason, it is also beneficial to include sorbitol and / or glycine.

[0289] In some embodiments, the pharmaceutical composition may further comprise a tonicity adjuster. The tonicity adjuster may be selected from the group consisting of glycerol, amino acids, sodium chloride, proteins, sugars, and sugar alcohols. In some embodiments, the tonicity adjuster is a sugar (e.g., sucrose). Tonicity adjusters, particularly sucrose, have been found to have beneficial effects on pharmaceutical compositions by reducing aggregation of the active agent, i.e., the mutant FGF-21 peptide (conjugate).

[0290] The tonicity adjusting agent, for example sucrose, may be present at a concentration of 50 mM to 200 mM, 100 mM to 175 mM, 135 mM to 160 mM, for example 150±2 mM.

[0291] Additionally, the pharmaceutical composition may, in some embodiments, include a surfactant, such as a non-ionic surfactant. In some embodiments, the surfactant or non-ionic surfactant is a polysorbate-based non-ionic surfactant, such as polysorbate 20 or polysorbate 80. Surfactants such as polysorbate 20 or polysorbate 80 have been found to reduce sub-visible particles less than 10 μm in size and appear to have a stabilizing effect on the pharmaceutical composition.

[0292] A surfactant or non-ionic surfactant, such as polysorbate 20 or polysorbate 80, is optionally present at a concentration of 0.01 mg / mL to 1 mg / mL, 0.05 to 0.5 mg / mL, e.g., 0.2±0.02 mg / mL. Polysorbate 20 or 80 has been found to stabilize the formulation against aggregation.

[0293] In some embodiments, the pharmaceutical composition comprises 0.1 to 50 mg / mL, e.g., 33±7 mg / mL, of a mutant FGF-21 peptide conjugate, 1 mM to 100 mM, e.g., 20±2 mM, of a buffering agent, e.g., Tris buffer, and 30 mM to 200 mM, e.g., 70±2 mM, of a salt, particularly NaCl, and has a pH of 7.5±0.3 (measured at 25±2°C).

[0294] In some embodiments, the pharmaceutical composition comprises 0.1 to 50 mg / mL, e.g., 26±4 mg / mL, of a mutant FGF-21 peptide conjugate; 1 mM to 100 mM, e.g., 16±2 mM, of a buffering agent, e.g., Tris buffer; 30 mM to 200 mM, e.g., 56±2 mM, of a salt, e.g., NaCl; 50 mM to 200 mM of a tonicity adjusting agent, e.g., sucrose; and 0.01 to 1 mg / mL, e.g., 0.2±0.02 mg / mL, of a surfactant or non-ionic surfactant (e.g., polysorbate 20), and has a pH of 7.5±0.3 (measured at 25±2°C).

[0295] In some embodiments, the pharmaceutical container comprises a mutant FGF-21 peptide conjugate of the present disclosure described herein or a pharmaceutical composition of the present disclosure described herein. In some embodiments, the pharmaceutical container is a syringe, a vial, an infusion bottle, an ampoule, a carpule, a syringe with a needle protection system, or a carpule in an injection pen.

[0296] After protein expression and optional purification, a PEG residue is attached to the mutant FGF-21 peptide, particularly at the newly introduced threonine residue, via at least one glycosyl moiety, and optionally via at least one amino acid residue located between the PEG and the glycosyl residue.

[0297] To obtain high-yield expression of nucleic acids encoding mutant FGF-21 of the present disclosure, polynucleotides encoding mutant Fibroblast Growth Factors are typically subcloned into expression vectors containing a strong promoter to direct transcription, a transcription / translation terminator, and a ribosome binding site for translation initiation. Suitable bacterial promoters are described, for example, in Sambrook and Russell (supra) and Ausubel et al. (supra). Bacterial expression systems for expressing native or mutant FGF-21 are available for, for example, Escherichia coli (E. coli), Bacillus, Salmonella, and Caulobacter. Kits for such expression systems are commercially available. Eukaryotic expression systems for mammalian cells, yeast, and insect cells are commercially available. In certain embodiments, the eukaryotic expression vector is an adenoviral vector, an adeno-associated vector, or a retroviral vector. In some embodiments, the variant FGF-21 peptide is produced recombinantly in E. coli cells, ie, the expression host is E. coli.

[0298] In some embodiments, the present disclosure also provides mutant FGF-21 peptide conjugates and / or pharmaceutical compositions for use as medicaments and for use in the treatment of diabetes and related diseases, particularly type 2 diabetes, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), and / or metabolic syndrome. For example, some embodiments also provide the use of a mutant FGF-21 peptide conjugate of the present disclosure and / or the use of a pharmaceutical composition of the present disclosure for the treatment of diabetes and related diseases, particularly type 2 diabetes, NAFLD, NASH, and / or metabolic syndrome.

[0299] In some embodiments, there is provided a method of treating diabetes and related diseases, particularly type 2 diabetes, NASH, nonalcoholic fatty liver disease (NAFLD), and / or metabolic syndrome, comprising administering to a subject in need thereof an amount of a mutant FGF-21 peptide conjugate according to the present disclosure or a pharmaceutical composition according to the present disclosure. In some embodiments, the subject is a human.

[0300] NAFLD is a chronic liver disease characterized histologically by hepatic steatosis and ballooning (ballooning) in 5% or more of hepatocytes. It is part of the spectrum of NAFLD, which includes NASH and cirrhosis resulting from fatty liver. NAFLD is a common chronic liver disease in Western countries and can progress to cirrhosis. It is associated with an increased risk of mortality in general, especially cardiovascular disease. In North America, the prevalence of NAFLD is estimated to be approximately 24%. Patients with NAFLD tend to be obese with insulin resistance and / or type 2 diabetes mellitus (T2DM), dyslipidemia, hypertriglyceridemia, and hypertension. NAFLD is increasingly recognized as a liver disease component of metabolic syndrome (MetS) (Chalasani, 2018).

[0301] NAFLD is usually asymptomatic unless progression to cirrhosis occurs. It is often diagnosed by liver imaging (e.g., ultrasound or magnetic resonance imaging (MRI)) demonstrating fatty liver in subjects with features of metabolic syndrome and no other identifiable etiology of hepatic fat accumulation (e.g., alcoholic liver disease, medications). Effective noninvasive tests for the diagnosis of steatohepatitis are currently unavailable, and liver biopsy remains necessary to diagnose the inflammation and cellular swelling characteristic of NASH (Torres, 2012).

[0302] The limited efficacy of current pharmacological treatments for NAFLD poses an urgent need for the development of more effective and safer drugs for this common and life-threatening disease. Obeticholic acid (OCA), a selective agonist of the farnesoid X receptor, appears to be a promising therapeutic agent for the management of NAFLD. The farnesoid X receptor ligand obeticholic acid in NASH Treatment (FLINT) trial revealed that OCA administration was associated with weight loss and blood pressure reductions as well as improved liver histology. Despite adverse effects on lipid profiles and insulin sensitivity, OCA may be considered for selected patients with NAFLD / NASH, especially those with adequately controlled glucose and lipid levels.

[0303] Various indicators are known to indicate the clinical effectiveness of the compounds and compositions described herein, including, but not limited to, reductions in HbA1c, glucose and insulin, body weight, serum lipids (total cholesterol, LDL, triglycerides), liver enzymes (ALT, AST), liver weight, relative liver weight (% body weight), NAFLD activity score (NAS), fibrosis score (e.g., liver fibrosis), proinflammatory cytokines (e.g., IL1β, MCP-1), fibrosis biomarkers (αSMA, collagen 1α), liver cholesterol, liver triglycerides, and liver fatty acids. An increase in at least one of adiponectin (e.g., high molecular weight (HMW)) or HDL is also an indicator of the clinical effectiveness of the compounds and compositions described herein. Therefore, a change (as described above) in at least one indicator reflects the clinical effectiveness of the compounds or compositions described herein.

[0304] In some embodiments, the therapeutic effect of a compound or composition described herein is determined based on a reduction in at least one of serum triglyceride levels or serum insulin levels.

[0305] HOMA-IR, for example, is an indicator of the presence and degree of insulin resistance in a subject. It is a dynamic and accurate indicator of the relationship between baseline (fasting) blood glucose and the corresponding insulin levels. It is also referred to as an insulin resistance calculator. In humans, the healthy range is 1.0 (0.5-1.4). A value below 0 indicates that the subject is insulin sensitive, which is ideal. A value above 1.9 indicates that the subject is experiencing early insulin resistance, and a value above 2.9 indicates that the subject is experiencing significant insulin resistance. The HOMA-IR blood code is calculated as follows: insulin μIU / mL (mU / L) x glucose mg / dL = HOMA-IR. Calculations require US Standard Units. To convert from International SI units, divide (÷) by 6 to convert from pmol / L to uIU / mL for insulin, or multiply (X) by 8 to convert from mmol / L to mg / dL for glucose.

[0306] Some embodiments relate to a dosing regimen in which an effective amount of a mutant FGF-21 peptide conjugate described herein or a pharmaceutical composition comprising a therapeutically effective amount of a mutant FGF-21 peptide conjugate is administered to a subject in need thereof. In some embodiments, about 3 mg to about 30 mg of a mutant FGF-21 peptide conjugate described herein or a pharmaceutical composition comprising about 3 mg to about 30 mg of a mutant FGF-21 peptide conjugate is administered to a subject in need thereof once a week. In some embodiments, the effective amount of the mutant FGF-21 peptide conjugate is in the range of about 3 mg to about 30 mg, about 9 mg to about 30 mg, about 15 mg to about 30 mg, about 18 mg to about 30 mg, about 3 mg to about 27 mg, about 9 mg to about 27 mg, about 15 mg to about 27 mg, about 18 mg to about 27 mg, about 3 mg to about 9 mg, about 9 mg to about 15 mg, about 9 mg to about 18 mg, about 18 mg to about 27 mg, about 18 mg to about 30 mg, or about 3 mg to 18 mg, administered once weekly. For example, an effective amount of a variant FGF-21 peptide conjugate can be 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, or about 30 mg.

[0307] In some embodiments, about 18 mg to about 44 mg of a mutant FGF-21 peptide conjugate described herein or a pharmaceutical composition comprising about 18 mg to about 44 mg of a mutant FGF-21 peptide conjugate is administered to a subject in need thereof once every two weeks. For example, an effective amount of a mutant FGF-21 peptide conjugate may be 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.

[0308] In some embodiments, a therapeutically effective amount of a mutant FGF-21 peptide conjugate described herein or a pharmaceutical composition comprising a therapeutically effective amount of a mutant FGF-21 peptide conjugate is administered twice daily, once daily, every other day, three times a week, once a week, once every two weeks, once every three weeks, or once a month.

[0309] The long-term efficacy of the mutant FGF-21 peptide conjugates described herein is evidenced by the surprisingly long half-lives (between about 55 and about 100 hours) measured for these conjugates in animal model systems. Native FGF21 has a short lifespan (about 2 hours), limiting its potential for use as a therapeutic agent. In humans, the effects of pegbelfermin (a PEGylated FGF-21 with a half-life of 19-24 hours) showed lower efficacy in lipid measures (% change from baseline) when administered weekly than when administered daily.

[0310] The long-term efficacy of the mutant FGF-21 peptide conjugates described herein allows for the mutant FGF-21 peptide conjugates to be administered less frequently, for example, once per week or once every two weeks. Thus, in some embodiments, a mutant FGF-21 peptide conjugate described herein or a composition comprising same is administered to a subject in need thereof no more frequently than once per week or once per two weeks. For example, a mutant FGF-21 peptide conjugate described herein or a composition comprising same may be administered to a subject in need thereof once every 7 days, once every 8 days, once every 9 days, once every 10 days, once every 11 days, once every 12 days, once every 13 days, once every 14 days, once every 15 days, once every 16 days, once every 17 days, once every 18 days, once every 19 days, once every 20 days, once every 21 days, once every 22 days, once every 22 days, once every 23 days, once every 24 days, once every 25 days, once every 26 days, once every 27 days, once every 28 days, once every 29 days, once every 30 days, or once every 31 days.

[0311] In an exemplary embodiment, the compounds described herein and compositions comprising the same are administered to a subject in need thereof once per week. In an exemplary embodiment, the compounds described herein and compositions comprising the same are administered to a subject in need thereof once every two weeks.

[0312] In another exemplary treatment regimen, induction therapy is performed with the compounds described herein and compositions containing the same, followed by maintenance therapy. The induction therapy is administered more frequently, such as twice a week or once a week, at the beginning of the treatment regimen, and the maintenance therapy is administered every other week or once a month. Such a regimen is effective in that the initial treatment improves the subject's condition to an acceptable and manageable level, achieving a clinical condition acceptable for maintaining the disease / condition. Thereafter, maintenance therapy is used to maintain the health level at a maintenance level.

[0313] The therapeutic efficacy of compounds and / or compositions for treating diabetes and related diseases, particularly type 2 diabetes, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH) and / or metabolic syndrome can be assessed using various parameters and assays known to those skilled in the art and described herein (see Examples 1 and 4). The therapeutic efficacy of compounds and / or compositions for treating non-alcoholic steatohepatitis (NASH) can be assessed using various parameters and assays known to those skilled in the art and described herein (see Examples 5 and 6).

[0314] Measurement of HbA1C is considered the standard assay for determining a subject's glycemic index over time. It is therefore a stable indicator of glycemic index and reflects glucose levels over the last approximately 3-4 months. Therefore, subjects with diabetes (e.g., type 2 diabetes) can be defined by the HbA1C rate determined by an appropriate assay.

[0315] For healthy people without diabetes, the normal range for hemoglobin A1c levels is 4% to 5.6%. A hemoglobin A1c level of 5.7% to 6.4% indicates a high likelihood of developing diabetes. A level of 6.5% or higher indicates a person has diabetes.

[0316] In some embodiments, HbA1c is measured by HPLC using a glycosylated hemoglobin test system (BIO-RAD, Hercules, CA, USA). Blood samples (e.g., 1.0 mL per sample) are collected from the cephalic or saphenous vein into BD Vacutainer® K2-EDTA tubes. The samples are immediately stored at 4°C or kept on wet ice and analyzed on the same day the blood is drawn. HbA1c levels in blood can be measured by those skilled in the art using HPLC with a glycosylated hemoglobin test system (BIO-RAD, Hercules, CA, USA).

[0317] Regarding NASH, this condition is currently diagnosed only by biopsy. However, there are several surrogate biomarkers that are considered to be predictive of NASH, such as liver fat (measured by MRI), liver enzymes (ALT and ALT / AST ratio), fibrosis biomarkers (e.g., pro-C3), and non-invasive tests (NITs) (e.g., VCTE, FAST, FIB-4) (see Examples 4-6).

[0318] In some embodiments, a subject in need thereof is receiving background GLP1 therapy and is administered a mutant FGF-21 peptide conjugate described herein once or twice weekly as described herein (see Example 12).

[0319] In some embodiments, the methods described herein result in a reduction in the incidence of HCC tumor nodules and tumor burden. [Example]

[0320] Example 1: Single Ascending Dose Study of Mutant FGF21-GalNAc-SA-PEG-20kDa (BIO89-100) in Humans

[0321] <Method> In one embodiment, a total of 58 healthy subjects were treated with subcutaneous (SC) BIO89-100 (seven levels) or placebo in a single-center study to evaluate safety, tolerability, PK, immunogenicity, and exploratory pharmacodynamics. Subjects were randomized to receive a single dose of BIO89-100 at 0.45 mg, 1.2 mg, 3 mg, 9.1 mg, 18.2 mg, 39 mg, or 78 mg, or in a 6:2 ratio with placebo (7:3 ratio for the 9.1 mg dose). Subjects were followed for 4 weeks, initially with frequent assessments, then weekly assessments from Days 8 through 29.

[0322] <Result> 1-14 show the results of a single ascending dose (SAD) study in humans of some embodiments of the present disclosure. Baseline demographics were similar between pooled BIO89-100-treated subjects (N=43) and pooled placebo (N=15), as were mean baseline laboratory parameters (within normal ranges). The mean age was 39.3 years, and the mean BMI was 26.7 kg / m. 2 The study included 86% of subjects, 86% of whom were male. There were no deaths, serious adverse events, discontinuations due to adverse events, or dose modifications related to adverse events. Among subjects receiving BIO89-100, the most common treatment-related adverse events occurring in two or more subjects in the pooled BIO89-100 group were injection site reactions and headache, all of which were reported as mild. No clinically meaningful trends were observed in laboratory or other safety-related parameters. The PK of BIO89-100 was generally dose-proportional, with a mean elimination half-life of approximately 53 to 100 hours. At a single dose of 9.1 mg or higher, BIO89-100 demonstrated significant improvements over baseline in key lipid parameters measured on days 8 and 15 after dosing. Mean changes from baseline included a decrease in triglycerides (by 51%, Table 1), a decrease in LDL-C (by 37%), and an increase in HDL-C (by 36%).

[0323] [Table 1]

[0324] <Additional Evaluation> Body weight was assessed only at baseline and at the end of the study. No significant changes were observed. There was no information about the potential for weight loss. ALT and AST: baseline values ​​in the normal range (mean = 21.3 and 20.1 U / L). No significant reduction was observed. Fasting glucose and insulin: baseline values ​​in the normal range (mean = 88.2 mg / dL and 7.3 μIU / mL) No significant reduction was observed. · It is highly likely to be related to the mechanism of action of FGF21 as an insulin-sensitizing substance.

[0325] <Overview of SAD study> · BIO89-100 has good tolerance at single doses up to 78 mg SC. · The incidence of overall adverse events and treatment-related adverse events showed no significant difference between treatment groups across the dose range. · Exception: Adverse events (AEs) at the injection site occurred more frequently in the high-dose groups (39 mg and 78 mg). · The tendency to show clinically important adverse effects of BIO89-100 was not evident in laboratory or other safety-related clinical parameters. · The PK profile was generally dose-proportional within the range of about 53 - 100 hours T 1 / 2 range. · BIO89-100-related effects on lipid PD parameters were observed at doses of 9.1 mg or higher. · A dose-dependent tendency was observed for the main PD parameters in the 39 mg dosing group, but no further benefit was observed in the 78 mg dosing group. · The PD effects were consistent across all lipid parameters and were generally consistent among subjects. · The data support both once-weekly and once-every-two-week dosing.

[0326] <Conclusion> BIO89-100 was safe and highly tolerable in healthy subjects at single doses up to 78 mg, showed a good PK profile, and was associated with a significant improvement in triglycerides, LDL, and HDL.

[0327] <Example 2: Subcutaneous administration of BIO89-100, a novel glycoPEGylated fibroblast growth factor 21 (FGF-21) analog, once a week suppresses the preference for sweetness in obese cynomolgus monkeys>

[0328] <Background> BIO89-100, a novel glycoPEGylated analog of FGF-21, is being developed for the treatment of nonalcoholic steatohepatitis (NASH). FGF-21 regulates carbohydrate and lipid metabolism. FGF-21 and long-acting analogs have been shown to modulate sweet and alcohol preferences in mice and sweet taste preferences in monkeys. The purpose of this study was to evaluate the effect of BIO89-100 on sweet taste preference in obese cynomolgus monkeys.

[0329] <Method> Obese cynomolgus monkeys (mean age approximately 12.6 years; body weight approximately 10.8 kg) were trained in a two-bottle test before dosing. After a 2-week acclimation period to two drinking water bottles, the monkeys were acclimated to a drinking water bottle and a bottle of sweetened water (3% sucrose) for an additional 2 weeks. After baseline data were collected, BIO89-100 (N=3) was administered subcutaneously at a dose of 1 mg / kg or vehicle (N=3) weekly (qW) for 3 weeks, followed by a 2-week washout period. Preference for sweetened water versus unsweetened water was monitored by measuring daily water intake. Clinical evaluations and laboratory tests were also performed.

[0330] <Result> Before the introduction of sweetened water, mean water consumption was 235 mL / day. After the introduction of sweetened water, mean fluid consumption increased significantly to 650 mL / day and consisted almost exclusively of sweetened water. After BIO89-100 administration, preference for sweetened water significantly decreased within 1 day and continued to decline to the point where negligible amounts of sweetened water were consumed (average 40 mL / day). After the end of treatment (washout), preference for sweetened water gradually reappeared. Control animals preferred sweetened water throughout the study period. Compared to baseline, BIO89-100-treated monkeys exhibited decreases in body weight (up to -13%), food intake (up to -60%), triglycerides (up to -78%), and alanine transaminase (up to -44%), as well as increases in high-density lipoprotein (up to 39%). Control animals did not exhibit decreases in body weight, food intake, or other changes in blood lipids.

[0331] <Conclusion> When BIO89-100 was administered weekly to obese monkeys, there was a significant reduction in sweet taste preference and improvement in metabolic and liver-related test parameters. These results demonstrate that FGF-21 analogs such as BIO89-100 can be used as a treatment modality for NASH. The reduction in sugar preference in humans can further provide additional significant benefits in NASH patients.

[0332] <Example 3 - Mechanism of Action by Potent FGF Receptor Agonism>

[0333] Figures 15-37G show the results of the potency of mutant fibroblast growth factor-21 (FGF-21) peptide conjugates.

[0334] <PERK Function Assay> 1. L6 cells expressing KLB / FGFR1 were seeded in a 96-well plate format (104 cells / well) 24 hours prior to treatment. 2. On the day of the assay, the cells were starved in serum-free medium for 2 hours and then treated with the indicated concentrations and at the indicated time points (3 times) with the ligand (or vehicle only). 3. All tubes, plates, and pipette tips used in the assay were pre-coated overnight with 1% BSA (in PBS). 4. Dilutions of FGF-21 and BIO89-100 were made in PBS1X pH 7.4 containing 0.1% BSA. 5. After treatment, the cells were transferred to ice and washed twice with ice-cold PBS and lysed with the manufacturer's protocol MSD * lysis buffer. 6. Samples were processed for pERK / ERK levels by Mesoscale.

[0335] <Results> Data showed that in cells expressing only KLB and cells expressing KLB-FGFR4, FGF21 and BIO89-100 had only a slight increase in pERK at the highest concentration tested (3,000 nM) and were mostly inactive.

[0336] FGF21 and BIO89-100 were highly active in cells expressing FGFR1, FGFR2, and FGFR3.

[0337] BIO89-100 was more potent than FGF21 in KLB-FGFR2- and KLB-FGFR1-expressing cells.

[0338] BIO89-100 had potency comparable to FGF21 in KLB-FGFR3-expressing cells.

[0339] FGF-19 was highly active in cells expressing FGFR4, but was also active on FGFR1 and FGFR3.

[0340] The negative control, FGF-23, was not active in all cells, and there was no difference between KLB alone and the four receptors.

[0341] The positive control EGF was highly active in cells transfected with the four receptors.

[0342] <Conclusion> Preclinical data demonstrate that BIO89-100 has activity similar to native FGF21 at FGF receptors 1c, 2c, and 3c, suggesting that BIO89-100 may be able to recapitulate the beneficial metabolic benefits of the native hormone, which could translate into clinical benefit for patients with NASH.

[0343] Activation of FGF receptors 1c, 2c, and 3c, along with the co-receptor β-Klotho, is important for FGF21 signaling and is thought to be responsible for the observed beneficial metabolic effects. In vitro receptor agonism studies showed that BIO89-100 was active at very low nanomolar concentrations in cells coexpressing β-Klotho and either FGF receptor 1c, 2c, or 3c (Figures 15-37G). The EC50 (the concentration at which half of the maximal FGF receptor agonist effect is observed) of BIO89-100 was similar across FGF receptors 1c, 2c, and 3c in this functional assay and was comparable to or superior to that of native FGF21. The EC50 could not be determined for native FGF21 or BIO89-100 at FGF receptor R4.

[0344] Example 4

[0345] Study BIO89-100-002 is a randomized, double-blind, placebo-controlled, multiple ascending dose (MAD) study evaluating the safety, tolerability, PK and PD profile, and immunogenicity of subcutaneously administered BIO89-100 in approximately 83 subjects with NASH or NAFLD at high risk for NASH. This multisite study consists of six cohorts evaluating two dosing schedules: weekly (QW; Cohorts 1-4) and every 2 weeks (Q2W; Cohorts 5 and 6) (Table 2).

[0346] There are two dose escalation decisions: After Cohort 1 completes the Day 36 visit, subjects can be randomized into Cohorts 2 and 5 (both cohorts will start simultaneously). At least 8 subjects from both Cohort 2 and Cohort 5 will have at least one subject receiving placebo in each cohort, and after completing the Day 36 visit, subjects can be randomized into Cohorts 3, 4, and 6 (all three cohorts will start simultaneously).

[0347] Cohorts 1-4 (Weekly Regimen): On Day 1, eligible subjects will be randomized (as above) and will receive the study intervention via subcutaneous injection every week (QW) from Day 1 through Day 85.

[0348] Cohorts 5 and 6 (every 2 weeks regimen): On Day 1, eligible subjects will be randomized (as above) and treated with the study intervention every other week (Q2W) from Day 1 through Day 85 by subcutaneous injection.

[0349] Subjects in all cohorts will be followed on Day 92 (1 week after the last dose of study intervention) and Day 113, 4 weeks after the last dose of study intervention (end of study visit).

[0350] [Table 2]

[0351] Change and percent change from baseline in the following biomarkers / PD parameters: Anthropomorphic measurements: -body weight -Waist circumference -Waist / Hip Ratio Laboratory parameters triglycerides Non-high-density lipoprotein (non-HDL) cholesterol High-density lipoprotein (HDL-c) Low-density lipoprotein (LDL-c) Hemoglobin A1c (HbA1c) Homeostasis Model Assessment of Insulin Resistance (HOMA-IR) Liver function tests: alanine transaminase (ALT), aspartate transaminase (AST) Adiponectin N-terminal propeptide of type III collagen (Pro-C3) Free fatty acids and Adipo-IR (fasting free fatty acids x fasting insulin) Inflammatory marker high-sensitivity C-reactive protein (hs-CRP) Total cholesterol Oral glucose tolerance test (OGTT) including C-peptide, glucose, and insulin IGF-1, total CK-18 Enhanced liver fibrosis (ELF) panel · Imaging means - Magnetic resonance imaging - whole liver proton density fat fraction (MRI-PDFF) - Liver volume - Intra-abdominal visceral fat - Abdominal subcutaneous fat - FibroScan CAP score - FibroScan VCTE score

[0352] <Enhanced liver fibrosis (ELF) panel> The enhanced liver fibrosis (ELF) blood test has recently been recommended by the National Institute for Health and Care Excellence (NICE) to test for advanced fibrosis in non-alcoholic fatty liver disease (NAFLD). In the ELF test, a score is calculated from the concentrations of serum biomarkers. Serum biomarkers include tissue inhibitor of matrix metalloproteinase 1 (TIMP-1), amino-terminal propeptide of type III procollagen (P3NP), and hyaluronic acid (HA).

[0353] <N-terminal propeptide of type III collagen (Pro-C³)> The N-terminal propeptide (P3NP) neoepitope (Pro-C³) of type III procollagen cleaved by N-protease is derived from the synthesis of type III collagen. Pro-C³ appears to correlate with the stage of liver fibrosis, regression of fibrosis, and response to treatment, both as a single test and as part of an algorithm.

[0354] <Magnetic resonance imaging - whole liver proton density fat fraction (MRI-PDFF)> MRI-PDFF is a non-invasive, quantitative, and accurate measure of liver fat content (image-based biomarker) for assessing treatment response in clinical studies of NASH. This technology allows post-processing of MRI data into a parametric map of PDFF (Antaros Medical, Sweden), providing an accurate and quantitative measurement of liver fat.

[0355] <Liver volume> A dedicated axial three-dimensional (3D) T1-weighted scan, with or without fat suppression, is placed to cover the entire liver. Analysis is performed using semi-automated software to delineate the outer border of the liver, and liver volume is calculated in liters.

[0356] <Visceral abdominal fat (VAT) / Subcutaneous abdominal fat (SAT)> Two- or three-point gradient-echo Dixon imaging is performed in the axial plane centered at the L4-L5 boundary, with a range of approximately ±10 cm in the foot-cephalad direction. Water and fat images are reconstructed, and abdominal visceral and subcutaneous adipose tissue volumes are quantified using semi-automated software, which reports adipose tissue volume in liters.

[0357] <pdff> PDFF is determined using a six-echo gradient-echo pulse sequence that covers the liver axially. Analysis is performed by semi-automatically contouring the liver slice by slice, avoiding major blood vessels and bile ducts. The method combines a multi-peak lipid spectral model and T2 * Simultaneous quantification and revision of the liver fat function (PDFF) is applied. The liver fat function (PDFF) is the average value of all voxels within the identified volume of interest.

[0358] Example 5: Phase 1b / 2a proof-of-concept study evaluating pegosafermin (formerly BIO89-100) for the treatment of NASH

[0359] NASH is a serious liver condition associated with significant comorbidities. See Table 3. There is currently no available treatment. The number of cases is 16.5 million and is projected to increase to 27 million by 2030. NASH is expected to become the leading cause of liver transplants.

[0360] [Table 3]

[0361] The Phase 1b / 2a NASH study design is shown in Figure 38A. Key inclusions for Cohorts 1-6 and Cohort 7 are shown in Figure 38B. Cohort 7 includes NASH patients with fibrosis stages F2 and F3 who have a NAFLD Activity Score (NAS) score of ≥4 and an MRI-PDFF of ≥8%. NAS can range from 0 to 8 and is calculated by the sum of the scores for steatosis (0-3), intralobular inflammation (0-3), and hepatocellular ballooning (0-2). Figure 38B is a table showing baseline characteristics.

[0362] In a single-arm cohort, patients with biopsy-proven NASH and fibrosis stages F2 and F3 were treated with 27 mg of pegosafermin weekly for 20 weeks. At baseline, 65% of patients had fibrosis stage F3.

[0363] Of the 20 patients enrolled, 19 underwent end-of-treatment biopsies, and the results from these 19 patients were as follows:

[0364] [Table 4]

[0365] [Table 5]

[0366] Figures 39A and 39b show that BIO89-100 demonstrated robust reduction in liver fat with a high responder rate.

[0367] Figures 40A and 40B show that BIO89-100 significantly reduced alanine aminotransferase (ALT), with greater reductions in patients with elevated baseline ALT.

[0368] Figures 41A and 41B show that BIO89-100 significantly improved the NAFLD Activity Score (NAS) and all components of the NAS. 63% of patients had a ≥ 2-point improvement in NAS and no worsening of fibrosis (≥ 1-point improvement in ballooning or inflammation) (primary endpoint). 100% of patients had improvement or no change in ballooning and inflammation.

[0369] Figure 42 shows that BIO89-100 demonstrated clinically meaningful changes in key histologic efficacy endpoints. By three-panel read, NASH resolution was up to 47% (range: 26-47%), fibrosis improvement was up to 42% (range: 12-42%), and a 2-point NAS improvement was up to 79% (range: 68-79%).

[0370] Results also demonstrated clinically meaningful and significant changes across key non-invasive tests (NITs) related to fibrosis, risk of fibrosis, or resolution of NASH.

[0371] Figure 43A shows that BIO89-100 substantially improved scores across non-invasive tests (NITs) that correlated with advanced fibrosis. The NIT descriptions are as follows: VCTE: Measurement of liver stiffness using FibroScan® FAST score: liver stiffness FibroScan® and AST were used to assess (VCTE) and steatosis (CAP); 0-1 scale FIB-4 score: combined serum marker / age measurement Pro-C3: a serum biomarker of collagen deposition

[0372] Clinically relevant thresholds are as follows: · VCTE: A reduction of >20% correlates with improvement in fibrosis. FAST score: A score ≦0.35 predicts fibrosis stage F0 / F1 and NAS<4. FIB-4 score: A score <1.3 predicts fibrosis stage F0 / F1. · Pro-C3: A reduction of >15% correlates with improvement in fibrosis.

[0373] Figure 43B shows that Bio89-100 had a high responder rate based on clinically relevant thresholds for NIT.

[0374] In addition to significantly improving liver health, treatment with pegosafermin also had a significant positive effect on glycemic control, lipids, and body weight.

[0375] [Table 6]

[0376] Figures 44A, 44B and 44C show that BIO89-100 showed clinically significant improvements on HbA1c and adiponectin, and demonstrated significant weight loss.

[0377] FIG. 45 shows that BIO89-100 exhibits clinically meaningful reductions in lipid parameters such as TG, LDL-C, non-HDL-C, and implies a complete increase in HDL-C.

[0378] Figures 46A, 46B, and 46C show that BIO89-100 improves many markers of liver health and comorbidities associated with NASH.

[0379] In the 83 patients treated with BIO89-100 throughout the Phase 1b / 2a study, BIO89-100 continued to have a favorable safety profile and was generally well tolerated. There were no drug-related serious adverse events, only one treatment-related discontinuation, and no tremor or hypersensitivity reactions were observed. There were no adverse effects on blood pressure or heart rate. In the open-label histology cohort, the most commonly reported treatment-related adverse events were nausea, diarrhea, vomiting, and injection site reactions, most of which were graded as mild and / or short-lived.

[0380] Example 6: Phase 2b NASH Study Design

[0381] Figure 47 shows the Phase 2b NASH study design.

[0382] A randomized, double-blind, placebo-controlled study to evaluate the efficacy, safety, and tolerability of pegosafermin in subjects with confirmed nonalcoholic steatohepatitis (NASH). Doses tested: placebo, pegosafermin 30 mg weekly, pegosafermin 15 mg weekly, pegosafermin 44 mg every other week Duration: 24 weeks of treatment until the primary endpoint, followed by 24 weeks of blinded extension treatment Number of subjects: Approximately 200 patients Key inclusion criteria: F2-F3 NASH; NAS ≥ 4 Selected primary endpoints: Improvement of stage 1 fibrosis without worsening of NASH Resolution of NASH without worsening fibrosis NAS ≥ 2 point improvement Combined histological and metabolic endpoints Biopsy evaluation method: Baseline and end-of-treatment biopsies (24 weeks) were evaluated by three pathologists using consensus reading.

[0383] Example 7: Safety, pharmacokinetics, and pharmacodynamics of pegosafermin, a glycopegylated analog of fibroblast growth factor 21, in patients with nonalcoholic steatohepatitis: a multicenter, randomized, double-blind, placebo-controlled, phase 1b / 2a, multiple-ascending dose study

[0384] <Summary> <Background> The objective of this study was to evaluate the safety and efficacy of pegosafermin (BIO89-100), a glycoPEGylated fibroblast growth factor analog, in participants with biopsy-confirmed nonalcoholic steatohepatitis (BC-NASH) or nonalcoholic fatty liver disease and high risk for NASH (referred to in this study as phenotypic NASH [PNASH]).

[0385] <Method> This multicenter, randomized, double-blind, placebo-controlled, phase 1b / 2a, multiple-ascending-dose study enrolled adults (21-75 years) with BC-NASH / PNASH at 12 clinical sites in the United States. Patients were randomized to receive pegosafermin (3, 9, 18, or 27 mg once weekly [QW]; 18 or 36 mg once every 2 weeks [Q2W]) or placebo subcutaneously for 12 weeks. Primary endpoints were safety, tolerability, and pharmacokinetics. Secondary endpoints consisted of the effect of immunogenicity (anti-drug antibodies [ADA]) and changes from baseline to week 13 in liver parameters (magnetic resonance imaging proton density fat fraction [MRI-PDFF], alanine aminotransferase [ALT], aspartate transaminase [AST], N-terminal propeptide of type III collagen [PRO-C3]), lipids (non-high-density lipoprotein cholesterol [non-HDL-D], high-density lipoprotein cholesterol [HDL-D], low-density lipoprotein cholesterol [LDL-C]), adiponectin, free fatty acids, insulin resistance, adipose tissue insulin resistance, and body weight (ClinicalTrials.gov number: NCT04048135).

[0386] <Insights> Between July 29, 2019, and March 18, 2020, 275 participants were screened, and 81 (15 [18.5%] patients with BC-NASH) were randomized (62 received pegosafermin and 19 received placebo). The most common treatment-related adverse event (AE) was mild increased appetite (10 / 63 [15.9%] for pooled pegosafermin and 0 / 18 [0.0%] for pooled placebo), which was not associated with weight gain. No treatment-related serious AEs or deaths occurred. Dose-proportional pharmacokinetics was observed. ADAs were detected in 41 of 63 participants (65.1%) treated with pegosafermin. Occurrence appeared to be dose-dependent, with no evidence of an impact on the PK, PD, or safety profile. Pegozafermin statistically significantly improved liver parameters versus pooled placebo, with the greatest reductions in the 27 mg QW cohort (n=7) observed in liver fat fraction (MRI-PDFF) (least squares mean absolute reduction -13.5% vs. +1.4%, difference: -14.9% [95% CI: -20.1, -9.7], p<0.0001), ALT levels (least squares mean relative reduction -43.7% vs. -4.2%, difference: -39.5% [95% CI: -59.9, -19.2], p=0.0002), and AST levels (least squares mean relative reduction -37.9% vs. -4.2%, difference: -33.5% [95% CI: -51.4, -15.5], p=0.0004), and 85.7% (6 / 7) achieved a relative reduction of 30% or more in MRI-PDFF, compared with no reduction in the pooled placebo group (p<0.0001).Pegozafermin 27mg The LS mean differences for QW vs. pooled placebo were -25.4% (95% CI: -48.4, -2.4; p=0.0308) for triglycerides, -17.5% (95% CI: -30.6, -4.4; p=0.0095) for non-HDL-C, -17.6% (95% CI: -32.7, -2.6; p=0.0224) for LDL-C, +65.1% (95% CI: +36.6, +93.7; p<0.0001) for adiponectin, -30.9% (95% CI: -57.4, -4.5; p=0.0227) for PRO-C3, and -2.2% (95% CI: -4.2, -0.12; p=0.0380) for weight. No statistically significant effects were observed for other secondary endpoints evaluated in the 27 mg QW cohort.

[0387] <Interpretation> Pegozafermin was generally well tolerated and associated with clinically meaningful reductions in measures of liver fat, liver function, and lipids. Further evaluation of pegosafermin in NASH is warranted.

[0388] Research in context <Evidence before this study> It is estimated that 27 million people in the United States (approximately 8% of the general population) will have nonalcoholic steatohepatitis (NASH) by 2030. NASH is a significant risk factor for cirrhosis, hepatocellular carcinoma, and cardiovascular events, and a leading cause of liver transplantation. Management strategies for NASH are primarily based on lifestyle changes, and no approved disease-modifying drugs exist yet. Fibroblast growth factor 21 (FGF21), an endogenous metabolic hormone, is a key regulator of glucose and lipid metabolism. Administration of FGF21 analogs has emerged as a potential therapeutic strategy for NASH. On November 22, 2021, we searched PubMed for randomized controlled trials of FGF21 analog-based therapies for the treatment of NASH using the following search string: (nonalcoholic fatty liver disease [MeSH] OR nonalcoholic fatty liver disease OR AFLD OR steatohepatitis OR ASH OR fatty liver) AND (fibroblast growth factor receptor [MeSH] OR fibroblast growth factor 21 OR FGF21 OR FGF-21). Two randomized, double-blind, placebo-controlled phase 2a trials were identified evaluating the effects of pegbelfermin (polyethylene glycol-conjugated [PEGylated] recombinant human FGF21) and effluxifermin (a fusion of the human IgG1 Fc domain with modified human FGF21), respectively, administered subcutaneously at least once weekly for 12 to 16 weeks in patients with biopsy-confirmed NASH (BC-NASH). Each study reported statistically significant reductions in hepatic fat fraction, hepatic transaminases (alanine aminotransferase and aspartate aminotransferase), and lipid metabolism (triglycerides, low- and high-density lipoprotein cholesterol, and adiponectin) with study drug versus placebo. Statistically significant reductions in PRO-C3 (a marker of fibrosis) levels were also reported in both studies. The most common adverse events (AEs), occurring more frequently with pegbelfermin or effluxifermin than with placebo, were gastrointestinal (GI) related in nature.Although the absolute reductions in hepatic fat fraction reported in the effluxifermin trials (-12.3% to -14.1% [effluxifermin] vs. +0.3% [placebo]) were higher than those reported in the pegbelfermin trials (-6.8% to -5.2% pegbelfermin vs. -1.3% [placebo]), gastrointestinal AEs appeared to occur significantly more frequently with effluxifermin than with pegbelfermin.

[0389] <Added value of this research> In this randomized, double-blind, placebo-controlled, phase 1b / 2a proof-of-concept study, participants with nonalcoholic fatty liver disease (NAFLD) at high risk for NASH (referred to in this study as phenotypic NASH) or BC-NASH received multiple ascending doses of the glycopegylated FGF21 analog pegosafermin (BIO89-100) once daily (QW) or once every two weeks (Q2W) for 12 weeks. The most common treatment-related AE was mild increased appetite unrelated to weight gain. No other GI-related AEs were reported more frequently in the pegosafermin group compared with placebo. Statistically significant absolute reductions in hepatic fat fraction of -13.5% and -9.7% were observed at week 13 with pegosafermin QW and Q2W administration, respectively, compared with a +1.4% increase with placebo. A high proportion of participants (up to 88%) experienced a reduction in liver fat content of at least 30%, a threshold previously shown to correlate with histological improvement and reduced fibrosis progression. Similar to previous FGF21 analog studies, improvements in liver transaminases, the fibrosis marker PRO-C3, and lipid metabolism were observed in patients treated with pegosafermin compared with placebo. These data suggest that pegosafermin may combine the promising efficacy of FGF21 analogs in the treatment of NASH with the potential additional benefits of a milder AE profile and the possibility of Q2W dosing.

[0390] <What all available evidence means> These results further support the therapeutic potential of FGF21 analogs in patients with NASH, a disease with a high unmet medical need. The beneficial effects of these molecules on liver-related parameters, coupled with the reduction of metabolic abnormalities underlying NASH pathology and risk factors for cardiovascular disease (the leading cause of death in these patients), are promising. The efficacy and safety of pegosafermin 15 mg QW, 30 mg QW, and 44 mg Q2W are currently being evaluated in patients with NASH (NAFLD Activity Score [NAS] ≥ 4) and fibrosis (stage 2 or 3) in the ongoing Phase 2 ENLIVEN trial (ClinicalTrials.gov number: NCT04929483).

[0391] <Introduction> Nonalcoholic steatohepatitis (NASH) is an advanced form of nonalcoholic fatty liver disease (NAFLD), a chronic disease characterized by steatosis in at least 5% of hepatocytes, intralobular inflammation, and hepatocyte ballooning, with or without fibrosis. 1 The current global prevalence is 1.5-6.5% in the general population. In the United States, the prevalence of NASH is expected to increase from 16.5 million cases in 2015 (approximately 5% of the general population) to 27 million (approximately 8%) by 2030. 2,3 NASH is predicted to become the leading cause of liver transplants in the near future (currently the leading cause in women). 4 Approximately 20% of patients progress to fibrosis and cirrhosis, and 45% of patients with cirrhosis progress to decompensated cirrhosis within 10 years. 5-7

[0392] Despite advances in understanding the pathomechanisms of NASH, there are currently no approved disease-modifying pharmacological interventions, and lifestyle modifications remain the recommended strategy for disease management. 8 Progression of fibrosis is a strong predictor of mortality and liver-related morbidity in patients with NASH, and amelioration of fibrosis is an important goal of treatment. 9 Notably, a relative reduction in liver fat of at least 30% as assessed by magnetic resonance imaging proton density fat fraction (MRI-PDFF) in NASH clinical trials correlates with histological improvement and reduced fibrosis progression. 10-12

[0393] Fibroblast growth factor 21 (FGF21) is an endogenous metabolic hormone secreted by the liver and is a key regulator of energy expenditure, as well as glucose and lipid metabolism, through activation of various FGF receptors (FGFRs) in metabolically active organs. 13-15 In patients with insulin resistance and NASH (and possibly some genetic variants such as rs499765) (Jiang, 2014), circulating and tissue FGF21 levels are increased and correlate with disease severity, suggesting FGF21 resistance, which may be overcome by administering pharmacological doses of FGF21. On this basis, administration of exogenous FGF21 is being investigated as a treatment for obesity-related insulin resistance disorders, including NASH. 16-18

[0394] Pharmacological administration of FGF21 has been shown to have beneficial effects in patients with NASH, including increasing hepatic insulin sensitivity, stimulating fatty acid oxidation, inhibiting de novo lipogenesis, and reducing the delivery of triglyceride-rich very low-density lipoproteins (VLDL) via downregulating VLDL receptor expression in hepatocytes. 19 However, the short half-life of native FGF21 (approximately 2 hours) limits its therapeutic potential. In animals, glycoPEGylation of FGF21 significantly increases its in vivo half-life while maintaining efficacy similar to endogenous FGF21 with lower dosing frequency and total cumulative exposure. 20,21

[0395] Pegozafermin (BIO89-100) is a glycopegylated FGF21 analogue with an N-terminal methionine residue, two point mutations, and a single 20 kDa linear polyethylene glycol (PEG) covalently attached via a glycosyl moiety. In two NASH animal models (mice and spontaneously diabetic monkeys), pegosafermin conferred liver-related and metabolic benefits, including improved transaminase levels and liver histology, weight loss, improved glycemic parameters and lipid profiles, and elevated adiponectin levels. 21,22 A first-in-human, Phase 1, single-ascending-dose study in healthy volunteers demonstrated that subcutaneous administration of pegosafermin has a half-life of 55-100 hours, supporting the investigation of both once-weekly (QW) and once-every-2-weekly (Q2W) dosing. 23 The study also observed statistically significant beneficial changes in triglycerides, low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and adiponectin levels. 23 These early studies suggested that patients with NASH may benefit from treatment with pegosafermin.

[0396] Here, we present data from a Phase 1b / 2a study aimed at evaluating the safety, tolerability, pharmacokinetic (PK), and pharmacodynamic (PD) effects of multiple ascending doses of pegosafermin in participants with biopsy-confirmed NASH (BC-NASH) or NAFLD and at high risk for NASH (hereafter referred to as phenotypic NASH [PNASH]).

[0397] <Method> <Research design and participants> This was a randomized, double-blind, placebo-controlled, multiple-ascending-dose, proof-of-concept, Phase 1b / 2a study conducted at 12 clinical sites in the United States from July 29, 2019, to August 3, 2020. The study protocol and amendments (Figure 51) were approved by the Institutional Review Board or Independent Ethics Committee at each site. All participants provided written informed consent. The trial was registered with ClinicalTrials.gov (NCT04048135).

[0398] MRI-PDFF ≥ 10% and body mass index (BMI) of at least 25 kg / m 2 Adults aged 21 to 75 years who were aged 21 to 75 years were enrolled. Participants were required to have either BC-NASH with NASH Clinical Research Network (CRN) fibrosis stage 1, 2, or 3 based on biopsy performed within the 24 months prior to screening, or PNASH if biopsy was not available. PNASH was defined as obesity (BMI > 30 kg / m ) with either type 2 diabetes mellitus (T2DM; fasting plasma glucose ≥ 126 mg / dL, 2-hour plasma glucose ≥ 200 mg / dL on a 75-g oral glucose tolerance test, or glycated hemoglobin [HbA1c] ≥ 6.5% [48 mmol / mol]) or evidence of liver injury (elevated alanine aminotransferase [ALT] ≥ 40 U / L in men or ≥ 30 U / L in women, and / or Fibroscan [Echosens, Waltham, MA, USA] vibration-controlled transient elastography score ≥ 7 kPa). 2 ) Individuals were excluded if they had liver disease other than NASH, evidence of cirrhosis, cardiovascular or cerebrovascular disease, or any condition that, in the investigator's opinion, could confound the results of the study or pose additional risk to the patient. Individuals with any clinically significant abnormalities at screening in laboratory parameters, electrocardiogram (ECG), or vital signs were also excluded.

[0399] Randomization and Masking Investigators at each study site enrolled participants. Eligible individuals were randomly assigned (allocation concealment) to one of six cohorts in order of enrollment using an interactive web response system (IWRS; developed, deployed, and supported by ProSciento). Randomization and inventory schedules for "dummy" subjects were tested in the IWRS to ensure the system performed according to protocol requirements. The final, actual subject randomization and inventory schedules were then imported into the IWRS by an unblinded statistician. Periodic reviews of randomization were performed by the unblinded statistician throughout the study.

[0400] Participants, investigators, other study personnel, and the study sponsor were blinded to treatment assignment throughout the study. Pegozafermin and placebo were prepared in syringes at each study site by unblinded pharmacists. The unblinded pharmacists were not involved in any other study-related procedures. Syringes containing pegosafermin or placebo were identical in appearance, and the study medication was administered by blinded site staff.

[0401] Two dosing regimens were evaluated: weekly (QW; 3 mg [Cohort 1], 9 mg [Cohort 2], 18 mg [Cohort 3], 27 mg [Cohort 4]) and once every 2 weeks (Q2W; 18 mg [Cohort 5], 36 mg [Cohort 6]) (randomization ratios and block sizes are reported in Figure 52). Within each cohort, participants were randomized to pegosafermin or placebo and treated for 12 weeks. The first dose was administered on day 1, and the last on day 85 (13 doses for the QW regimen and 7 doses for the Q2W regimen). A safety monitoring committee (SMC) was established to review participant safety and for dose escalation decisions. The SMC consisted of the sponsor's medical monitor (MM), the clinical research site's medical monitor (LM), and at least one principal investigator (BBF). There were two planned SMC meetings for dose escalation decisions, with additional follow-up meetings possible if necessary. A blinded safety review was conducted by the SMC after participants in Cohort 1 completed the Day 36 visit. If no safety concerns were identified, randomization of additional participants into Cohorts 2 and 5 began. If at least eight participants from both Cohort 2 and Cohort 5 completed the Day 36 visit and no safety concerns were identified after at least one participant in each cohort received placebo, randomization of additional participants into Cohorts 3, 4, and 6 began.

[0402] <Procedure> Patients received QW (Cohorts 1, 2, 3, and 4) or Q2W (Cohorts 5 and 6) treatment with one or two subcutaneous injections of pegosafermin or placebo in the abdomen from Day 1 through Day 85. Administration of pegosafermin or placebo was performed by qualified study personnel (acceptable reasons for study discontinuation, withdrawal, or interruption are provided in Figure 52).

[0403] AEs were continuously monitored throughout the study (Figures 51 and 52) and coded using the Medical Dictionary for Regulatory Activities version 23.0. The SMC reviewed blinded safety data (AEs, clinical laboratory parameters, vital signs, and electrocardiograms [ECGs]).

[0404] Blood sampling for PK analysis began on Day 1 (the day of the first dose) and Day 29, when steady-state serum pegosafermin concentrations were achieved (Day 5 for the QW cohort; Day 3 for the Q2W cohort). Samples were collected pre-dose (Days 1 and 29), and 6, 12, and 24 hours (Days 2 and 30), 48 hours (Days 3 and 31), 72 hours (Days 4 and 32), 96 hours (Days 5 and 33), and 168 hours (Days 8 and 36) post-dose. In the Q2W cohort, an additional PK blood sample was collected 36 hours post-dose on Day 43 (but not on Day 29) and incorporated into the steady-state PK analysis on Day 29 as a "trough" value for pegosafermin exposure.

[0405] PD assessments (MRI-PDFF, liver and metabolic biomarkers, etc.), clinical tests, 12-lead electrocardiograms, and vital signs were assessed according to a schedule (Figures 53 and 54).

[0406] <Result> The primary endpoints of the study were the safety and tolerability of pegosafermin, as assessed by the frequency and severity of adverse events (AEs) and serious adverse events (SAEs), and the number of patients who discontinued treatment due to AEs and treatment-related AEs, and the PK of pegosafermin, which was assessed by the maximum serum concentration (C) within the dosing interval. max ), the area under the serum drug concentration-time curve from time zero to the last measurable concentration within the dosing interval (AUC last ), Cmax achievement time (t max ), terminal elimination half-life (t 1 / 2 ), and accumulation ratio (AUC last [Steady state] / AUC on day 1 last ) was determined.

[0407] Absolute and percent changes from baseline in hepatic fat fraction (MRI-PDFF) were key secondary endpoints. Additional secondary endpoints included absolute and percent changes from baseline in body weight, triglycerides, HDL-C, non-HDL-C, LDL-C, HbA1c, homeostasis model assessment of insulin resistance (HOMA-IR), adipose insulin resistance (Adipo-IR), liver function tests (ALT and aspartate transaminase [AST]), adiponectin, and the N-terminal propeptide of type III collagen (PRO-C3). The immunogenicity of pegosafermin, as measured by the incidence and characteristics of post-dose anti-drug antibodies (ADAs) (e.g., potency and / or binding specificity to the PEG moiety of pegosafermin, and neutralizing immunogenicity), and the potential effect of ADAs on serum pegosafermin concentrations and safety, were also evaluated as secondary endpoints. Additionally, absolute and percent change from baseline in liver volume (by MRI) was a key exploratory endpoint.

[0408] Other safety endpoints included the incidence and clinically significant changes in vital signs, physical examination findings, electrocardiogram data, and laboratory values ​​(including complete blood count, biochemistry, cortisol, and urinalysis).

[0409] <Statistical analysis> A formal sample size calculation was not performed for the primary endpoint because the number of participants (n = 81) was deemed sufficient to meet safety, tolerability, and PK goals. Regarding the change in liver fat fraction (a key secondary endpoint), a power analysis indicated that comparing 9, 12, or 14 subjects in the treatment group with 19 subjects in the pooled placebo group would have approximately 89%, 93%, and 95% power, respectively, to detect a 30% difference between treatment groups in the mean percent change from baseline in MRI-PDFF, assuming a 25% standard deviation for this endpoint in each group. These calculations were based on a 2-sample t-test with a one-sided 5% (two-sided 10%) Type I error probability.

[0410] Statistical analyses were performed using SAS version 9·4 or higher (SAS Institute Inc., Cary, NC, USA). Six population analysis sets were defined (Figure 55). The placebo group from each cohort was pooled for analysis. Summary descriptive statistics were used to present demographic and baseline characteristics, safety endpoints, and PK and PD parameters. Mixed-model repeated measures (MMRM) analysis was used to analyze change from baseline and / or rate of change from baseline in PD endpoints. MMRM included baseline as a covariate and treatment group, visit, and the interaction between treatment group and visit as factors. Analyses were performed using SAS PROC MIXED, and interaction terms were examined in the primary analysis. Covariance was unstructured. If the model did not converge, other structures, such as compound symmetry, were considered. Least squares (LS) means and LS mean differences were presented for each visit, along with corresponding standard errors, p-values, and two-sided Wald intervals with 90% and 95% CIs (data reported here are relative to 95% CIs; all 90% and 95% CIs are shown in Figures 56-72). Nonparametric methods, such as the Wilcoxon rank-sum test, were considered when strong evidence existed that the assumption of normality was violated. Response rates were calculated with Miettinen-Nurminen 95% CIs. Differences in response rates between placebo and pegosafermin were analyzed using Fisher's exact test. No adjustment for multiplicity or imputation of missing values ​​was performed for study outcomes.

[0411] The incidence and number of AEs were summarized by system organ and preferred term, treatment group, and combined pegosafermin or placebo group using descriptive statistics.

[0412] Data processing procedures, including case report forms and data management activities such as data collection, data review, data reconciliation, and database locking, were managed by IBM® Clinical Development using an electronic data capture system.

[0413] <Result> Of the 275 individuals screened for the study, 81 met all eligibility criteria and were randomized to study treatment (pegozafermin, n = 62; placebo, n = 19) (Figure 47). Of the patients randomized to pegosafermin, 6 were assigned to 3 mg QW, 12 to 9 mg QW, 11 to 18 mg QW, 10 to 27 mg QW, 14 to 18 mg Q2W, and 9 to 36 mg Q2W. One participant assigned to placebo in the randomized analysis set inadvertently received a single dose of 3 mg QW pegosafermin. Therefore, the safety analysis set included 63 subjects in the pegosafermin group and 18 subjects in the pooled placebo group. Reasons for early discontinuation by 10 participants (12.3%) included AEs (n = 2), non-compliance with the study protocol (n = 1), and withdrawal of consent (n = 7). Study interruptions due to COVID-19 infection occurred in 11 participants (2 receiving placebo [1 after treatment completion] and 9 receiving pegosafermin [4 after treatment completion]; duration, 6–21 days).

[0414] Overall, baseline characteristics were similar between the pooled placebo and pegosafermin cohorts (Figure 73). Nearly all participants (95.1% [77 / 81]) met at least one of five criteria associated with a high risk of having NASH, and 64.2% (52 / 81) met at least two of these criteria (Table 1). Baseline characteristics were also similar between participants with BC-NASH (n = 15) and those with PNASH (n = 66), although T2DM was less prevalent in the BC-PNASH subpopulation (26.7% [4 / 15] vs. 50.0% [33 / 66]), and the proportion of male participants was lower in the BC-PNASH subpopulation (20.0% [3 / 15] vs. 42.4% [28 / 66]). Within the BC-NASH subgroup, 11 participants were treated with pegosafermin (4 in the 18 mg QW cohort and 7 in the 18 mg Q2W cohort) and 4 participants were treated with placebo. See Figures 73-75.

[0415] Overall, treatment-emergent adverse events (TEAEs) occurred in 40 of 63 participants (63.5%) receiving pegosafermin and 8 of 18 participants (44.4%) receiving placebo (Table 2). Most (27 / 40 [68%]) were mild in severity. Two serious non-drug-related TEAEs due to COVID-19 infection occurred in the pegosafermin group; neither led to treatment or study discontinuation. Treatment discontinuation due to AEs occurred in two participants. One participant in the 27 mg QW cohort had a grade 2 skin rash that was considered possibly related to treatment. One participant in the 18 mg Q2W cohort (with T2DM) experienced grade 3 acute hyperglycemia, grade 1 chest pain, and grade 1 visual impairment, all of which were assessed as unrelated to treatment. The most common AEs (pooled pegosafermin vs. pooled placebo) were increased appetite (15.9% [10 / 63] vs. 0%), diarrhea (12.7% [8 / 63] vs. 22.2% [4 / 18]), headache (11.1% [7 / 63] vs. 5.6% [1 / 18]), and nausea (7.9% [5 / 63] vs. 16.7% [3 / 18]). Of these, increased appetite and headache occurred more frequently with pegosafermin than with placebo. Increased appetite was not associated with weight gain. Treatment-related TEAEs were reported in 24 of 63 participants (38.1%) in the pooled pegosafermin group and in 5 of 18 participants (27.8%) in the pooled placebo group. No deaths were reported.

[0416] Gastrointestinal AEs, including diarrhea, nausea, and abdominal pain / discomfort, occurred with similar frequency between the pooled pegosafermin (19 / 63; 30.2%) and pooled placebo (6 / 18; 33.3%) groups, with no notable differences observed between treatment groups.Mild, transient, and self-limited injection site events (erythema, pain, pruritus, or reactions) were reported in 4 of 63 participants (6.3%) in the pooled pegosafermin group and none in the pooled placebo group.

[0417] Overall, ADAs were detected at presentation in 41 (65.1%) of 63 participants treated with pegosafermin (range across cohorts: 14.3-78.6%). The incidence of treatment-induced ADAs in the pegosafermin group was 63.9% (39 / 61). Two participants in the pooled pegosafermin group were ADA-positive at baseline. Specificity was mostly directed against the FGF21 domain (63.5% [40 / 63]). Specificity against PEG was present in 4.8% (3 / 63) of pegosafermin-treated participants. The occurrence of ADAs appeared to be dose-related, with higher antibody titers elicited at doses greater than 3 mg QW, and the number of ADA-positive subjects and ADA antibody titers increased with increasing treatment duration. No differences in ADA response were observed between the QW and Q2W regimens. There was no evidence that the pegosafermin PK, PD, or safety profile was altered in participants with ADAs. No neutralizing antibodies were observed.

[0418] No clinically significant findings were identified based on laboratory parameters, vital signs, ECG, or physical examination. Specifically, no hypersensitivity reactions or tremors were reported, and no clinically relevant changes in blood pressure or heart rate were observed. No clinically significant findings were observed for bone biomarkers (C-terminal telopeptide, N-terminal propeptide of procollagen type 1, osteocalcin, and bone-specific alkaline phosphatase) or 24-hour urinary cortisol assessment.

[0419] At steady state on day 29, the terminal phases of the concentration-time profiles were roughly parallel on semi-log plots (Figures 48A and 48B), suggesting dose-proportional PK, with a median t of approximately 46 to 68 hours across cohorts. 1 / 2 Exposure (AUC last ) was also roughly dose-proportional across cohorts, with dose-normalized AUC last The median C varied over the dose range tested, with no discernible dose-related pattern (Table 2). max Values ​​ranged from 103 ng / mL to 1674 ng / mL, with t values ​​at 48 to 72 hours post-dose across cohorts. max The median accumulation ratios ranged from 1.0 to 1.4 for the QW regimen and 1.0 to 1.1 for the Q2W regimen.

[0420] At week 13, liver fat fraction was statistically significantly reduced from baseline for all evaluated pegosafermin doses compared with pooled placebo, with the greatest effect observed in the 27 mg QW cohort (-13.5% vs. +1.4% LS mean absolute change in MRI-PDFF; difference: -14.9% [95% CI: -20.1, -9.7]; p<0.0001). There was also an overall trend toward greater absolute reductions in liver fat fraction with increasing pegosafermin dose. The LS mean placebo-adjusted relative change in liver fat fraction from baseline to week 13 was -70.2% (95% CI: -92.5, -47.9; p<0.0001 vs. pooled placebo) in the 27 mg QW cohort. Liver volume also decreased statistically significantly from baseline at week 13 in most pegosafermin cohorts compared with pooled placebo, with a mean placebo-adjusted relative decrease in LS of -16.4% (95% CI: -24.8, -7.9; p=0.0003 vs. pooled placebo) and a mean absolute decrease in LS of -0.33 L (95% CI: -0.52, -0.15; p=0.0007 vs. pooled placebo) observed in the 27 mg QW cohort.

[0421] In the 27 mg QW cohort, 85.7% (6 / 7) of participants achieved a relative reduction of 30% or greater in liver fat fraction at week 13 compared with 0 in the pooled placebo group (p<0.0001). Additionally, 71.4% (5 / 7) of participants achieved a relative reduction of at least 50% compared with 0 in the pooled placebo group (p=0.0004) (range across the pegosafermin cohort: 60.0–87.5% for a relative reduction of 30% or greater, 20.0–71.4% for a relative reduction of 50% or greater; see Figure 50). Baseline characteristics of participants who achieved a relative reduction of 30% or greater in liver fat fraction and those who did not are shown in Figure 75. In the 27 mg QW cohort, liver fat fraction normalized (<5%) in 42.9% (3 / 7) of participants compared with none in the pooled placebo group (p=0.0152). Changes in all MRI-assessed liver parameters were similar between BC-NASH and PNASH participants treated with pegosafermin or placebo.

[0422] Pegozafermin treatment resulted in statistically significant reductions in ALT levels from baseline at week 13 in most dose cohorts, with the largest LS mean relative change observed in the 27 mg cohort being -43.7% (-30 U / L absolute change) compared with -4.2% (-3.4 U / L absolute change) for pooled placebo (differences from pooled placebo were -39.5% [95% CI: -59.9, -19.2]; p=0.0002 and -26.6 U / L [95% CI: -39.2, -13.9]; p<0.0001, respectively). An overall trend of greater ALT reductions with increasing pegosafermin dose was also observed. The reduction in ALT levels from baseline at week 13 was particularly pronounced in participants with elevated baseline ALT levels (defined as >45 U / L by central laboratory) (least squares mean absolute change, pooled pegosafermin -34.6 U / L [n=17], pooled placebo -10.3 U / L [n=5]; difference -24.3 U / L [95% CI: -47.6, -0.9]; p=0.0426). A reduction in ALT levels of at least 17 U / L was observed in 71.4% (5 / 7) of participants in the 27 mg QW group and 57.1% (4 / 7) of participants in the 36 mg Q2W group, compared with 16.7% (3 / 18) of participants in the pooled placebo group (p=0.0169 and p=0.0664, respectively, vs. pooled placebo). In participants treated with pegosafermin, a relative decrease of at least 30% in liver fat fraction was statistically significantly correlated with a relative decrease of at least 30% in ALT levels (r=0.540; p<0.001) (see Figure 49).

[0423] The LS mean relative change from baseline in AST at week 13 was -37.9% (-14.5 U / L absolute change) in the 27 mg QW cohort compared with -4.4% (-5.2 U / L absolute change) in the pooled placebo cohort (differences -33.5% [95% CI: -51.4, -15.5]; p=0.0004 and -9.3 U / L [95% CI: -16.8, -1.8]; p=0.0158, respectively).

[0424] In the 27 mg QW group, the LS mean relative decrease from baseline in PRO-C3 levels at week 13 was -27.7% compared with +3.3% for pooled placebo (difference -30.9% [95% CI: -57.4, -4.5], p=0.0227). Numerical decreases in PRO-C3 levels were also observed in the other pegosafermin cohorts, except for the 18 mg QW cohort.

[0425] The reduction in ALT, AST, and PRO-C3 levels with pegozafermin treatment was similar in the BC-NASH and PNASH subpopulations.

[0426] At week 13, a mean reduction in triglyceride levels from baseline of -27.6% was observed in the 27 mg QW cohort (range across pegosafermin cohorts: 17.7-28.5%) compared with -2.2% for pooled placebo (difference -25.4% [95% CI: -48.4, -2.4], p=0.0308). In participants with elevated baseline triglyceride levels (≥200 mg / dL; n=15 for pooled pegosafermin), triglycerides decreased by 33.1-48.9% at week 13, with 53.3% achieving triglyceride normalization (<150 mg / dL). Statistically significant relative reductions from baseline in LS mean LDL-C (-16.5% in the pegosafermin 27 mg QW group vs. +1.2% in pooled placebo; difference -17.6% [95% CI: -32.7, -2.6]; p=0.0224) and increases in LS mean HDL-C levels (maximum increase of 20.1% in the 18 mg Q2W group vs. +2.0% in pooled placebo; difference -18.2% [95% CI: -8.6, -27.7]; p=0.0003) were also observed. In the 27 mg QW cohort, a LS mean reduction in non-HDL-C levels versus baseline of -16.3% was observed compared to +1.1% for pooled placebo (difference of -17.5% [95% CI: -30.6, -4.4]; p=0.0095) (Figure 65).

[0427] At week 13, greater improvements in insulin sensitivity, fasting plasma glucose, HbA1c, and body weight were observed in the cohort receiving the higher dose of pegosafermin than in the pooled placebo cohort (Figures 66-69). These changes were not statistically significant except for body weight, which decreased by only 2.2% of the LS mean from baseline in the 27 mg QW cohort compared to pooled placebo at week 12 (95% CI: -4.2, -0.12; p=0.0380). Additionally, a 65.1% increase in the LS mean of adiponectin was observed in the 27 mg QW cohort compared to pooled placebo (95% CI: +36.6, +93.7; p<0.0001). A 36.0% increase in adiponectin was observed in the pooled pegosafermin group compared to the pooled placebo (95% CI: +17.7, +54.3, p=0.0002). At week 13, no significant improvements from baseline were observed comparing pegosafermin with placebo in terms of changes in free fatty acid levels or Adipo-IR (Figures 70-71).

[0428] <Consideration> In this proof-of-concept, multiple-ascending-dose Phase 1b / 2a study, treatment with pegosafermin resulted in significant improvements in multiple liver-related and metabolic parameters in patients with BC-NASH or PNASH and was generally well tolerated. These benefits were observed at all doses tested in the QW and Q2W dosing cohorts, with the most pronounced effects observed at the highest doses tested, 27 mg QW and 36 mg Q2W.

[0429] Regarding the liver, 12 weeks of treatment with pegosafermin resulted in a significant reduction in hepatic fat fraction as assessed by MRI-PDFF. High proportions of participants had relative reductions in hepatic fat fraction of greater than 30% and 50%, which have been shown to correlate with clinically relevant histologic outcomes (e.g., a ≥ 2-point reduction in NAFLD Activity Score [NAS] and resolution of NASH). 10-12 Liver volume was also statistically significantly reduced in participants treated with pegosafermin. Previously, liver volume has been shown to be highly correlated with MRI measures of liver fat burden (including MRI-PDFF and total liver fat index) and histological steatosis in patients with NASH. 24 The changes in liver volume observed in pegosafermin-treated individuals are consistent with a significant decrease in the hepatic fat fraction. Whether the reduction in liver volume translates into further clinically meaningful outcomes, such as a decrease in right upper quadrant discomfort, is unknown. Treatment with pegosafermin also resulted in statistically significant decreases in ALT and PRO-C3 levels. Elevated ALT levels have been correlated with the incidence of steatohepatitis and fibrosis in patients with NASH. 25 , an ALT reduction of at least 17 U / L has been shown to correlate with histologic improvement. 26 This ALT threshold was achieved by 71% (5 / 7) of participants in the pegosafermin 27 mg QW group, with statistically significant reductions in PRO-C3, a neoepitope marker of type III collagen formation, and a novel noninvasive biomarker of fibrogenesis and fibrosis. 27-29 These data suggest that pegosafermin has important benefits across multiple liver-related parameters that may predict beneficial effects on clinically important histological and other endpoints in NASH. The effect of pegosafermin on NASH histological endpoints (NAS≧4, NASH CRN fibrosis stage 2 or 3) is currently being evaluated in a Phase 2 trial (ENLIVEN, NCT04929483).

[0430] NASH is generally considered the hepatic manifestation of metabolic syndrome, and the nomenclature metabolically associated fatty liver disease (MAFLD) has been proposed as a more accurate term to describe this condition. 30 Therefore, an ideal treatment for NASH would simultaneously address liver-related parameters (e.g., hepatocyte stress, immune cell infiltration, and fibrosis) and the underlying metabolic overload that drives liver pathology. Indeed, NASH patients often have multiple cardiovascular risk factors and are at increased risk for cardiovascular events and death, and NAFLD / NASH itself may confer additional cardiovascular risk, especially in patients with advanced fibrosis. 31 Importantly, cardiovascular mortality is the leading cause of death in patients with NASH. 31 Given these considerations, it is encouraging that 12 weeks of pegosafermin treatment resulted in clinically meaningful metabolic improvements, including reductions in triglycerides, LDL-C, and non-HDL-C, and increases in HDL-C, in addition to significant liver-related benefits. The simultaneous reductions in hepatic fat fraction and triglycerides are noteworthy, as it has been suggested that fibrates, which are approved to treat hypertriglyceridemia, may increase hepatic fat content and volume. 32 Notably, these benefits occurred in the absence of clinically significant safety concerns and confirm previous findings in healthy volunteers. 23

[0431] During the 12-week treatment period, trends toward improvement in additional metabolic parameters (HOMA-IR, fasting plasma glucose, and HbA1c) were observed with higher doses of pegosafermin compared to placebo, but these benefits did not reach statistical significance. A small but statistically significant 2.2% weight loss was observed in the 27 mg QW group, which was not secondary to gastrointestinal AEs. This weight loss is unlikely to have significantly contributed to the beneficial effects on serum lipids observed in other treatment groups, in which weight loss did not occur. The mechanism of weight loss was not investigated in this study; notably, pegosafermin has been shown to increase energy expenditure in CD-1 mice {Rosenstock, 2020 #19} and decrease sweet taste preference in cynomolgus monkeys {Rosenstock, 2019 #59}. Treatment with pegosafermin resulted in a strong increase (up to 61%) in adiponectin, an insulin-sensitizing, anti-inflammatory, anti-fibrotic, anti-atherosclerotic, and hepatoprotective factor produced primarily by adipocytes. 33 FGF21 potently induces adiponectin gene expression in adipocytes through a peroxisome proliferator-activated receptor (PPAR) gamma-dependent mechanism in mice. Various findings suggest that adiponectin is an important downstream mediator of FGF21, promoting its pleiotropic effects in major peripheral organs, including the liver, via abundantly expressed adiponectin receptors. 34 .

[0432] Two other FGF21 analogs, pegbelfermin (a PEGylated FGF21 analog) and effluxifermin (an Fc-FGF21 analog), are in clinical development for the treatment of NASH. FGF21 and its cofactor β-Klotho signal through their cognate receptors, FGFR1c, FGFR2c, and FGFR3, which are expressed throughout multiple organs, including the liver, adipose tissue, muscle, pancreas, and brain, contributing to the systemic effects observed in response to FGF21 agonism. Unlike FGF19 analogs, FGF21 does not signal through the FGFR4 / β-Klotho complex, the activation of which induces bile acid suppression and increases LDL-C levels. 35,36 Gastrointestinal AEs are treated with pegbelfermin 17 and effluxifermin 37 It was the most frequently reported event with pegosafermin. In contrast, the frequency of gastrointestinal AEs with pegosafermin in this study was similar to that with placebo. Tremor, which has been reported with effluxifermin, was not observed.

[0433] Overall, the magnitude of the anti-lipid deposition effect observed with FGF21 analogs is greater than that of other therapies under clinical evaluation, including thyroid hormone receptor β, PPAR agonists, farnesoid X receptor (FXR) agonists, FGF19, and glucagon-like peptide 1 (up to approximately 70% relative reduction, compared with 18–58% of other agents), highlighting the potential of this drug class to target intrahepatic lipid deposition, a major cause of NASH. 16,17,37-41 Furthermore, unlike FGF21 analogs, other treatments in development for NASH include LDL-C (FXR agonists) 38,42,43 or FGF19 analogues 44 ) and elevated triglyceride levels (acetyl-CoA carboxylase inhibitors) 45 , or promoting weight gain and fluid retention (PPAR agonists) 46 Some drugs may increase cardiovascular risk.

[0434] The main limitations of this study are the small sample size and relatively short treatment period. Furthermore, only a subset of participants had BC-NASH at baseline. Although the mixed histological and noninvasive inclusion criteria may have increased the heterogeneity of the study population, it should be noted that baseline characteristics, such as liver fat fraction assessed by MRI-PDFF, were similar in the BC-NASH and PNASH subpopulations, as were the observed treatment effects. Finally, when biopsies were available for BC-NASH determination, they were classified by a local pathologist. An ongoing Phase 2b study (ENLIVEN, NCT04929483), with centrally-read biopsies performed at baseline and after 24 weeks of treatment, followed by a blinded extension phase for a total of 48 weeks of treatment, will further evaluate the efficacy, safety, and tolerability of pegosafermin (15 mg QW, 30 mg QW, and 44 mg Q2W doses) in participants with BC-NASH (NAS ≥ 4, NASH CRN fibrosis stage 2 or 3).

[0435] In summary, in populations with BC-NASH and PNASH, 12 weeks of pegosafermin treatment resulted in clinically meaningful improvements in liver parameters assessed by MRI-PDFF, transaminases, and PRO-C3, as well as metabolic improvements indicated by decreases in triglycerides and LDL-C and increases in HDL-C and adiponectin. These beneficial effects were observed in participants treated with pegosafermin at both QW and Q2W dosing. Overall, treatment was associated with a favorable safety and tolerability profile. Collectively, these data suggest that pegosafermin has great potential as a treatment for NASH and other metabolic diseases, with the potential added benefit of an every-2-week dosing option.

[0436] Example 8: Variability in liver biopsy assessment: Data from a Phase 1b / 2a study of pegosafermin in subjects with nonalcoholic steatohepatitis (NASH)

[0437] <Summary> <Background> Variability in liver biopsy interpretation is increasingly recognized as a major challenge to drug development in NASH. Biopsy variability was evaluated in the open-label cohort of a Phase 1b / 2a study in NASH. This study demonstrated that pegosafermin (PGZ), a long-acting glycopegylated recombinant human FGF21 analog, resulted in histologic improvement and significant liver-related benefits (MRI-PDFF, ALT, and multiple fibrosis-related noninvasive tests) and cardiometabolic benefits, with a favorable safety and tolerability profile.

[0438] <Method> Twenty subjects with biopsy-confirmed NASH (NAS ≥ 4, fibrosis stage F2 / F3) received PGZ 27 mg QW subcutaneously for 20 weeks. Baseline (BL) and week 20 (W20) biopsies were performed in 19 of the 20 subjects. The primary analysis was performed by a single central reader (CR), a liver pathologist with extensive experience reading NASH studies. Post-hoc evaluation by a panel of three additional board-certified, expert NASH pathologists [Pathologists A (PA), B (PB), and C (PC)] was compared with the original reading of the same slides by the CR. Biopsy reading by the PA, PB, and PC was independent, and biopsies were scored for intralobular inflammation, ballooning, steatosis, and fibrosis. The proportions of subjects achieving an NAS ≥ 2, NASH resolution without worsening fibrosis, and fibrosis improvement ≥ 1 stage without worsening NASH were compared.

[0439] <Result> In BL, mean NAS scores were 5.4, 5.0, and 4.9 points for PA, PB, and PC assessments, respectively, compared with 5.4 points for CR assessments. At week 20, a ≥2-point reduction in NAS occurred in 79%, 79%, and 68% of subjects (PA, PB, and PC, respectively) compared with 74% of CR patients. The mean absolute reduction in NAS score was 2.3, 2.4, and 2.2 points, respectively, compared with 2.4 points for CR patients. Resolution of NASH without worsening of fibrosis occurred in 26%, 42%, and 47% of subjects, respectively, compared with 32% of CR patients. Improvement of fibrosis without worsening of NASH occurred in 42%, 32%, and 12% of controls, respectively, compared with 26% of CR patients. Furthermore, either NASH resolution or improvement of fibrosis was observed in 58%, 63%, and 53%, respectively, compared with 47% of CR patients.

[0440] <Conclusion> PGZ 27 mg QW for 20 weeks resulted in significant changes in key histologic endpoints in a cohort of NASH subjects with advanced fibrosis. Although limitations of the analysis include post-hoc reading and small sample size, the percentage of subjects meeting the guidance-recommended endpoints for NASH pivotal trials varied substantially across four pathologists' assessments, ranging from 26% to 47% for resolution of NASH without worsening fibrosis and 12% to 42% for improvement of fibrosis without worsening NASH. PGZ is currently being evaluated in NASH in the ongoing phase 2b ENLIVEN study, which will be read by a three-person expert panel according to a consensus charter. Alternatives to histologic endpoints are urgently needed in NASH clinical trials, as variability in endpoint assessments can increase placebo response and weaken efficacy signals.

[0441] <Introduction> Fibroblast growth factor 21 (FGF21) is an endogenous hormone that regulates lipid and glucose metabolism and energy expenditure.

[0442] Pegozafermin (PGZ) is a glycoPEGylated FGF21 analogue with an extended half-life compared to native FGF21, and is currently in development for the treatment of nonalcoholic steatohepatitis (NASH) and severe hypertriglyceridemia (SHTG).

[0443] In a randomized, placebo-controlled, phase 1b / 2a proof-of-concept study in subjects with NASH, PGZ had significant liver- and metabolic-related benefits.

[0444] In the open-label cohort of this proof-of-concept study, which included subjects with biopsy-confirmed NASH (NAS ≥ 4, fibrosis stage F2 or F3; N = 20), 20 weeks of PGZ 27 mg resulted in clinically meaningful histologic improvements and significant liver-related (MRI-PDFF, ALT, multiple fibrosis-related noninvasive tests) and cardiometabolic benefits, and was safe and well-tolerated.

[0445] Intra- and inter-reader variability in liver biopsy interpretation is increasingly recognized as a major challenge to drug development in NASH.

[0446] If biopsy slides from a NASH study are reviewed by different pathologists (or by the same pathologist at different time points), this may affect the assessment of subject eligibility and the proportion of subjects who met the histologic endpoint.

[0447] <Purpose> The objective of the study was to evaluate the impact of assessment by four expert NASH pathologists on histologic endpoints in a phase 1b / 2a proof-of-concept trial.

[0448] <Method> The study design is shown in Figure 76.

[0449] <Biopsy reading> For the primary analysis, a single liver pathologist with extensive experience reading NASH trials centrally read biopsies at baseline (BL) and end of treatment (EOT).

[0450] In a post-hoc exploratory analysis, a panel of three additional expert NASH pathologists—Pathologist A, Pathologist B, and Pathologist C—evaluated the same BL and EOT slides evaluated by the central pathologist. Slides from BL and EOT were mixed, and the pathologists were blinded to the time points.

[0451] Biopsies were scored for intralobular inflammation, ballooning, steatosis, and fibrosis.

[0452] The proportions of subjects achieving a ≥2-point reduction in NAS, resolution of NASH without worsening of fibrosis, and improvement of ≥1 stage of fibrosis without worsening of NASH were compared.

[0453] <Result> The baseline characteristics of all subjects are shown in the table below.

[0454] [Table 7]

[0455] Baseline Biopsy Reading: The proportion of subjects in NAS grade / stage categories across the study population was generally consistent. All subjects were deemed eligible by a central reader (primary analysis). Three panel pathologists assessed the fibrosis stage as more advanced than that determined by the central reader.

[0456] [Table 8]

[0457] 6 / 19 (32%) subjects were assessed as having F4 fibrosis by two or more panel pathologists (probable F4), and 4 / 19 (21%) subjects were assessed as having F4 fibrosis by all three panel pathologists.

[0458] Figure 77 shows the primary analysis (central reader). PGZ reliably improved the NAFLD Activity Score (NAS) and all components of the NAS.

[0459] 63% of patients had a ≥2-point improvement in NAS and no worsening of fibrosis (primary endpoint) (ballooning or inflammation improved ≥1 point).

[0460] 100% of patients had improvement or no change in ballooning and inflammation.

[0461] Figure 78 shows the primary analysis (reader). PGZ demonstrated clinically meaningful changes to the key histologic efficacy endpoint.

[0462] Week 20 Biopsy Reading: The mean EOT scores and mean changes from baseline for most parameters were similar when assessed by four pathologists. For the entire study population, the proportion of subjects assigned to a particular histologic grade / stage category by the four pathologists was more variable at EOT compared to baseline.

[0463] The table below shows the week 20 biopsy endpoints by individual pathologist.

[0464] [Table 9]

[0465] The table below shows the median reader endpoints at week 20, excluding estimated F4 at baseline ( ** Sensitivity analysis excluding subjects with F4 fibrosis assessed by 2+ panel pathologists (n=6). Cirrhosis was an exclusion criterion in this study.

[0466] [Table 10]

[0467] The table below shows baseline characteristics - estimated F4 fibrosis * ( * Subjects with F4 fibrosis assessed by a panel pathologist of 2+ are shown.

[0468] [Table 11]

[0469] Table shows estimated F4 fibrosis at baseline - Non-invasive liver testing (NIT) at 20 weeks * ( * Subjects with F4 fibrosis assessed by 2+ panel pathologists ** N=5; one outlier with poor measurement quality was excluded *** VCTE >20% reduction; FAST score ≤0.35).

[0470] [Table 12]

[0471] In putative F4 subjects (n=6), ≥1 stage fibrosis improvement without worsening of NASH ranged from 17% to 57%, and NASH resolution without worsening of fibrosis ranged from 20% to 50%.

[0472] <Consideration> Eligibility Impact: Thirty-two percent (6 / 19) of subjects failed baseline screening as F4 in post-hoc analysis by 3 panel pathologists, rather than F3 as determined by the central study reader. A consensus approach using two or more readers may reduce variability.

[0473] Impact on study endpoints: The proportion of subjects meeting guideline-recommended endpoints for NASH clinical trials varied among the four pathologists, ranging from 26% to 47% for resolution of NASH without worsening fibrosis and 12% to 42% for improvement of ≥1 stage fibrosis without worsening NASH. In this post-hoc analysis, excluding subjects assessed (by 2+ panel pathologists) as having probable F4 fibrosis at baseline, the study histologic endpoint would have been met by a higher proportion of subjects in the primary analysis. - NAS ≥ 2 points 75%-77%; - Resolution of NASH without worsening of fibrosis 32%-46%; - Fibrosis improvement ≥ 1 stage without worsening of NASH 26%-38%

[0474] Subjects with presumed F4 fibrosis at baseline * ( * Subjects with F4 fibrosis assessed by a panel pathologist of 2+): All subjects with presumed F4 fibrosis had well-compensated cirrhosis (Child-Pugh A). There were no clinical or laboratory findings suggestive of clinically significant portal hypertension or other complications of cirrhosis. At 20 weeks, there was a significant improvement in most liver-related and metabolic NITs in subjects with presumed F4 fibrosis. -Safety and tolerability were good. These findings are reassuring regarding the safety and potential benefits of PGZ in the F4 population.

[0475] <Conclusion> This analysis demonstrates inter-reader variability in biopsy scoring. In this post-hoc analysis, if subjects with presumed F4 fibrosis at baseline (by 2+ panel pathologists) were excluded, the proportion of subjects meeting the histologic endpoint in the primary analysis would likely have been even higher. Significant beneficial effects on hepatic NIT and metabolic markers, with good safety and tolerability, were observed in subjects with estimated F4 fibrosis at baseline. The presumed F4 subjects were well compensated and no safety issues suggestive of cirrhosis-related complications were reported. Study limitations include post-hoc analysis and small sample size.

[0476] Example 9: Pegozafermin inhibits NASH-induced hepatocellular carcinoma in the STAM™ mouse model

[0477] <Background and Objectives> Pegozafermin (PGZ), a long-acting glycoPEGylated recombinant human FGF21 analog, demonstrated significant histological and other liver-related benefits, as well as cardiometabolic benefits, with a well-tolerated safety profile in a Phase 1b / 2a study in NASH. PGZ does not activate FGFR4 and is not mitogenic. NASH-associated HCC, previously considered a complication of cirrhotic NASH, is increasingly being diagnosed in precirrhotic NASH. In the STAM® model, which recapitulates the human NASH-HCC sequence, HCC appears at 16 weeks of age and develops universally by 20 weeks of age. In HCC prevention studies in this model, treatment usually begins between 6 and 12 weeks of age. PGZ significantly improved NASH features, including liver histology, liver transaminases, and various metabolic parameters, in STAM® mice. The purpose of this study was to evaluate its effect on HCC development in this model.

[0478] <Method> STAM™ mice (12- or 13-week-old, male, N=20 per group) were treated with vehicle, pegosafermin (formerly BIO89-100) (0.3 mg / kg, 1.0 mg / kg, or 3.0 mg / kg), or the positive control, sorafenib (30 mg / kg, once daily), three times per week for 9 weeks, starting at week 12 (N=16 per group) or week 13 (N=4 per group). Sorafenib (a protein kinase inhibitor) delayed tumor progression and reduced tumor burden in a mouse model of HCC. All surviving animals were sacrificed at 20 or 21 weeks of age. At the time of sacrifice, the number of surviving animals, liver weight, liver weight / body weight ratio, and the number of visible tumor nodules on the liver surface of surviving mice were assessed.

[0479] <Result> PGZ dose-dependently reduced the number of macroscopic tumor nodules: the mean (±SD) number of visible tumor nodules per mouse was 9±7, 10±4, 7±4, 2±2, and 5±4 in the vehicle, PGZ 0.3 mg / kg, PGZ 1.0 mg / kg, and sorafenib groups, respectively (Figure 79; p<0.05 for PGZ 3 mg / kg). PGZ-treated animals showed decreased liver weights and liver / body weight ratios, with mean liver weights and liver / body weight ratios of 2391 ± 473 mg and 9.6 ± 2.2 mg, 2498 ± 1120 mg and 11.0 ± 4.6 mg, 1802 ± 391 mg and 7.8 ± 1.7 mg, 1252 ± 210 mg and 6.2 ± 1.1 mg, and 2013 ± 916 mg and 8.4 ± 3.8 mg in the vehicle, PGZ 0.3 mg / kg, PGZ 1.0 mg / kg, and sorafenib groups, respectively (p < 0.05 for liver weights and liver / body weight ratios in the PGZ 3 mg / kg group). Survival rates were 4 / 20, 4 / 20, 9 / 20, 8 / 20, and 12 / 20 in the vehicle, PGZ 0.3 mg / kg, PGZ 1.0 mg / kg, and sorafenib groups, respectively. The effect on survival was not statistically significant with either PGZ or sorafenib treatment. Immunohistochemical staining for cleaved caspase 3 (AC3) and proliferating cell nuclear antigen (PCNA) was not significantly different between vehicle-treated and PGZ- or sorafenib-treated animals.

[0480] <Conclusion> In STAM mice, PGZ treatment reduced HCC tumor nodule development and tumor burden, comparable to that achieved with sorafenib. These data suggest that PGZ has beneficial effects on NASH and fibrosis, has significant metabolic benefits, and is a promising therapeutic option for NASH. If these preclinical data are translated to human use, an additional benefit of PGZ treatment may be reduced risk of HCC. The mechanism of this antitumor effect remains to be elucidated. PGZ is currently being studied in the ENLIVEN phase 2b clinical trial for NASH.

[0481] Example 10: Phase 2b ENLIVEN trial of pegosafermin in nonalcoholic steatohepatitis (NASH)

[0482] Figure 80 shows the Phase 2b study design.

[0483] In this study, both 44 mg every 2 weeks (Q2W) and 30 mg weekly (QW) administration of a mutant fibroblast growth factor-21 (FGF-21) peptide conjugate met both primary histological endpoints per the U.S. Food and Drug Administration (FDA) guidance for endpoints and statistical analysis with high statistical significance (Figures 82-94C).

[0484] Both the 44 mg Q2W and 30 mg QW treatment groups demonstrated at least one stage of fibrosis improvement without worsening of NASH (27% and 26%, respectively) at 3.5 times the placebo rate (7%) and NASH resolution without worsening of fibrosis at 12-14 times the placebo rate (2%) (26% and 23%, respectively).

[0485] The ENLIVEN study biopsies were independently scored by three expert blinded pathologists to minimize individual reader bias and inter-reader variability. See Figure 81.

[0486] Results were consistent and achieved statistical significance for the 44 mg Q2W and 30 mg QW dose groups using multiple imputation analysis, completer analysis (patients who underwent baseline and end-of-treatment biopsies at week 24), and intention-to-treat (ITT) analysis (Phase 3 analysis design). Using completer analysis for the fibrosis endpoint, the placebo-adjusted effect sizes for the 44 mg Q2W and 30 mg QW dose groups were 20% and 19%, respectively (p=0.008 and p=0.009, respectively; Figure 85). For the NASH resolution endpoint, the placebo-adjusted effect sizes for the 44 mg Q2W and 30 mg QW dose groups were 24% and 21%, respectively (p=0.0004 and p=0.0009, respectively; Figure 88). Using an ITT analysis, which considered patients in whom biopsies were not performed as non-responders, results were statistically significant for both primary histologic endpoints at both doses.

[0487] Significant changes compared to baseline were observed in liver fat and other key non-invasive tests ("NITs") of liver inflammation and fibrosis. Improvements were also observed in HbA1c and overall key lipid markers, factors important for the effective treatment of NASH.

[0488] The study also included 14 NASH patients with biopsy-confirmed compensated cirrhosis (F4 patients), who were not included in the primary analysis but continued to participate in the study, 12 of whom underwent follow-up biopsies at week 24. A descriptive analysis of these data showed that 5 of 11 patients receiving pegosafermin had at least one stage improvement in liver fibrosis and no worsening of NASH by week 24, compared with 0 of 1 patient receiving placebo. An additional 4 pegosafermin-treated patients experienced at least one stage improvement in liver fibrosis.

[0489] Gozafermin demonstrated a favorable safety and tolerability profile consistent with previous studies. Across dose groups, the most frequently reported treatment-related adverse events (AEs) were grade 1 or 2 gastrointestinal events (diarrhea, nausea, and increased appetite), most of which were mild to moderate in nature. The observed rate of treatment-related AEs was less frequent with the Q2W dosing regimen. See Figure 95.

[0490] Example 11: Randomized controlled trial of the FGF21 analogue pegozafermin in NASH

[0491] <Summary> <Background> Pegozafermin is a long-acting glycoPEGylated FGF21 analogue under development for nonalcoholic steatohepatitis (NASH) and severe hypertriglyceridemia. We aimed to evaluate the efficacy and safety of pegosafermin in patients with non-cirrhotic NASH.

[0492] <Method> ENLIVEN was a phase 2b, 24-week, multicenter, double-blind, randomized, placebo-controlled trial comparing subcutaneous pegosafermin 15 mg or 30 mg once weekly or 44 mg every other week with placebo in patients with biopsy-confirmed NASH and F2 / F3 fibrosis. The two primary endpoints were the proportion of patients achieving: 1. Fibrosis improvement (≥1 stage) without worsening of NASH at week 24; 2. NASH resolution without worsening of fibrosis at week 24.

[0493] <Result> Of 222 randomized patients, 219 received treatment. The proportions of patients achieving the fibrosis improvement endpoint were 7% in the placebo group, 22% in the 15 mg once-weekly pegosafermin group (difference from placebo: 14.5 [95% CI: -8.7 to 37.6]), 26% in the 30 mg once-weekly pegosafermin group (difference: 18.9 [5.2 to 32.5]; P = 0.009), and 27% in the 44 mg every other week group (difference: 20.3 [5.3 to 35.4]; P = 0.008). The proportions of patients achieving the NASH resolution endpoint for pegosafermin 15 mg weekly, 30 mg weekly, and 44 mg every other week versus placebo (2%) were 37% (difference 34.7 [10.0 to 59.3]), 23% (difference 20.9 [9.1 to 32.7]), and 26% (difference 23.6 [10.0 to 37.2]), respectively. The most common adverse events associated with pegosafermin were nausea and diarrhea.

[0494] <Conclusion> In this Phase 2 study, treatment with pegosafermin resulted in improvement of fibrosis. These results support advancing pegosafermin into Phase 3 development.

[0495] Nonalcoholic steatohepatitis (NASH) is characterized by excessive fat accumulation, liver inflammation, and cellular injury, with or without fibrosis (Rinella et al., Hepatology 2023;77:1797-835; Loomba et al., Cell 2021;184:2537-64). It is associated with metabolic syndrome and an increased risk of cardiovascular disease (Pouwels et al., BMC Endocr Disord 2022;22:63; Shroff et al., Curr Hepatol Rep 2020;19:315-26; Younossi et al., Hepatology 2016;64:73-84). The development of significant fibrosis in NASH is associated with increased liver-related outcomes (e.g., cirrhosis and its complications, progression to hepatocellular carcinoma), cardiovascular events, and mortality (Loomba et al., Cell 2021;184:2537-64; Dulai et al. Hepatology 2017;65:1557-65). The prevalence of NASH in adults is reported to be 5.3% worldwide and 14% in middle-aged Americans (Younossi et al., Hepatology 2023;77:1335-47; Harrison et al., J Hepatol 2021;75:284-91) and is rising (Riazi et al., Lancet Gastroenterol Hepatol 2022;7:851-61; Estes et al., Hepatology 2018;67:123-33), but there are no approved pharmacological treatments (Loomba et al., Gastroenterology 2022;162:680-8).

[0496] Fibroblast growth factor 21 (FGF21) regulates lipid and glucose metabolism and energy expenditure (Lee et al., Clin Endocrinol (Oxf) 2014;80:57-64). Pegozafermin, a long-acting glycoPEGylated recombinant FGF21 analog, is being developed for the treatment of NASH and severe hypertriglyceridemia (Rosenstock et al., J Hepatol 2019;70:e155; Margalit et al., J Clin Lipidol 2020;14:585-6; Loomba et al. Lancet Gastroenterol Hepatol 2023;8:120-32). A phase 1b / 2a study in patients with NASH revealed no safety concerns and suggested that pegosafermin may improve hepatic steatosis, markers of inflammation and fibrosis, circulating lipids, and glycemic control (Loomba et al., Lancet Gastroenterol Hepatol 2023;8:120-32). A benefit on liver histology was observed in an open-label cohort of patients with biopsy-confirmed NASH (Loomba et al. J Hepatol 2022;77:S730).

[0497] The objective of this study was to evaluate the efficacy and safety of pegosafermin in patients with non-cirrhotic NASH.

[0498] <Method> <Test Design and Monitoring> This randomized, placebo-controlled, double-blind, phase 2b study was conducted at 61 sites in the United States to evaluate the efficacy, safety, and tolerability of pegosafermin over a 24-week period. It included a 12-week screening period and a 24-week treatment period. A placebo-controlled, single-blind, 24-week extension study is ongoing under the same protocol. The study was conducted in accordance with the Declaration of Helsinki, the International Council for Medical Organizations' International Ethical Guidelines, the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICPHA), and applicable laws and regulations. The study protocol and amendments were approved by the Institutional Review Board or independent ethics committee at each site. All participants provided written informed consent. The sponsor (89bio), together with the academic steering committee, designed the study and conducted on-site monitoring, data collection, and data analysis. All authors had access to the data and participated in data interpretation. They vouched for the protocol and data analysis, and ensured the study's fidelity to the protocol, available at NEJM.org. The steering committee and the first author decided to publish the paper. The first draft was written by Dr Loomba and further developed with the assistance of a medical writer (funded by the sponsor) under the guidance of the authors.

[0499] <patient> Eligible patients were 21 to 75 years of age and had confirmed NASH (Clinical Research Network [CRN] fibrosis stage F2 or F3, Nonalcoholic Fatty Liver Disease Activity Score [NAS] ≥ 4, steatosis, ballooning, or intralobular inflammation ≥ 1 point) at screening or on a biopsy performed within 6 months prior to screening. There was no minimum liver fat content requirement. Key exclusion criteria were liver disease other than NASH, cirrhosis, uncontrolled or newly diagnosed type 2 diabetes, and any disease that, in the investigator's opinion, could affect study results or pose additional risk to participants. Clinically significant laboratory, electrocardiogram, or vital sign abnormalities also excluded participants.

[0500] <Procedure> Patients were initially randomized (centrally using interactive response technology) in a 2:1:3:3:3 ratio to receive placebo once weekly (QW) or once every 2 weeks (Q2W) or pegosafermin 15 mg QW, 30 mg QW, or 44 mg Q2W. Dose selection was based on a maximum effect model using magnetic resonance imaging proton density fat fraction (MRI-PDFF) data from the Phase 1b / 2a study. After protocol amendment 2, the randomization ratio was updated to 16:8:6:24:15, and randomization was limited to the 15 mg QW treatment group due to concerns that histological efficacy may not be optimal at this dose. Randomization was stratified by type 2 diabetes status and fibrosis stage (F2 vs. F3). Patients, investigators, and site personnel were blinded to treatment assignment but not to dosing frequency. Details of study drug administration and lifestyle counseling are provided (Glass et al., J Hepatol 2020;73:680-93; Pais et al., J Hepatol 2023;S0168-8278(23)00189-7). Follow-up biopsies were performed at week 24. Biopsies were initially reviewed by a single central pathologist. In response to advances in consensus reading methods in NASH clinical trials (Sanyal et al., Hepatology 2021;74:968A; Sanyal et al., AASLD The Liver Meeting 2022:5008 (Abstract)), a central three-panel consensus scoring method replaced the original biopsy adjudication approach. Biopsies were evaluated by three expert liver pathologists blinded to patient, treatment, and sequence and scored using the NAS and CRN fibrosis staging systems (Kleiner et al., Hepatology 2005;41:1313-21). A consensus score was derived from the scores of individual readers using an algorithm designed to minimize interreader interaction. Baseline biopsies, initially evaluated by a single pathologist, were reread by the panel. Protocol-specified reasons for study withdrawal, discontinuation, or interruption were provided.

[0501] <endpoint> The two primary endpoints assessed at week 24 versus baseline were the proportion of patients with a ≥1-stage improvement in fibrosis without worsening NASH (increase in either ballooning, inflammation, or steatosis) and the proportion of patients with NASH resolution (complete absence of ballooning and absence or mild inflammation) without worsening fibrosis (increase in ≥1 stage). Key secondary endpoints included the proportion of patients with a ≥2-point improvement in NAS and no worsening of fibrosis. Other secondary endpoints included changes from baseline to week 24 in liver parameters (MRI-PDFF, liver chemistry tests, and N-terminal type III collagen propeptide [Pro-C3]) and metabolic parameters (adiponectin, serum triglycerides, high-density lipoprotein cholesterol [HDL-C], non-HDL-C, low-density lipoprotein cholesterol [LDL-C], and glycated hemoglobin [HbA1c]). Safety endpoints included the frequency and severity of adverse events (AEs) classified according to the Medical Dictionary for Regulatory Activities version 23.0. Further safety assessments included safety laboratory parameters, vital signs, electrocardiograms, and dual energy x-ray absorptiometry (DXA) scans.

[0502] <Statistical analysis> Based on the assumption of a placebo response and a 15% dropout rate, a sample size of approximately 184 patients was planned to provide 83–94% power to detect a 30% treatment difference in the two primary endpoints. To match the intended target population defined by regulatory authorities, the prespecified primary efficacy analysis included all patients with baseline F2 / F3 fibrosis and NAS ≥4 who received ≥1 dose of study treatment (full analysis set [FAS]). For the primary and key secondary endpoints, results were analyzed for the three randomized but untreated patients with F2 / F3 fibrosis and NAS ≥4, as well as all randomized patients. Safety analyses included all patients who received ≥1 dose of study treatment. The placebo group was pooled in all analyses.

[0503] Multiple imputation and stratified Cochran-Mantel-Haenszel methods were used to analyze the primary and key secondary endpoints. Sensitivity analyses were performed to assess the robustness of the primary analysis results. A series of efficacy endpoints were analyzed using mixed-model repeated-measures analysis. There was no prespecified plan to adjust for multiple comparisons. For the first primary endpoint (improvement of fibrosis), comparisons of the 30 mg and 44 mg dose groups with placebo are reported with P values ​​at a two-sided 0.05 significance level. All other results are reported with 95% confidence intervals (CIs) only. The 95% CI widths were not adjusted for multiplicity and should not be used to infer definitive treatment effects. Statistical analyses were performed using SAS software, version 9.4.

[0504] <Result> <patient> 222 patients were randomized, of whom 219 received study treatment and comprised the safety analysis set. Demographic and disease characteristics are shown in Table 7.

[0505] [Table 13-1] [Table 13-2]

[0506] Most patients were Caucasian, with a minority of African Americans. Mean baseline body mass index and FibroScan vibration-controlled transient elastography (VCTE) scores were somewhat higher in the placebo group than in the pegosafermin group. Of the 222 randomized patients, 27 were initially assessed by a single reader as having F2 / F3 fibrosis and NAS ≥ 4 but were later assessed by a three-panel reader as not meeting the study's histological inclusion criteria. These patients, as well as three patients who were randomized but not treated, were excluded from the FAS. Thus, 192 patients were included in the FAS. Full agreement or mode determined 91–99% of the final biopsy scores.

[0507] <Effectiveness> Baseline and week 24 biopsy results were available for 164 patients, and results were imputed to the FAS for the remaining 28 patients. After 24 weeks, the proportion of patients achieving ≥1 stage improvement in fibrosis without worsening of NASH was significantly higher with pegosafermin 30 mg QW (26%; difference [95% Cl] 18.9 [5.2-32.5]; P = 0.009) and 44 mg Q2W (27%; difference 20.3 [5.3-35.4]; P = 0.008) than with placebo (7%) (Figure 96A). For pegosafermin 15 mg QW, the proportion was 22% (difference 14.5 [-8.7-37.6]). The proportion of patients with NASH resolution without worsening of fibrosis was also superior for pegosafermin 30 mg QW (23%; difference 20.9 [9.1-32.7]) and 44 mg Q2W (26%; difference 23.6 [10.0-37.2]) versus placebo (2%) (Figure 96B). For pegosafermin 15 mg QW, the proportion was 37% (difference 34.7 [10.0-59.3]). Results were consistent in prespecified sensitivity analyses (imputation analysis and imputation of missing biopsies as non-response) and in analyses of all randomized patients with F2 / F3 fibrosis and NAS ≥ 4 who did not receive treatment, in addition to the FAS. Post-hoc analysis showed that 89% of patients treated with pegosafermin who achieved the primary fibrosis endpoint had an NAS improvement of ≥ 2 points. In post-hoc analysis, positive results for fibrosis regression in patients with F4 fibrosis were observed.

[0508] Analysis of key secondary endpoints generally supported the primary endpoint results: the proportion of patients with a ≥2-point NAS improvement and no worsening of fibrosis was 37% for pegosafermin 15 mg QW, 65% for pegosafermin 30 mg QW, 62% for pegosafermin 44 mg Q2W, and 24% for placebo.

[0509] At week 24, the least-squares mean percent change from baseline in liver fat fraction (MRI-PDFF) was -27%, -48%, and -42% for pegosafermin 15 mg QW, 30 mg QW, and 44 mg Q2W, respectively, compared with -5% for placebo (Tables 2 and S7). A ≥50% reduction in liver fat from baseline occurred in 63% and 58% of patients receiving pegosafermin 30 mg QW and 44 mg Q2W, respectively, compared with 12% of patients receiving placebo.

[0510] Twenty-four weeks of pegosafermin treatment was associated with reductions in liver chemistry tests (Table 8).

[0511] [Table 14-1] [Table 14-2]

[0512] Alanine aminotransferase (ALT) normalization occurred in 59% of patients in the pegosafermin 30 mg QW group and 65% of patients in the 44 mg Q2W group (ALT <30 U / L at end of study in patients with baseline ALT ≥ 30 U / L) compared with 24% in placebo (post-hoc). The results suggested improvement in corrected T1, which assesses fibroinflammation, and fibrosis markers including the Enhanced Liver Fibrosis (ELF) test score, Fibroscan VCTE, Fibroscan-aspartate transaminase (FAST) score, Pro-C3, and Fibrosis-4 (FIB-4) index score, as well as reductions in liver and spleen volumes (Table 8).

[0513] Results also suggested that pegosafermin treatment was associated with decreased serum triglycerides and increased HDL-C at pegosafermin 30 mg QW compared to placebo (Table 8), and increased adiponectin levels in all pegosafermin treatment groups compared to placebo. HbA1c, LDL-C, and HDL-C results are shown in Table 8. No clear effect on body weight was observed.

[0514] <Safety> Treatment-emergent AEs were reported in 95%, 85%, and 67% of patients with pegosafermin 15 mg QW, 30 mg QW, and 44 mg Q2W, respectively, compared with 68% with placebo (Table 9).

[0515] [Table 15-1] [Table 15-2]

[0516] The most frequent AEs were nausea, diarrhea, and injection site erythema. Grade 3 serious adverse events were reported in 10% of patients in the pegosafermin 15 mg QW group, 4% in the 30 mg QW group, and 9% in the 44 mg Q2W group, compared with 9% in the placebo group. No adverse events of severity grade >3 or deaths were reported. Serious AEs were reported in 5%, 4%, and 11% of patients in the pegosafermin 15 mg QW, 30 mg QW, and 44 mg Q2W groups, respectively, compared with 4% in the placebo group. The only serious adverse event determined by the investigator to be related to study treatment was acute pancreatitis, which occurred in a patient receiving a single dose of pegosafermin 44 mg and whose imaging revealed gallbladder sludge. The clinical course was typical of uncomplicated acute pancreatitis.

[0517] Drug-related AEs leading to treatment discontinuation were reported in 5% (diarrhea, N=1), 6% (diarrhea, N=2; nausea, N=1; injection site erythema, N=1), and 2% (pancreatitis, N=1) of patients with pegosafermin 15 mg QW, 30 mg QW, and 44 mg Q2W, respectively, compared with 0% with placebo.

[0518] No consistent patterns were observed for safety-related laboratory parameters, and there were no clinically relevant findings for vital signs or electrocardiograms. No clinically relevant changes were observed for insulin-like growth factor 1, thyroid-stimulating hormone, or bone biomarkers. No adverse changes were observed on DXA scans after 24 weeks, and all traumatic fractures were reported in 3 / 69 patients in the placebo group and 1 / 150 patients in the pegosafermin group. No incidences of possible drug-induced liver injury or tremors were reported.

[0519] <Consideration> In this study, treatment with the FGF21 analog pegosafermin at 30 mg QW and 44 mg Q2W for 24 weeks resulted in significant improvement in fibrosis compared to placebo, without worsening of NASH. Results also supported a benefit in resolving NASH without worsening fibrosis. Fibrosis progression is an important predictor of clinical outcomes in NASH, including liver-related and all-cause mortality (Dulai et al., Hepatology 2017;65:1557-65). The improvement of both steatohepatitis and fibrosis indicates that pegosafermin may affect important aspects of the pathophysiology of NASH.

[0520] Significant variability in biopsy interpretation may contribute to the large variability in placebo response rates in NASH clinical trials (Ng et al., Hepatology 2022;75:1647-61). In this study, we used an objective consensus biopsy interpretation methodology, in which consensus scores based on individual scores submitted by three expert pathologists were determined by a prespecified algorithm rather than by consensus discussion or the use of an adjudicator. The low placebo rate for the primary endpoint in our study may reflect the high accuracy of our biopsy interpretation method, which allowed for a more accurate estimation of the actual rate of spontaneous regression of NASH (i.e., placebo response). Using this method may also result in a lower response rate in the pegosafermin treatment group.

[0521] Confirmation of biopsy findings by noninvasive testing increases the reliability of histological findings. No formal hypothesis testing was performed on secondary and exploratory outcomes. However, the results suggest that pegosafermin is associated with improvements in liver fat and noninvasive markers of liver injury, inflammation, and fibrosis, including corrected T1, which assesses fibroinflammation, and fibrosis markers ELF, Fibroscan VCTE, FAST, Pro-C3, and FIB-4.

[0522] NASH is highly associated with metabolic syndrome, as metabolic abnormalities contribute to the progression of NASH and increase cardiovascular risk, which is an important cause of morbidity and mortality in this population. 3,4 NASH drugs would ideally ameliorate metabolic comorbidities, but this has not been achieved with several classes of therapeutic agents under development for NASH (Harrison et al., Gastroenterology 2021;160:219-31.e1; Neuschwander-Tetri et al., Lancet 2015;385:956-65; Pockros et al., Liver Int 2019;39:2082-93; Younossi et al., Lancet 2019;394:2184-96; Kim et al., Cell Metab 2017;26:394-406.e6; Francque et al., N Engl J Med 2021;385:1547-58). Consistent with findings from previous studies, the results of the present study suggest that pegosafermin may have positive effects on adiponectin, serum triglycerides, and HDL-C.

[0523] Nausea and diarrhea were the most common AEs with pegosafermin. One serious adverse event, acute pancreatitis, was determined by the investigator to be related. Effects on bone turnover have been reported in preclinical studies with FGF21 analogs and in early clinical trials with two FGF21 analogs (Rader et al., J Clin Endocrinol Metab 2022;107:e57-e70; Talukdar et al., Cell Metab 2016;23:427-40; Kim et al., Diabetes Obes Metab 2017;19:1762-72). There was no evidence of bone mineral density loss or fractures in this study, but longer-term studies are needed to fully evaluate this potential risk. No hepatotoxicity was observed.

[0524] One limitation of this study was its short duration; a further 24-week single-blind extension study will provide data on longer-term safety and noninvasive biomarker assessments. Another limitation was the lack of diversity in the patients, most of whom were Caucasian, potentially limiting the generalizability of the data.

[0525] In conclusion, 24 weeks of pegosafermin treatment resulted in improvement of fibrosis in patients with biopsy-confirmed NASH, with both weekly and biweekly dosing. The availability of biweekly dosing may improve patient convenience and compliance. The results of the current study will be useful for guiding dose selection for larger, longer-term phase 3 trials in NASH.

[0526] Example 12 In this study, subjects were treated with GLP-1 standard of care therapies (such as semaglutide, dulaglutide, and liraglutide) before initiating treatment with a mutant fibroblast growth factor-21 (FGF-21) peptide conjugate administered at 44 mg every other week (Q2W) or 30 mg weekly (QW).

[0527] Figure 97 shows that in patients receiving background GLP-1 therapy, significant reductions in key markers of liver fibrosis (ELF score, VCTE), liver damage (ALT), glycemic index (HbA1c), and liver fat content (MRI-PDFF) were observed when pegosafermin was added compared to placebo. Results were consistent in both groups, so pooled results include the 30 mg and 44 mg groups.

[0528] Tolerability was acceptable, with nausea being the most common adverse event. There were no treatment-related discontinuations.

[0529] Specific examples of methods and kits are described herein for illustrative purposes. These are examples only. The techniques provided herein can be applied to systems other than the exemplary systems described above. Many changes, modifications, additions, omissions, and substitutions are possible within the scope of the present disclosure. The present disclosure includes variations on 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 of the embodiments described herein with features, elements, and / or acts of other technologies, and / or omitting to combine features, elements, and / or acts of the described embodiments.

[0530] The disclosed embodiments described above are for purposes of example only, and those skilled in the art will appreciate that various modifications of detail may be made to these embodiments, all of which fall within the scope of the present disclosure.

[0531] All publications mentioned herein are incorporated herein by reference in their entirety. Although the foregoing disclosure has been described in some detail for purposes of clarity and understanding, those skilled in the art will understand upon reading this disclosure that various changes in form and detail can be made without departing from the true scope of the disclosure in the appended claims.

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Claims

1. 1. A method of treating non-alcoholic steatohepatitis (NASH) in a subject in need thereof, comprising: administering to a subject in need thereof once weekly a pharmaceutical composition comprising 15 mg to 30 mg of a mutant fibroblast growth factor-21 (FGF-21) peptide conjugate and a pharmaceutically acceptable carrier; The mutant FGF-21 peptide conjugates are i) a mutant FGF-21 peptide comprising the amino acid sequence of SEQ ID NO: 2; ii) a glycosyl moiety; and iii) 20 kDa polyethylene glycol (PEG); It contains the variant FGF-21 peptide is linked to a 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 linked to a 20 kDa PEG by a covalent bond between a second site of the glycosyl moiety and the 20 kDa PEG; The administration of the pharmaceutical composition comprises: reduction of liver fat, Improvement in liver fibrosis score, Resolution of NASH, a ≥ 2 point improvement in NAFLD activity score; Improved VCTE score, Improvement in FAST scores, Improvement in FIB-4 score, Magnetic resonance imaging - decreased liver size assessed by proton density fat fraction; a reduction in the levels of one or more biomarkers including Pro-C3, alanine transaminase (ALT), enhanced liver fibrosis (ELF) panel, CK-18, the inflammatory marker high-sensitivity C-reactive protein (hs-CRP), hemoglobin A1c (HbA1c), non-HDL-c, LDL-c, and triglycerides; and Increased levels of HDL-c and / or adiponectin, The method of claim 1, wherein the method results in at least one of the following:

2. The method of claim 1 , wherein the subject is a human.

3. 10. The method of claim 1, wherein the pharmaceutical composition is administered subcutaneously.

4. 2. The method of claim 1, wherein the glycosyl moiety 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.

5. 2. The method of claim 1, wherein the glycosyl moiety 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.

6. 6. The method of claim 5, wherein at least one Sia residue is a 9-carbon carboxylated sugar.

7. 7. The method of claim 6, wherein the at least one Sia residue is N-acetyl-neuraminic acid (2-keto-5-acetamido-3,5-dideoxy-D-glycero-D-galactonunulopyranos-1-onic acid (Neu5Ac), N-glycolylneuraminic acid (Neu5Gc), 2-keto-3-deoxy-nonulosonic acid (KDN), or a 9-substituted sialic acid.

8. 8. The method of claim 7, 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.

9. The method of claim 1, wherein the glycosyl moiety comprises the structure -GalNAc-Sia-.

10. 2. The method of claim 1, wherein the 20 kDa PEG moiety is attached to the glycosyl moiety by a covalent bond to a linker, the linker comprising at least one amino acid residue.

11. 11. The method of claim 10, wherein at least one amino acid residue is glycine (Gly).

12. 2. The method of claim 1, wherein the mutant FGF-21 peptide conjugate comprises the structure -GalNAc-Sia-Gly-PEG(20 kDa).

13. The mutant FGF-21 peptide conjugate comprises the following structure: 【Chemical Formula 21】 2. The method of claim 1, wherein n is an integer selected from 450 to 460.

14. The method of claim 1 , wherein the 20 kDa PEG is a linear or branched PEG.

15. 2. The method of claim 1, wherein the 20 kDa PEG is a 20 kDa methoxy-PEG.

16. 2. The method of claim 1, wherein the mutant FGF-21 peptide conjugate exhibits equal or greater potency than wild-type FGF-21 when tested in vitro in KLB-FGFR1, KLB-FGFR2, and KLB-FGFR3 expressing cells.

17. 10. The method of claim 1, comprising administering to a subject in need thereof once weekly a pharmaceutical composition comprising about 25 to about 30 mg of the mutant FGF-21 peptide conjugate.

18. 10. The method of claim 1, comprising administering to a subject in need thereof once weekly a pharmaceutical composition comprising 30 mg of the mutant FGF-21 peptide conjugate.

19. 1. A method of treating non-alcoholic steatohepatitis (NASH) in a subject in need thereof, comprising: administering to a subject in need thereof, once every two weeks, a pharmaceutical composition comprising 18 mg to 44 mg of a mutant fibroblast growth factor-21 (FGF-21) peptide conjugate and a pharmaceutically acceptable carrier; The mutant FGF-21 peptide conjugates are i) a mutant FGF-21 peptide comprising the amino acid sequence of SEQ ID NO: 2; ii) a glycosyl moiety; and iii) 20 kDa polyethylene glycol (PEG); It contains the variant FGF-21 peptide is linked to a 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 linked to a 20 kDa PEG by a covalent bond between a second site of the glycosyl moiety and the 20 kDa PEG; The administration of the pharmaceutical composition comprises: reduction of liver fat, Improvement in liver fibrosis score, Resolution of NASH, a ≥ 2 point improvement in NAFLD activity score; Improved VCTE score, Improvement in FAST scores, Improvement in FIB-4 score, Magnetic resonance imaging - decreased liver size assessed by proton density fat fraction; a reduction in the levels of one or more biomarkers including Pro-C3, alanine transaminase (ALT), enhanced liver fibrosis (ELF) panel, CK-18, the inflammatory marker high-sensitivity C-reactive protein (hs-CRP), hemoglobin A1c (HbA1c), triglycerides, non-HDL-c, and LDL-c; and Increased levels of HDL-c and / or adiponectin, The method of claim 1, wherein the method results in at least one of the following:

20. 20. The method of claim 19, wherein the subject is a human.

21. 20. The method of claim 19, wherein the pharmaceutical composition is administered subcutaneously.

22. 20. The method of claim 19, wherein the glycosyl moiety 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.

23. 20. The method of claim 19, wherein the glycosyl moiety 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.

24. 24. The method of claim 23, wherein at least one Sia residue is a 9-carbon carboxylated sugar.

25. 25. The method of claim 24, wherein the at least one Sia residue is N-acetyl-neuraminic acid (2-keto-5-acetamido-3,5-dideoxy-D-glycero-D-galactonunulopyranos-1-onic acid (Neu5Ac), N-glycolylneuraminic acid (Neu5Gc), 2-keto-3-deoxy-nonulosonic acid (KDN), or a 9-substituted sialic acid.

26. 26. The method of claim 25, 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.

27. 20. The method of claim 19, wherein the glycosyl moiety comprises the structure -GalNAc-Sia-.

28. 20. The method of claim 19, wherein the 20 kDa PEG moiety is attached to the glycosyl moiety by a covalent bond to a linker, the linker comprising at least one amino acid residue.

29. 29. The method of claim 28, wherein at least one amino acid residue is glycine (Gly).

30. 20. The method of claim 19, wherein the mutant FGF-21 peptide conjugate comprises the structure -GalNAc-Sia-Gly-PEG(20 kDa).

31. The mutant FGF-21 peptide conjugate comprises the following structure: 【Chemical 22】 20. The method of claim 19, wherein n is an integer selected from 450 to 460.

32. 20. The method of claim 19, wherein the 20 kDa PEG is a linear or branched PEG.

33. 20. The method of claim 19, wherein the 20 kDa PEG is a 20 kDa methoxy-PEG.

34. 20. The method of claim 19, wherein the mutant FGF-21 peptide conjugate exhibits equal or greater potency than wild-type FGF-21 when tested in vitro in KLB-FGFR1, KLB-FGFR2 and KLB-FGFR3 expressing cells.

20. The method of claim 19, comprising administering to a subject in need thereof once a week a pharmaceutical composition comprising about 40 to about 50 mg of the mutant FGF-21 peptide conjugate.

35. 20. The method of claim 19, comprising administering to a subject in need thereof once weekly a pharmaceutical composition comprising 44 mg of the mutant FGF-21 peptide conjugate.

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