Fusion compounds and their uses

Abstract

This invention relates to a fusion protein comprising a GLP-1 polypeptide and an FGF21 polypeptide separated by a spacer. The invention also relates to a fusion compound comprising the fusion protein and at least one substituent. Furthermore, the invention relates to pharmaceutical applications of the fusion compound.

Description

Technical Field

[0001] The present invention relates to a fusion compound comprising a GLP-1 polypeptide and an FGF21 polypeptide. Further, the present invention relates to a pharmaceutical composition comprising such a fusion compound.

[0002] Sequence Listing This application is filed together with an electronic form of the sequence listing. The entire content of the sequence listing is incorporated herein by reference.

Background Art

[0003] Fusion proteins can be produced by genetically engineering the joining of two or more genes that originally encoded separate proteins. As a result, a single polypeptide having the functional properties of both parental proteins, i.e., a bifunctional fusion protein, is obtained. Combinations of unrelated proteins can be difficult because it can be demonstrated that production is difficult due to incompatible properties. This can, for example, cause aggregation or misfolding in one domain, while the conditions are perfect for the other domain. Further, two or more proteins (or polypeptides) fused together can have conflicting stability requirements that make formulation difficult. Further, it can be difficult to control and adjust the relative amounts of each component, and thus administration becomes complicated for optimal effectiveness and safety. In addition, the different polypeptide portions in a fusion protein can affect each other such that the biological activity is reduced, which is undesirable. Direct fusion of polypeptide portions without a linker can result in many undesirable consequences, including misfolding of the fusion protein, low yields of protein production, or impairment of biological activity.

[0004] GLP-1 is an incretin hormone produced by intestinal endocrine cells after food intake. GLP-1 is a regulator of glucose metabolism, controlling insulin secretion from the beta cells of the islets of Langerhans in the pancreas. GLP-1 also triggers insulin secretion in diabetic conditions. Furthermore, through its ability to enhance satiety, GLP-1 can reduce food intake, thereby limiting weight gain and potentially even causing weight loss. In summary, these actions confer a unique profile to GLP-1, particularly its glucose-dependent anti-hyperglycemic effect, which is considered highly desirable for antidiabetic drugs due to the need to minimize the risk of severe hypoglycemia. However, its pharmacokinetic / pharmacodynamic profile makes natural GLP-1 less therapeutically useful. GLP-1 is highly sensitive to enzymatic degradation in vivo, and cleavage by dipeptidyl peptidase IV (DPP-IV) is perhaps the most relevant, as this occurs rapidly and produces non-insulin-secreting metabolites. Therefore, methods to extend the half-life of GLP-1 in vivo have attracted considerable attention. Various different approaches have been used to modify the structure of glucagon-like peptide 1 (GLP-1) compounds to provide a longer duration of action in vivo, which has led to the development of once-weekly dosing products such as albiglutide, dulaglutide, and semaglutide.

[0005] FGF21 belongs to the FGF19 subfamily of metabolic atypical fibroblast growth factors (FGFs), rather than those with mitotic effects. FGF21 binds to and activates FGF receptors (FGFR1c, FGFR2c, and FGFR3c), but only in the presence of the non-signaling coreceptor beta-cloto (BKL). Tissue-specific expression of BKL determines the metabolic activity of FGF21. FGF21 transgenic mice are resistant to diet-induced obesity and have an extended lifespan. FGF21 is a metabolic regulator of energy expenditure, glucose, and lipid metabolism. FGF21 may improve body weight, hyperglycemia, and dyslipidemia in obese patients with diabetes and dyslipidemia.

[0006] FGF21 suffers from in vivo instability due to proteolytic degradation, and half of endogenous circulating human FGF21 is inactive. The loss of activity is due to C-terminal degradation, and most of these metabolites terminate at P171 rather than S181. Therefore, protection against metabolic degradation of the C-terminal region is desirable for therapeutic FGF21 molecules. Various methods have been reported to extend the in vivo half-life of recombinant FGF21 proteins. For example, one example is PEGylation. However, PEGylation at position 179 of [-1M, 179C]FGF21 results in a dramatic decrease in in vitro activity (J. Xu et al., Bioconjugate Chemistry (2013), 24, 915-925). Fc fusion technology has also been used. However, Fc fusion proteins obtained by attaching Fc to the C-terminus of FGF21 are far less active than natural FGF21 and FGF21 with N-terminal Fc fusion (Hecht et al., PLoS One 2012, 7(11), e49345). The Fc moiety has a molecular weight of approximately 50 kDa. Therefore, incorporating Fc into a fusion protein increases its molecular weight by at least 50 kDa. However, this increase in molecular weight is often undesirable, for example, by increasing viscosity, which makes the corresponding pharmaceutical formulation more difficult. Furthermore, the incorporation of the Fc domain may cause steric hindrance between polypeptide moieties, which can lead to decreased biological activity, as well as changes in the in vivo distribution and metabolism of the protein moiety due to interference between domains.

[0007] Co-administration of GLP-1 and FGF21 has been reported (see International Publication 2010 / 142665). Co-administration of FGF21 protein and GLP-1 compounds requires either injection of two separate products or a single injection of a co-formulation of two different compositions. Two injections allow for flexibility in dosage and timing, but are inconvenient for patients in terms of both compliance and pain. Co-formulations may also offer some flexibility in dosage, but due to the different molecular properties of the two different products, finding formulation conditions that allow for the chemical and physical stability of both compositions is often very difficult or impossible.

[0008] Therefore, there is a need for fusion compounds containing GLP-1 polypeptides and FGF21 polypeptides that have improved bioavailability, extended half-life, and / or increased potency. [Overview of the Initiative] [Problems that the invention aims to solve]

[0009] The present invention relates to a fusion compound comprising a GLP-1 polypeptide and an FGF21 polypeptide, wherein the GLP-1 polypeptide and the FGF21 polypeptide are separated by a spacer. [Means for solving the problem]

[0010] In the first aspect, the fusion compound comprises a GLP-1 polypeptide, an analog of SEQ ID NO: 1, and an FGF21 polypeptide, an analog of FGF21(1-181) of SEQ ID NO: 2, wherein the GLP-1 polypeptide and the FGF21 polypeptide are separated by a spacer containing 1 to 257 amino acids. The fusion compound may contain substituents. In the second aspect, the present invention relates to a pharmaceutical composition comprising a fusion protein or fusion compound. In the third aspect, the present invention relates to the use of a fusion protein or fusion compound for medical use. In the fourth aspect, the present invention relates to the medical use of a fusion protein or fusion compound, for example, in the treatment of obesity and / or to improve lipid parameters and / or non-alcoholic fatty liver disease (NAFLD), such as non-alcoholic steatohepatitis (NASH). In the fifth aspect, the present invention relates to a method for preparing a fusion protein or fusion compound. [Brief explanation of the drawing]

[0011] [Figure 1] Figure 1 shows the cumulative food intake (g) after a single intravenous administration of a vehicle or active compound in lean mice. [Figure 2] Figure 2 shows baseline-adjusted body weight data in LDLr- / - mice treated for 21 days with chemical formulas 24, 25, 14, 16, 19, 12, and 23. Data: mean + / - SEM (n=9~10 / group) [Modes for carrying out the invention]

[0012] In the following, as used herein, "a" may mean "one or more" or "at least one." Greek letters may also be represented by their symbols or corresponding names, for example, α = alpha, β = beta, ε = epsilon, γ = gamma, ω = omega, and so on. The Greek letter μ may also be represented by "u," for example, μl = ul, μM = uM.

[0013] Asterisk in chemical formula ( * ) indicates a bonding point. Hereafter, unless otherwise indicated herein, terms presented in the singular form include plural contexts; for example, when referring to a "fusion compound," it should be understood that it encompasses all individual variants included within the broader definition of that fusion compound.

[0014] As used herein, the terms “about” or “approximately,” when used with a number (e.g., 5, 10%, 1 / 3), refer to a range of numbers that may be smaller or larger than that number. For example, “about 5” refers to a range of numbers that are 10%, 5%, 2%, or 1% smaller or larger than 5, e.g., 4.5–5.5, or 4.75–5.25, or 4.9–5.1, or 4.95–5.05. In some examples, “about 5” refers to a range of numbers that are 2% or 1% smaller or larger than 5, e.g., 4.9–5.1 or 4.95–5.05. In some embodiments, as used herein, the term “about” means ±10% of the value being referred to, including that value.

[0015] Unless otherwise indicated by the context, all ranges described herein should be interpreted as including their endpoints, and unrestricted ranges should be interpreted as including only commercially useful values. Similarly, unless otherwise specified by the context, all lists of values ​​should be considered to include intermediate values.

[0016] In the first aspect, the present invention relates to chemical formula 1: ABC (wherein, A is the GLP-1 polypeptide, which is an analog of Sequence ID No. 1. B is a spacer consisting of 1 to 257 amino acids. C is sequence number 2 This relates to a fusion protein containing the polypeptide of FGF21, which is an analog of FGF21, or to a pharmaceutically acceptable salt, amide, or ester of FGF21.

[0017] Similarly, or alternatively, the present invention, Chemical formula 1: ABC (wherein, A is the GLP-1 polypeptide, which is an analog of Sequence ID No. 1. B is a spacer consisting of 1 to 257 amino acids. C is the FGF21 polypeptide, an analog of Sequence ID No. 2. This relates to fusion compounds containing a substituent, or pharmaceutically acceptable salts, amides, or esters thereof.

[0018] Similarly, or alternatively, the present invention relates to fusion compounds that can activate the human GLP-1 receptor and / or the FGFR complex.

[0019] In a second aspect, the present invention relates to a pharmaceutical composition comprising a fusion protein or fusion compound (i.e., a fusion protein or fusion compound as defined in the first aspect of the present invention, including all embodiments and their specific features), and optionally one or more pharmaceutically acceptable excipients.

[0020] In a third aspect, the present invention relates to a fusion protein or fusion compound (i.e., a fusion protein or fusion compound as defined in the first aspect of the present invention, including all embodiments and their specific features) or a pharmaceutical composition (i.e., a composition as defined in the third aspect of the present invention, including all embodiments and their specific features) for use as a pharmaceutical or for use in medical applications.

[0021] In a fourth aspect, the present invention relates to a fusion protein or fusion compound (i.e., a fusion protein or fusion compound as defined in the first aspect of the present invention, including all embodiments and their specific features) or a pharmaceutical composition (i.e., a composition as defined in the third aspect of the present invention, including all embodiments and their specific features), (i) prevention and / or treatment of any form of diabetes, (ii) delaying or preventing the progression of diabetic disease and / or delaying the progression from insulin-free type 2 diabetes to insulin-requiring type 2 diabetes, (iii) improving β-cell function, (iv) prevention and / or treatment of cognitive impairment and / or neurodegenerative disorders, (v) prevention and / or treatment of eating disorders and / or prevention and / or treatment of comorbidities of obesity, (vi) prevention and / or treatment of diabetic complications, (vii) improvement of lipid parameters, (viii) prevention and / or treatment of cardiovascular disease, and (ix) prevention and / or treatment of gastrointestinal disorders, (x) prevention and / or treatment of serious illnesses, prevention or reduction of the likelihood of patients having bacteremia, sepsis and / or septic shock in hospital, and / or stabilization of blood glucose, insulin balance and, if applicable, metabolism in intensive care unit patients with acute illness, (xi) prevention and / or treatment of polycystic ovary syndrome (PCOS), (xii) prevention and / or treatment of cerebral disorders, such as cerebral hemorrhage including cerebral ischemia, and / or traumatic brain injury, (xiii) prevention and / or treatment of sleep apnea syndrome, (xiv) prevention and / or treatment of abuse, such as alcohol abuse and / or drug abuse, (xv) prevention and / or treatment of dyslipidemia, (xv) treatment and / or prevention of hepatic steatosis, and / or (xvi) use in non-alcoholic fatty liver disease (NAFLD) and / or acute and chronic pancreatitis.

[0022] In a fifth aspect, the present invention relates to a method for preparing a fusion protein or fusion compound (i.e., a fusion protein or fusion compound as defined in the first aspect of the present invention, including all embodiments and their specific features).

[0023] General definition As used herein, the term “compound” refers to a molecular entity, and therefore a “compound” may have different structural elements other than the minimum elements defined for each compound or group of compounds. Fusion compounds may be referred to as “compounds.” The term “compound” is also intended to include pharmaceutically appropriate forms thereof, i.e., the compounds as defined herein, or their pharmaceutically acceptable salts, amides, and esters.

[0024] As used herein, the terms “polypeptide” or “polypeptide sequence” refer to a compound comprising a series of two or more amino acids interconnected via amide (or peptide) bonds. The term polypeptide is used interchangeably with the terms “peptide” and “protein.”

[0025] The term "part" refers to a fragment or portion of a molecule, such as a fusion compound or fusion protein.

[0026] The term "polypeptide moiety" refers to a polypeptide fragment, or a part of a molecule such as a fusion compound or fusion protein. For example, "GLP-1 polypeptide moiety" refers to A in chemical formula 1, and "FGF21 polypeptide moiety" refers to C in chemical formula 1. For example, the term "spacer moiety" refers to B in chemical formula 1. In other words, the term "GLP-1 polypeptide moiety" refers to a "GLP-1 polypeptide fragment" or a part of chemical formula 1.

[0027] As used herein, the term “analog” generally refers to a polypeptide having one or more amino acid changes in its sequence compared to a reference amino acid sequence. Such amino acid changes may include amino acid additions, amino acid deletions, and / or amino acid substitutions. Amino acid “substitutions” may also be called “mutations.” In certain embodiments, an analog “contains” a specified change. In other particular embodiments, an analog “consists of” a particular change or “has” a change. When the terms “comprises” or “comprising” are used in reference to amino acid changes in an analog, the analog may, of course, have further amino acid changes compared to its reference sequence. When the terms “consists of” or “has” are used in reference to amino acid changes in an analog, of course, a specified amino acid mutation is the only amino acid change in the analog compared to the reference sequence. In the context of this application, the term “analog” refers to analogs of human glucagon-like peptide-1 GLP-1(7-37) (SEQ ID NO: 1), analogs of human endogenous FGF21 (FGF21(1-181) (SEQ ID NO: 2)), and / or analogs of fusion compounds.

[0028] The term “derivative” generally refers to a polypeptide that may be prepared from a natural polypeptide or its analogues by chemical modification, particularly by the covalent bonding of one or more substituents. Derivatives may also be called alkylated analogues. For example, the fusion compounds defined herein are derivatives of fusion proteins derived herein.

[0029] As used herein, the term “amino acid” refers to any amino acid, i.e., both proteogenic and non-proteogenic amino acids. As used herein, the term “proteogenic amino acid” refers to the 20 standard amino acids encoded by the human genetic code. As used herein, the term “non-proteogenic amino acid” refers to all amino acids that are not proteogenic. Generally, amino acid residues, as used herein, may be identified, for example, in relation to polypeptide sequences, by their full names, their one-letter codes, and / or their three-letter codes. These three methods are entirely equivalent and interchangeable. Hereafter, each amino acid in the peptides of the present invention for which optical isomers are not described should be understood to mean the L-isomer (unless otherwise specified).

[0030] As used herein, the terms “fused” and “fused” refer to a compound comprising two or more individually defined polypeptides covalently linked by peptide bonds or by spacers. As used herein, the term “spacer” refers to a molecular portion separating two individually defined polypeptides.

[0031] The term "bifunctional" means being active in two ways. For example, in a bifunctional fusion compound, both polypeptides of a bifunctional fusion protein, such as GLP-1 and FGF21, are active.

[0032] As used herein, the term “sequence identity” refers to the degree to which two amino acid sequences (e.g., polypeptides) have the same residues at the same positions in alignment. This may also be simply called “identity.” Sequence identity is conveniently expressed as a percentage; i.e., if 85 of the 100 aligned positions between two sequences are identical, the degree of identity is 85%. For the purposes of this invention, sequence identity between two amino acid sequences is determined by simple handwriting and visual estimation, and / or by using a standard protein or peptide alignment program such as “Align” based on the Needleman-Wunsch algorithm. This algorithm is described in Needleman, S.B. and Wunsch, CD, (1970), Journal of Molecular Biology, 48:443-453, and in the alignment program by Myers and W. Miller, “Optimal Alignments in Linear Space,” CABIOS (computer applications in the biosciences) (1988) 4:11-17. For alignment, the default score matrix BLOSUM62 and the default identity matrix may be used, the penalty for the first residue in the gap may be set to -12, or preferably -10, and the penalty for additional residues in the gap may be set to -2, or preferably -0.

[0033] As used herein, the term “FGFR complex” refers to the FGF receptor β-cloto (FGFR-BKL) complex, such as FGFR1c, for example, FGFR3, for example, FGFR2.

[0034] GLP-1 polypeptide As used herein, the term “GLP-1 polypeptide” refers to an analog (or variant) of human glucagon-like peptide-1 (GLP-1(7-37)), whose sequence is listed in the sequence listing as SEQ ID NO: 1 HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG. The polypeptide of SEQ ID NO: 1 may also be referred to as “natural human GLP-1”. The GLP-1 polypeptide has one or more amino acid changes compared to natural human GLP-1, and such amino acid changes may be in the form of amino acid additions, amino acid deletions, and / or amino acid substitutions. The numbering of amino acid residues in the GLP-1 polypeptide of the fusion compound or fusion protein of the present invention follows established experience in the art for natural human GLP-1. That is, the first (N-terminal) amino acid residue is numbered 7, and the amino acid residues that follow downstream toward the C-terminus are numbered 8, 9, 10, and so on, up to the last (C-terminal) amino acid residue. In natural human GLP-1, the C-terminal amino acid residue is Gly, which has the number 37. Numbering is done differently in sequence listings, where the first amino acid residue (His) of SEQ ID NO: 1 is assigned number 1, and the last (Gly) is number 31. However, in this specification, we follow the numbering practices established in the art, as described above, namely, the first (N-terminal) amino acid residue is given number 7 of SEQ ID NO: 1. The GLP-1 polypeptide of the fusion compound or fusion protein of the present invention, i.e., the GLP-1 polypeptide portion of the fusion compound or fusion protein, can be described by referring to i) the nature of the actual change and ii) the position of the native amino acid residue being changed. Thus, an amino acid change in the form of substitution may be referred to as "Xaa", where aa is the amino acid introduced at the substitution site and X is the number corresponding to the position of the amino acid residue of SEQ ID NO: 1 being substituted.

[0035] The GLP-1 polypeptide portion of the fusion compound or fusion protein of the present invention may be referred to, for example, as [8G, 22E, 27C, 36G]GLP-1(7-37) below, with reference to the amino acid changes relative to, for example, GLP-1(7-37)(SEQ ID NO: 1). In this example, the GLP-1 polypeptide is an analog of GLP-1(7-37)(SEQ ID NO: 1), the analog has Gly at the position corresponding to position 8 of GLP-1(7-37), Glu at the position corresponding to position 22 of GLP-1(7-37), Cys at the position corresponding to position 27 of GLP-1(7-37), and Gly at the position corresponding to position 36 of GLP-1(1-37)(SEQ ID NO: 1). When amino acid changes in the GLP-1 polypeptide are shown in comparison to GLP-1(7-37) (SEQ ID NO: 1), it is understood that this refers to changes in the GLP-1 polypeptide moiety alone and does not include any other parts of the fusion compound or fusion protein, such as the spacer or FGF21 polypeptide moiety.

[0036] The term "GLP-1 polypeptide" is used interchangeably with "GLP-1 analogue" and "GLP-1 variant." A GLP-1 polypeptide "containing" a specific designated variant may contain further variants when compared to GLP-1(7-37)(SEQ ID NO: 1).

[0037] In some embodiments, the GLP-1 polypeptide portion of the fusion protein or fusion compound is an analog of SEQ ID NO: 1. In some embodiments, the GLP-1 polypeptide portion is at least 50%, preferably at least 55%, preferably at least 60%, preferably at least 65%, preferably at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90% identical to SEQ ID NO: 1. In some embodiments, the GLP-1 polypeptide portion has a maximum of 12 amino acid changes, preferably a maximum of 11 amino acid changes, preferably a maximum of 10 amino acid changes, preferably a maximum of 9 amino acid changes, preferably a maximum of 8 amino acid changes, preferably a maximum of 7 amino acid changes, preferably a maximum of 6 amino acid changes, preferably a maximum of 5 amino acid changes, preferably a maximum of 5 amino acid changes, preferably a maximum of 4 amino acids, or preferably a maximum of 3 amino acid changes when compared to SEQ ID NO: 1. In some embodiments, the GLP-1 polypeptide portion has 3 to 7 changes when compared to SEQ ID NO: 1.

[0038] In some embodiments, the GLP-1 polypeptide has the amino acid sequence H-Xaa8-E-G-T-F-T-S-D-V-S-S-Y-L-E-Xaa 22 -Q-A-A-Xaa 26 -Xaa 27 -F-I-A-W-L-V-K-G-Xaa 36 -G (SEQ ID NO: 3), where Xaa8 is G, Xaa 22 is E, Xaa 26 is R, C, or K, Xaa 27 is C or E, Xaa 36 is G or C.

[0039] In some embodiments, the GLP-1 polypeptide can undergo Cys alkylation.

[0040] Non-limiting examples of GLP-1 polypeptides are provided in Table 1. [Table 1]

[0041] FGF21 polypeptide The terms "human FGF21," "natural FGF21," "wild-type FGF21," "human endogenous FGF21," and "FGF21(1-181)" are used interchangeably, as indicated by Sequence ID No. 2. This refers to a polypeptide consisting of HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS. In this formula, the numbering of amino acid residues follows that of FGF21(1-181) (SEQ ID NO: 2), with the first (N-terminal) amino acid residue (H) being numbered as position number 1, and subsequent amino acid residues being numbered 2, 3, 4, etc., towards the C-terminus, continuing until the last (C-terminal) amino acid residue (S). In FGF21(1-181), this S residue corresponds to position number 181. In the sequence listing, the first amino acid residue (H) of sequence number 2 is assigned the number 1, and the last residue (S) is assigned the number 181; as a result, the numbering in this specification and the sequence listing are identical. The same applies to other FGF21 polypeptide sequences.

[0042] The term "FGF21 polypeptide" is used interchangeably with "FGF21 analogue" and "FGF21 variant."

[0043] As used herein, the term “FGF21 polypeptide” refers to a polypeptide capable of activating the human FGF21 receptor. In some embodiments, the FGF21 polypeptide portion of a fusion compound or fusion protein can activate the FGF21 receptor. As used herein, the term “FGF21 analog” refers to a polypeptide that is an analog of FGF21(1-181). In some embodiments, the FGF21 polypeptide portion of a fusion compound is an analog of FGF21(1-181). In some embodiments, the FGF21 polypeptide portion of a fusion compound is an analog of SEQ ID NO: 2, "[121Q, 168L, 180C]FGF21(1-181)". In this example, the FGF21 polypeptide is an analogue of FGF21(1-181), the analogue having Gln at position 121 corresponding to FGF21(1-181), Leu at position 168 corresponding to FGF21(1-181), and Cys at position 180 corresponding to FGF21(1-181). The addition of Ala at the N-terminus corresponds to position -1 of FGF21(1-181), and may therefore also be called "-1A" or "-1Ala". When amino acid changes in the FGF21 polypeptide are shown in comparison to FGF21(1-181), it is understood that this refers to changes to the FGF21 polypeptide moiety alone and does not include any other parts in the fusion compound, such as spacers or the GLP-1 polypeptide moiety.

[0044] When amino acid changes in the FGF21 polypeptide are shown in comparison to FGF21(1-181) (SEQ ID NO: 2), it is understood that this refers to changes in the FGF21 polypeptide moiety alone and does not include any other parts of the fusion compound, such as spacers or the GLP-1 polypeptide moiety.

[0045] In some embodiments, the FGF21 polypeptide portion of the fusion compound or fusion protein is an analogue of SEQ ID NO: 2. In some embodiments, the FGF21 polypeptide is at least 50%, preferably at least 55%, preferably at least 60%, preferably at least 65%, preferably at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, or most preferably at least 95% identical to SEQ ID NO: 2. In some embodiments, the FGF21 polypeptide contains up to 15 amino acid changes, preferably up to 14, preferably up to 13, preferably up to 12, preferably up to 11, preferably up to 10, preferably up to 9, preferably up to 8, preferably up to 7, preferably up to 6, preferably up to 5, preferably up to 5, preferably up to 4, or preferably up to 3 amino acid changes compared to SEQ ID NO: 2. In some embodiments, the FGF21 polypeptide has 4 amino acid changes compared to SEQ ID NO: 2. In some embodiments, the FGF21 polypeptide contains 180C. In some embodiments, the FGF21 polypeptide contains or has the following amino acid changes compared to SEQ ID NO: 2: [121Q,168L,180C] or [121Q,168L,171G] or [121Q,168L,171G,180E]. In some embodiments, the FGF21 polypeptide portion of the fusion compound is selected from the list consisting of SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12. In some embodiments, the FGF21 polypeptide has FGF21 activity. In some embodiments, FGF21 can activate FGFR1c. In some embodiments, FGF21 can activate FGFR3c.

[0046] In some embodiments, the FGF21 polypeptide can undergo Cys alkylation.

[0047] Table 2 provides non-exclusive examples of FGF21 polypeptides. [Table 2]

[0048] Spacer Fusion compounds or fusion proteins often contain spacers that separate the bioactive portion of the compound, ensuring that any functionality present in the bioactive portion is not interfered with by the proximity of other bioactive portions.

[0049] As used herein, the term “spacer” refers to an element that covalently bonds the bioactive portion of the fusion compound or fusion protein of the present invention. The spacer may also be referred to as the “spacer polypeptide,” “spacer moiety,” or “spacer element.” In some embodiments, the spacer comprises an amino acid sequence whose N-terminus is bonded to the C-terminus of the GLP-1 polypeptide moiety via an amide bond, and whose C-terminus is bonded to the N-terminus of the FGF21 polypeptide moiety via an amide bond. In some embodiments, the spacer has the following chemical formula: 2(GAQP) x -C y -(GAQP) h -A j The formula contains repeating elements of (wherein x is an integer in the range of 0 to 64, y is an integer in the range of 0 to 1, h is an integer in the range of 1 to 64, and j is an integer in the range of 0 to 1). An example of the spacer nomenclature used herein is "[GAQP]8-A" or "(GAQP)x8,A", which are abbreviations for polypeptide sequences consisting of eight segments of GAQP followed by one segment of A. The sequence is described in its entirety as GAQPGAQPGAQPGAQPGAQPGAQPGAQPGAQPA.

[0050] Spacers may affect the pharmacokinetic properties of a fusion compound, for example, by increasing its half-life. In some embodiments, spacers may have the ability to improve the half-life of a fusion compound. The expression "improved half-life" of a fusion compound may mean extending the plasma half-life of the fusion compound so that it is suitable for once-daily or twice-weekly injections, preferably once-weekly injections.

[0051] In some embodiments, the spacer may consist of 1 to 257 amino acids, such as 5 to 257 or 9 to 129. In some embodiments, the spacer may include at least two segments of GAQP. In some embodiments, the spacer is (GAQP) 32 -May include A In some embodiments, the spacer is selected from a list consisting of SEQ ID NOs: 13, 14, 15, 16, 17, and 18. In some embodiments, the spacer includes or consists of SEQ ID NOs: 13, 14, 15, 16, 17, or 18.

[0052] In some embodiments, the spacer can undergo Cysalkylation.

[0053] Table 3 provides non-limiting examples of spacers. [Table 3]

[0054] substituent As used herein, the term “substituent” refers to a portion of the compound of the present invention that is covalently bonded to the GLP-1 polypeptide or FGF21 polypeptide or spacer. The terms “substituent” and “side chain” are used interchangeably. This substituent has the ability to form non-covalent bonds with albumin, thereby promoting the circulation of the derivative in the bloodstream, and also has the effect of extending the action duration of the fusion compound due to the slow detachment of the fusion compound from its albumin-bound state, which means that it takes time for the free form of the derivative to be released. Therefore, the entire substituent is sometimes referred to as the “albumin-binding portion.” The substituent may include a portion particularly related to albumin binding and the resulting extension, which may be referred to as the “protractor” or “extension portion.” The extension portion may be near the end (or distal end, or free end) of the substituent with respect to the binding site to the peptide, and preferably at its end. The substituent may include a portion between the extension portion and the binding site to the peptide, which may be referred to as the “linker.” The "substituent" may be lipophilic and / or negatively charged at physiological pH (7.4). The "protractor" or "substituent" may be covalently bonded to the thiol group of the cysteine ​​residue of the GLP-1 polypeptide, FGF21 analogue, or spacer by alkylation. The substituent may be synthesized and activated as a haloacetamide group and react with the thiol group of the cysteine ​​residue to form a covalent thiol-carbon bond (this process is called Cys-alkylation), which is also called a thioether bond. Thus, no halogen atom is present in the derivative, and the substituent is linked via a sulfur atom. When the thiol group is mentioned in relation to the derivative, it should be understood as the sulfur atom that is part of the thiol group of cysteine ​​before Cys-alkylation. Alternatively, the substituent may be activated with a maleimide group that reacts with the thiol group of the cysteine ​​residue under the formation of a covalent thiol-carbon bond. The substituent may function as an albumin binder or albumin-binding moiety. The substituent may be an albumin binder or an albumin-binding moiety.

[0055] The protractor may be located near the distal end of the side chain, or at that position, relative to the binding site to the protein. In one embodiment, each protractor is of the chemical formula 3: HOOC (CH2)x-CO- * It includes or consists of a protractor. The length of the carbon chain defined by x may vary from 8 to 18, for example, 14 to 18, or for example, 14 to 16.

[0056] The nomenclature is standard in the relevant technical field; for example, in the above formula, * CO * is carbonyl ( * -C(=O)- * This refers to any formula (R-CO-) as used herein. For example, any formula (R-CO-) as used herein. * )(wherein R is defined by each equation), R-CO- * is RC(=O)- * It refers to.

[0057] Linker: A linker may include at least one of the following linker elements: chemical formulas 4, 5, and 6. Chemical formulas 4 and 5 each have -NH- and CO- termini, respectively, and they can be linked to each other by amide bonds with the -CO- or -NH- group of chemical formula 3 or 6.

[0058] Chemical formula 6 has an -NH terminus (which can form an amide bond with any of chemical formulas 3, 4, or 5) and an -NH-CO-CH2 terminus, and its unreacted form is a haloacetamide, and has the ability to react with a cysteine ​​thiol group incorporated into the GLP-1 polypeptide moiety, spacer moiety, or FGF21 polypeptide moiety of the fusion protein or fusion compound of the present invention.

[0059] The linker element chemical formulas 4, 5, and 6 are as follows: Chemical formula 4 is * -NH-CH(COOH)-(CH2)2-CO- * And, Chemical formula 5 is * NH-(CH2)2-[O-(CH2)2] k -O-[CH2] m -CO- * And, In the formula, k is an integer in the range of 1 to 5, and m is an integer in the range of 1 to 5. Chemical formula 6 is, * -NH-(CH2) n -NH-CO-CH2- * In this equation, n is an integer in the range of 1 to 5.

[0060] Chemical formulas 4, 5, and 6 may be interconnected via amide bonds, and their * -NH terminus, protractor CO- according to chemical formula 3 * It is connected to the end, and those CH2- * At its terminal end, it may be linked to an amino acid such as cysteine ​​A, B, or C.

[0061] Chemical formula 4 may also be referred to herein as gGlu, gammaGlu, or γGlu. [ka] Chemical formula 5 may also be referred to as "Ado" in this specification. [ka] [ka]

[0062] In some embodiments, the substituent is of chemical formula 7: [ka]

[0063] In one embodiment, the substituent is [2-[2-[[2-[2-[[2-[[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoyl-amino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]-ethylamino]-2-oxoethyl] (Chemical Formula 7).

[0064] In some embodiments, the fusion compound includes one or more substituents, such as first and second substituents. In some embodiments, the fusion compound includes at least one substituent, e.g., one, e.g., two, e.g., three, e.g., seven substituents. The substituents may be bound to any amino acid residue in the fusion compound. In some embodiments, the substituents are bound to Cys residues incorporated into the fusion compound. In some embodiments, the substituents are bound to Cys residues in the GLP-1 polypeptide moiety of the fusion compound. In some embodiments, the substituents are bound to Cys residues in the FGF21 polypeptide moiety of the fusion compound. In some embodiments, the substituents are bound to Cys residues in the spacer moiety of the fusion compound. In some embodiments, the first substituent is bound to a Cys residue in the GLP-1 polypeptide moiety, and the second substituent is bound to a Cys residue in the FGF21 polypeptide moiety of the fusion compound. In some embodiments, the first substituent is bound to a Cys in the spacer, and the second substituent is bound to a Cys in the FGF21 polypeptide. In some embodiments, the substituents are bound to amino acid residues in the polypeptide backbone of the fusion compound. In some embodiments, the substituent is attached to a spacer portion or an FGF21 polypeptide portion of the fusion compound. In some embodiments, the substituent is attached to a Cys residue in the GLP-1 polypeptide portion of the fusion compound, located at position 27 or 26. In some embodiments, the substituent is attached to a Cys residue in the FGF21 polypeptide portion of the fusion compound, located at position 180.

[0065] Fusion protein A fusion protein is a protein created through the fusion of two or more genes originally encoding separate proteins. Translation of this fusion gene yields a single polypeptide with functional properties derived from each of the original proteins. A fusion protein may contain two or more bioactive sites, which exert their bioactivity primarily through interaction with two separate sites. Thus, a GLP-1 / FGF21 fusion protein contains a GLP-1 polypeptide and an FGF21 polypeptide. A GLP-1 / FGF21 fusion protein may further contain a peptide spacer. Thus, a GLP-1 / FGF21 fusion protein contains a GLP-1 polypeptide moiety, an FGF21 polypeptide moiety, and optionally a spacer moiety. A fusion protein containing a GLP-1 polypeptide and an FGF21 polypeptide may be fused such that the C-terminus of the GLP-1 polypeptide is fused to the N-terminus of the peptide spacer, and the C-terminus of the peptide spacer is fused to the N-terminus of the FGF21 polypeptide. GLP-1 peptide exerts its biological activity via GLP-1R, while the latter primarily exerts its biological activity through the activation of the FGFR complex.

[0066] A complete linear string of amino acids that forms a spacer for any biologically active polypeptide (e.g., GLP-1 polypeptide and FGF21 polypeptide) and any spacer separating the biologically active polypeptide may be referred to herein as the “polypeptide skeleton.” The terms “fusion protein” and “polypeptide skeleton” are used interchangeably herein. Thus, the polypeptide skeleton of a fusion protein is substituent-free. If substituents are present, the compound is referred to herein as the “fusion compound” instead of the “fusion protein.” The difference between a fusion protein as defined herein and a fusion compound as defined herein is that a fusion compound may contain one or more substituents. The fusion protein of the present invention may be incorporated into the fusion compound of the present invention.

[0067] In some embodiments, the fusion protein is a bifunctional fusion protein. In some embodiments, the fusion protein is a fusion protein of the form ABC (chemical formula 1), where A is a GLP-1 polypeptide which is an analog of SEQ ID NO: 1, B is a peptide spacer consisting of 1 to 257 amino acids, and C is an FGF21 analog which is an analog of SEQ ID NO: 2. In some embodiments, A is an amino acid sequence H-Xaa8-EGTFTSDVSSYLE-Xaa 22 -QAA-Xaa 26 -Xaa 27 -FIAWLVKG-Xaa 36 -G (SEQ ID NO: 3) is included in or consists of a GLP-1 polypeptide, where Xaa8 is G, and Xaa 22 is E and Xaa 26 is R, C or K, and Xaa 27 is C or E, Xaa 36 is G or C. In some embodiments, B is a peptide spacer, which is 5 to 257 amino acids long. In some embodiments, the C-terminus of the GLP-1 polypeptide is fused to the N-terminus of the peptide spacer, and the C-terminus of the peptide spacer is fused to the N-terminus of the FGF21 analog. In some embodiments, the fusion protein is selected from the list consisting of SEQ ID NOs: 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, and 34. In some embodiments, the fusion protein is selected from the list consisting of SEQ ID NOs: 26, 27, 32, and 33. In some embodiments, the fusion protein is SEQ ID NO: 27.

[0068] In some embodiments, a fusion protein of ABC (chemical formula 1) is incorporated into the fusion compound of the present invention.

[0069] Table 4 provides non-restrictive examples of fusion proteins. [Table 4-1] [Table 4-2] [Table 4-3]

[0070] fusion compound The term "fusion compound" refers to a derivative of a fusion protein. In some embodiments, the fusion compound includes a fusion protein and substituents. A "GLP-1 / FGF21 fusion compound" is a derivative of a GLP-1 / FGF21 fusion protein. In some embodiments, the compound of the present invention is a GLP-1 / FGF21 fusion compound. The fusion compound may be referred to, for example, by reference to a GLP-1 polypeptide, a spacer, an FGF21 polypeptide, and substituents. An example of the fusion compound nomenclature used herein is S{beta-27}-[2-[2-[[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]-ethylamino]-2-oxoethyl]-[8G, 22E, 27C, 36G]GLP-1(7-37)-(GAQP)x32, A-[121Q, 168L, 171G, 180E]FGF21(1-181). In this example, the fusion compound consists of the GLP-1 polypeptide of formula [8G,22E,27C,36G]GLP-1(7-37) and the FGF21 polypeptide of formula [121Q,168L,171G,180E]FGF21(1-181), where the GLP-1 polypeptide and the FGF21 polypeptide are separated by the spacer of formula (GAQP)x32,A, and the substituent of formula S{beta-27}-[2-[2-[[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]-ethylamino]-2-oxoethyl] is attached to the Cys residue at position 27 of the GLP-1 polypeptide.

[0071] In some embodiments, the fusion compound comprises a fusion protein of the form ABC (chemical formula 1), where A is a GLP-1 polypeptide analog of GLP-1(7-37) (SEQ ID NO: 1), B is a peptide spacer containing or consisting of 1 to 257 amino acids, and C is an FGF21 polypeptide analog analog of FGF21(1-180) (SEQ ID NO: 2), and is at least a substituent.

[0072] In some embodiments, the fusion compound has an improved half-life.

[0073] In some embodiments, the fusion compound is a bifunctional fusion compound. In some embodiments, the fusion compound includes substituents. In some embodiments, the fusion compound includes one or more substituents, such as two substituents or four substituents. In some embodiments, the fusion compound includes a substituent bonded to a Cys residue at a position corresponding to position 27 of GLP-1(7-37)(SEQ ID NO: 1). In some embodiments, the fusion compound includes a substituent bonded to a Cys residue at a position corresponding to position 26 of GLP-1(7-37)(SEQ ID NO: 1). In some embodiments, the fusion compound includes a substituent bonded to a Cys residue at a position corresponding to position 36 of GLP-1(7-37)(SEQ ID NO: 1). In some embodiments, the fusion compound includes a substituent bonded to a Cys residue of a spacer. In some embodiments, the fusion compound includes a substituent bonded to a Cys residue at a position corresponding to position 180 of FGF21(1-180)(SEQ ID NO: 2). In some embodiments, the fusion compound includes a first substituent bonded to a Cys residue in the GLP-1 polypeptide moiety of the fusion compound, for example, a Cys residue at the position corresponding to position 26 of GLP-1(7-37)(SEQ ID NO: 1), or for example, a Cys residue at the position corresponding to position 27 of GLP-1(7-37)(SEQ ID NO: 1), and a second substituent attached to the Cys residue at the position corresponding to position 180 of FGF21(1-180)(SEQ ID NO: 2). In some embodiments, the fusion compound includes a first substituent bonded to a Cys residue in the spacer moiety of the fusion compound and a second substituent bonded to a Cys residue in the FGF21 polypeptide moiety of the fusion compound, wherein the Cys is located at the position corresponding to position 180 of FGF21(1-180)(SEQ ID NO: 2).

[0074] In some embodiments, the fusion compound is selected from a list consisting of chemical formulas 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, and 23.

[0075] In some embodiments, the fusion compound is of chemical formula 23 [ka] , chemical formula 12 [ka] , chemical formula 14 [ka] , and chemical formula 16 [ka] It is selected from a list consisting of the following.

[0076] pharmaceutically acceptable salts, amides, or esters The fusion compound may be in the form of a pharmaceutically acceptable salt, amide, or ester.

[0077] Salts are formed, for example, by chemical reactions between a base and an acid, such as: 2NH3 + H2SO4 → (NH4)2SO4.

[0078] The salt may be a basic salt, an acidic salt, or neither (i.e., a neutral salt). Basic salts produce hydroxide ions in water, while acidic salts produce hydronium ions.

[0079] Salts of the fusion compounds can be formed by adding cations or anions between anionic groups or between cationic groups. These groups may be located on the polypeptide backbone and / or as substituents of the fusion compound.

[0080] Non-limiting examples of anionic groups in the fusion compounds of the present invention include, if present, free carboxylic acid groups in substituents, and free carboxylic acid groups in the GLP-1 polypeptide and / or FGF21 polypeptide. The amino acid sequence often contains a free carboxylic acid group at the C-terminus, and may also contain free carboxylic acid groups in internally acidic amino acid residues such as Asp and Glu.

[0081] Non-limiting examples of cationic groups in the fusion compound include, if present, a free N-terminal amino group, as well as any free amino groups of internal base amino acid residues such as His, Arg, and Lys. The N-terminal amino group of the fusion compound of the present invention may be free or acetylated.

[0082] The ester derivatives of the present invention may be formed, for example, by the reaction of a free carboxylic acid group with an alcohol or phenol, which leads to the substitution of at least one hydroxyl group with an alkoxy or aryloxy group.

[0083] Ester formation may involve a free carboxylic acid group at the C-terminus of the polypeptide skeleton, and / or any free carboxylic acid group within the substituent.

[0084] The amide derivatives of the present invention may be formed, for example, by the reaction of a free carboxylic acid group with an amine or a substituted amine, or by the reaction of a free or substituted amino group with a carboxylic acid.

[0085] Amide formation may involve a free carboxylic acid group at the C-terminus of the polypeptide skeleton, any free carboxylic acid group in the substituent, an amino group at the N-terminus of the polypeptide skeleton, and / or any amino group in the substituent.

[0086] In one embodiment, the fusion compound is in the form of a pharmaceutically acceptable salt. In one embodiment, the fusion compound is in the form of a pharmaceutically acceptable amide. In one embodiment, the fusion compound is in the form of a pharmaceutically acceptable ester.

[0087] GLP-1 activity In functional embodiments of the present invention, the fusion compound has GLP-1 activity. As used herein, the term "GLP-1 activity" refers to the ability to activate the GLP-1 receptor, and this activation may also be called "potency."

[0088] In some embodiments, this term refers to in vivo agonist activity / potency. In some embodiments, GLP-1 receptor activation is determined by measuring the cAMP response of cells stably expressing the GLP-1 receptor upon contact with the agonist in vitro. In some embodiments, cells according to Example 2. In some embodiments, the GLP-1 receptor is the human GLP-1 receptor. GLP-1 activity is measured in EC 50 The value, or reference compound having GLP-1 activity, for example, EC2 for chemical formula 24. 50 It can be expressed as a value. GLP-1 activity can be measured in the presence of HSA. GLP-1 activity is preferably measured as described in Example 2.

[0089] In some embodiments, GLP-1 activity is measured in baby hamster kidney (BHK) cells that stably express the CRE-luciferase reporter gene, enabling indirect measurement of cAMP formation by the human GLP-1 receptor and adenylyl cyclase. In some embodiments, GLP-1 activity is measured in the presence of 1% HSA. In some embodiments, GLP-1 activity is measured as activation of GLP-1R in the absence of HSA, as described in "General Methods for Measuring GLP-1 Activity" / Example 2, and EC 50 It is expressed as a value, EC 50 The concentration is less than 100 pM, preferably less than 70 pM, more preferably less than 50 pM, and most preferably less than 20 pM.

[0090] FGF21 activity In functional aspects of the present invention, the fusion compound has FGF21 activity. As used herein, the term FGF21 activity refers to the ability to activate the FGFR complex, and this activation may also be called "potency." Activity can be measured in an in vitro assay using HEK293 cells that endogenously express several FGF receptors, including FGFR1c, FGFR3c, and BKL. For example, the response of human FGFR can be measured using HEK (human embryonic kidney cells) that overexpress human beta-cloto (BKL). FGF21 activity is measured in EC 50 It may be expressed as a value. FGF21 activity may be measured in the presence of HSA. It is preferable to measure FGF21 activity as described in Example 3.

[0091] In some embodiments, FGF21 activity is measured in HEK293 cell lines overexpressing FGFR1c and human beta-cloto receptor (BKL). In some embodiments, FGF21 activity is measured in the presence of 0.1% HSA. In some embodiments, FGF21 activity is measured without the presence of HSA as activation of the FGFR complex, as described in Example 2, and EC 50 It is expressed as a value, EC 50 The concentration is less than 50 nM, preferably less than 10 nM, preferably less than 5 nM, and preferably 2 nM.

[0092] Pharmacokinetics In some embodiments, the fusion compounds according to the present invention may increase the mean residence time (MRT) compared to natural FGF21 and natural GLP-1, respectively.

[0093] In some embodiments, MRT may be measured in vivo using mice. The MRT measured in vivo using mice is preferably measured as described in Example 4.1. In some embodiments, MRT may be measured in vivo using miniature pigs. The MRT measured in vivo using miniature pigs is preferably determined as described in Example 4.2. In some embodiments, MRT may be measured in vivo using cynomolgus monkeys. The MRT measured in cynomolgus monkeys is preferably measured as described in Example 4.3.

[0094] In some embodiments, the half-life of the fusion compound is calculated using the “individual best fit” of log-linear regression for concentration versus time. In some embodiments, the MRT is calculated as MRT = AUMC / AUC, where AUC is AUMC and is calculated by the trapezoidal rule. In some embodiments, the MRT of the fusion compound is calculated based on non-partition analysis. In some embodiments, the MRT of the fusion compound, when measured in mice according to Example 4.1, is at least 2.5 hours, preferably at least 3 hours, preferably at least 4 hours, preferably at least 5 hours, preferably at least 6 hours, preferably at least 7 hours, more preferably at least 8 hours, preferably at least 9 hours, and preferably at least 9.4 hours. In some embodiments, the MRT of the fusion compound, when measured in miniature pigs according to Example 4.2, is at least 50 hours, preferably at least 60 hours, preferably at least 70 hours, preferably at least 80 hours, and preferably at least 90 hours. In some embodiments, the MRT of the fusion compound, when measured in cynomolgus monkeys according to Example 4.3, is at least 30 hours, preferably at least 40 hours, preferably at least 50 hours, and preferably at least 52 hours.

[0095] Pharmacodynamics FGF21 is a human hormone synthesized in the liver and involved in glucose, lipid, and energy homeostasis. Treatment with FGF21 efficiently lowers triglycerides, LDL-C, and VLDL-C, while increasing HDL-C. Therefore, those skilled in the art would expect that compounds possessing FGF21 activity can lower plasma levels of triglycerides, LDL-C, and VLDL-C. Treatment of obese rodents with FGF21 efficiently reduces body weight, which correlates with IGF-1, hepatic triglycerides, and liver enzymes such as ALT and AST. Thus, the PD effect of FGF21 can be tested in obese animal models.

[0096] GLP-1 is an incretin secreted in the intestines that is involved in glucose metabolism and satiety. Treatment with GLP-1 acutely and efficiently reduces food intake and leads to weight loss.

[0097] In one embodiment, the fusion compound can reduce acute food intake as measured in mice. In one embodiment, the fusion compound can efficiently reduce body weight as measured in mice. In one embodiment, the fusion compound can reduce hepatic triglycerides as measured in DIO mice or DIO-NASH models (GAN diet). In one embodiment, the fusion compound can reduce liver enzymes such as ALT as measured in DIO mice or DIO-NASH models (GAN diet).

[0098] In functional embodiments of the present invention, the fusion compound may lower the plasma concentration of low-density lipoprotein cholesterol (LDL-C). Similarly, or alternatively, the fusion compound of the present invention may lower the plasma levels of triglycerides. Similarly, or alternatively, the fusion compound of the present invention may lower the plasma levels of total cholesterol.

[0099] Pharmaceutical efficacy / medical use The present invention also relates to fusion compounds for use as pharmaceuticals. As used herein, the term “treatment” refers to any medical treatment for any human subject requiring treatment. Treatments may be preventive, prophylactic, palliative, symptomatic, and / or curative. The timing and purpose of such treatments may vary from individual to individual, depending on the health condition of the subject.

[0100] According to a second aspect of the present invention, a fusion compound as defined above (i.e., the compound as defined in the first aspect of the present invention, including all embodiments and their specific features) is provided for use as a pharmaceutical (or for use in pharmaceuticals).

[0101] To avoid any doubt, references to the compounds defined in the first aspect of the present invention include references to the fusion protein of chemical formula 1 (including all embodiments thereof) and its pharmaceutically acceptable salts, esters, and amides.

[0102] In some embodiments, the fusion compounds are for the treatment and / or prevention of eating disorders, cardiovascular diseases, diabetic complications, and / or improvement of lipid parameters, such as dyslipidemia, and may be particularly useful for lowering total serum lipids, increasing HDL, lowering small, high-density LDL, lowering VLDL, lowering triglycerides, lowering cholesterol, lowering plasma levels of lipoprotein a (Lp(a)) in humans, increasing plasma adiponectin in humans, inhibiting the production of apolipoprotein A (apo(A)), improving β-cell function, and / or delaying or preventing the progression of diabetic disease, and / or for the treatment and / or prevention of hepatic steatosis, and may be particularly useful for non-alcoholic fatty liver disease (NAFLD), metabolic dysfunction-related fatty liver disease (MAFLD), alcohol-related liver disease (ALD), MetALD, and non-alcoholic steatohepatitis (NASH), metabolic dysfunction-related fatty liver disease (MASH).

[0103] MASH is proposed as an alternative term to NASH to describe the same diagnostic entity (MERinella et al., j.aohep.2023.01133). As used herein, the terms "NASH" and "MASH" mean the same thing and are used interchangeably.

[0104] MAFLD is proposed as an alternative term to NAFLD to describe the same diagnostic entity (ME Rinella et al., j.aohep.2023.101133). As used herein, "NAFDL" and "MAFLD" mean the same thing and are used interchangeably.

[0105] In a third aspect of the present invention, compounds of the present invention as defined above are provided for use in the treatment and / or prevention of diseases. The diseases are selected from the group consisting of diabetes mellitus and related diseases, e.g., eating disorders, cardiovascular diseases, and diabetic complications; and / or for improvement of lipid parameters, improvement of β-cell function (such as prevention and / or treatment of dyslipidemia), reduction of total serum lipids; increase of HDL, decrease of small, dense LDL; reduction of VLDL, reduction of triglycerides; reduction of cholesterol; decrease of plasma lipoprotein a (Lp(a)) in humans; inhibition of apolipoprotein A (apo(A)) production; and / or for delaying or preventing the progression of diabetes; and / or for the treatment and / or prevention of fatty liver, NAFDL, and NASH.

[0106] In an alternative third aspect of the present invention, methods are provided for the treatment and / or prevention of diseases such as diabetes mellitus and related diseases such as eating disorders, cardiovascular disease, and complications of diabetes, and / or the prevention and / or treatment of dyslipidemia, for patients in need of treatment and / or prevention, for the purpose of improving lipid parameters such as a decrease in total serum lipids, an increase in HDL, a decrease in small, high-density LDL, a decrease in VLDL, a decrease in triglycerides, a decrease in cholesterol, and a decrease in plasma levels of lipoprotein a (Lp(a)) in humans, for the suppression of apolipoprotein A (apoA) production, for the improvement of β-cell function, and / or for delaying or preventing the progression of diabetes, and / or for the treatment and / or prevention of fatty liver, NAFDL, NASH, comprising administering a therapeutically effective amount of the compounds of the present invention as defined above.

[0107] A further alternative third aspect of the present invention provides for the use of the compounds of the present invention as defined above in the manufacture of pharmaceuticals for the treatment or prevention of diseases such as eating disorders, cardiovascular diseases, and diabetic complications; and / or use in the prevention and / or treatment of dyslipidemia, improvement of lipid parameters such as a decrease in total serum lipids, an increase in HDL, a decrease in small-molecule high-density LDL, a decrease in VLDL, a decrease in triglycerides, and a decrease in cholesterol; a decrease in lipoprotein a (Lp(a)) plasma levels in humans; inhibition of apolipoprotein A (apo(A)) production; improvement of β-cell function; and / or delay or prevention of the progression of diabetic disease; and / or use in the treatment and / or prevention of fatty liver, NAFDL, and NASH.

[0108] In some embodiments, the compounds of the present invention may be used for the following medical procedures: (i) Prevention and / or treatment of all forms of diabetes, including hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, non-insulin-dependent diabetes, MODY (young-onset adult-onset diabetes), gestational diabetes, and / or reduction of HbA1c. (ii) Delaying or preventing the progression of diabetic disease, such as the progression of type 2 diabetes; delaying the progression from impaired glucose tolerance (IGT) to insulin-dependent type 2 diabetes; delaying or preventing insulin resistance; and / or delaying the progression from insulin-free type 2 diabetes to insulin-dependent type 2 diabetes. (iii) For example, prevention and / or treatment of eating disorders such as obesity by reducing food intake, weight loss, appetite suppression, or satiety induction; treatment or prevention of obesity induced by bulimia nervosa, bulimia nervosa, and / or antipsychotic drugs or steroids; reduction of gastric motility, delay of gastric emptying, increase of physical mobility, and / or prevention and / or treatment of comorbidities of obesity such as osteoarthritis and / or urinary incontinence. (iv) Maintaining weight after successful weight loss (either drug-induced or diet and exercise-induced), i.e., preventing weight gain after successful weight loss. (v) Prevention and / or treatment of liver disorders such as hepatic steatosis, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), hepatitis, or fatty liver.

[0109] In some embodiments, the present invention relates to compounds of the present invention for use in the prevention and / or treatment of acute and / or chronic pancreatitis.

[0110] In some embodiments, the present invention relates to compounds of the present invention for use in the prevention and / or treatment of type 2 diabetes, metabolic syndrome, obesity, insulin resistance, and diseases selected from the group consisting of prediabetes, diabetic retinopathy, diabetic neuropathy, diabetic nephropathy, chronic kidney disease, diabetic nephropathy, diabetic dyslipidemia, fatty liver disease including non-alcoholic steatohepatitis (NASH), and atherosclerosis.

[0111] In some embodiments, the present invention relates to compounds of the present invention for use in the prevention and / or treatment of hepatocellular carcinoma (HCC).

[0112] In some embodiments, the present invention relates to compounds of the present invention for use in the prevention and / or treatment of alcoholic steatohepatitis (ASH) or alcoholic fatty liver disease (AFDL).

[0113] In some embodiments, the present invention relates to compounds of the present invention for use in the prevention and / or treatment of NASH, wherein such use prevents and / or delays increases in relative liver weight, plasma alanine aminotransferase levels, liver triglyceride content, and / or liver cholesterol. Relative liver weight is defined as liver weight as a percentage of total body weight. In some embodiments, such use reduces relative liver weight, plasma alanine aminotransferase levels, liver triglyceride content, and / or liver cholesterol.

[0114] In some embodiments, the present invention relates to compounds of the present invention for use in the prevention and / or treatment of NASH, the use of which prevents, delays and / or reduces the histopathological signs of fatty liver.

[0115] In some embodiments, the present invention relates to compounds of the present invention for use in the prevention and / or treatment of NASH, the use of which prevents, delays, and / or reduces inflammation of the liver.

[0116] NASH is the most extreme form of NAFLD. NAFLD is a type of fatty liver disease that occurs when fat accumulates in the liver due to causes other than alcohol consumption. Fat deposition is also called steatosis. Patients with NASH often have obesity, type 2 diabetes, dyslipidemia, and / or metabolic syndromes. Symptoms of NASH include fatigue, discomfort, or right upper abdominal discomfort.

[0117] In some embodiments, the present invention relates to compounds for use in the prevention and / or treatment of NASH, the use of which prevents, delays, and / or reduces fibrosis in the liver. In some embodiments, the present invention relates to compounds for use in the prevention and / or treatment of NASH, the compounds being administered in the form of a pharmaceutical composition containing 1 to 50 mg / ml of the compound, for example 5 to 40 mg / ml, for example 10 to 30 mg / ml of the compound.

[0118] In some embodiments, the present invention relates to compounds of the present invention for use in the prevention and / or treatment of NASH, wherein the compounds are administered subcutaneously once a week.

[0119] In some embodiments, the present invention relates to a compound of the present invention for use in the prevention and / or treatment of NASH, the compound being administered subcutaneously daily, or every two days, or every three days, or every four days, or every five days, or every six days. In some embodiments, the compound is administered for at least 12 months.

[0120] In some embodiments, the present invention relates to compounds of the present invention for use in the prevention and / or treatment of NASH, wherein the compounds are administered in a therapeutically effective dose to subjects requiring the prevention and / or treatment of NASH.

[0121] In some embodiments, the subject is obese and / or has diabetes. In some embodiments, the subject suffers from being overweight, obese, hyperglycemia, type 2 diabetes, impaired glucose tolerance, and / or type 1 diabetes.

[0122] In some embodiments, the indications may be type 2 diabetes and / or dyslipidemia and / or obesity. In some embodiments, the present invention relates to a method for weight management. In some embodiments, the present invention relates to a method for reducing appetite. In some embodiments, the present invention relates to a method for reducing food intake.

[0123] Generally, all subjects suffering from obesity are also considered to be overweight. In some embodiments, the present invention relates to methods for treating or preventing obesity. In some embodiments, the present invention relates to the use of derivatives of the present invention for treating or preventing obesity. In some embodiments, subjects suffering from obesity are adult humans or paediatric humans (including infants, children, and adolescents). Body Mass Index (BMI) is a measure of body fat based on height and weight. The formula is BMI = weight (kilograms) / height (meters). Human subjects suffering from obesity may have a BMI greater than 30, and these subjects may also be referred to as obese. In some embodiments, human subjects suffering from obesity may have a BMI greater than 35 or a BMI in the range of 30 to less than 40. In some embodiments, obesity is severe obesity or morbid obesity in which human subjects may have a BMI greater than 40.

[0124] In some embodiments, the present invention relates to methods for treating or preventing overweight, optionally in the presence of at least one weight-related comorbidity. In some embodiments, the present invention relates to the use of the compounds of the present invention for treating or preventing overweight, optionally in the presence of at least one weight-related comorbidity. In some embodiments, the overweight subject is a human being, such as an adult human or a child human (including infants, toddlers, and adolescents). In some embodiments, the overweight human subject may have a BMI greater than 25, such as greater than 27. In some embodiments, the overweight human subject has a BMI in the range of 25 to less than 30 or 27 to less than 30. In some embodiments, the weight-related comorbidity is selected from the group consisting of hypertension, diabetes (such as type 2 diabetes), dyslipidemia, hypercholesterolemia, and obstructive sleep apnea.

[0125] In some embodiments, the present invention relates to a method for weight loss. In some embodiments, the present invention relates to the use of the compounds of the present invention for weight loss. A person enjoying weight loss according to the present invention may have a BMI greater than 25, such as a BMI greater than 27 or greater than 30. In some embodiments, a person enjoying weight loss according to the present invention may have a BMI greater than 35 or greater than 40. The term “weight loss” may include the treatment or prevention of obesity and / or overweight.

[0126] Those skilled in the art will understand that references to the treatment of a particular condition (or similarly, references to the treatment of that condition) take on their usual meaning in the medical field. In particular, the term may refer to achieving a reduction in the severity and / or frequency of one or more clinical symptoms associated with the condition, as determined by a physician examining a patient who has or is susceptible to the condition. For example, in the case of NASH, the term may refer to increased liver enzymes, increased plasma lipids, increased liver rigidity, increased hepatic steatosis, loss of liver function, and increased hepatotoxicity.

[0127] As used herein, a reference to a patient (or more patients) refers to a living subject being treated, including a mammal (e.g., a human) patient. In particular, a reference to a patient refers to a human patient.

[0128] To avoid any doubt, a person skilled in the art will understand that such treatment [or prevention] is performed in a patient (or subject) who is in need of treatment or prevention. The need of a patient (or subject) for such treatment [or prevention] can be assessed by a person skilled in the art using routine techniques. As used herein, the terms disease and disorder (as well as similar terms such as condition, illness, medical problem) can be used interchangeably. As used herein, the term effective dose refers to the amount of compound that gives a therapeutic effect to the treated patient. The effect can be observed in an objective (i.e., measurable by several tests or markers) or subjective (i.e., the subject gives an indication of the effect and / or feels the effect). In particular, the effect can be observed (e.g., measured) in an objective manner using appropriate tests known to a person skilled in the art.

[0129] Pharmaceutical composition The present invention also relates to pharmaceutical compositions comprising a fusion compound. In one embodiment, the pharmaceutical composition comprising a fusion compound comprises at least one pharmaceutically acceptable excipient. The pharmaceutical compositions / formulations described herein may be prepared according to standard and / or accepted pharmaceutical practices.

[0130] The term “excipient” broadly refers to any component other than the active therapeutic component. Excipients may be inert, inactive, and / or non-pharmaceutically active substances. Excipients may serve various purposes, such as carriers, vehicles, diluents, and tablet adjuvants, and / or may function to improve the administration and / or absorption of the active substance. Formulations of pharmaceutically active components with various excipients are publicly known in the art; see, for example, Remington: The Science and Practice of Pharmacy (e.g., 19th edition (1995), and any subsequent editions). Additional optional components of a pharmaceutical composition include, for example, wetting agents, emulsifiers, antioxidants, bulking agents, metal ions, oily excipients, and proteins. Non-limiting examples of excipients include solvents, diluents, buffers, preservatives, isotonic agents, chelating agents, surfactants, and stabilizers.

[0131] A fourth aspect of the present invention provides a pharmaceutical composition comprising the fusion compound as defined above, and optionally comprising one or more pharmaceutically acceptable excipients. The injectable composition comprising the fusion compound can be prepared using the prior art in the pharmaceutical industry, which includes steps of dissolving and mixing the components as necessary to obtain the desired final product. Thus, according to one procedure, the fusion compound is dissolved in a suitable buffer at a suitable pH such that precipitation is minimized or avoided. In embodiments, the pharmaceutical composition may include wetting agents, emulsifiers, antioxidants, fillers, tonicity modifiers, chelating agents, metal ions, oily excipients, proteins (e.g., human serum albumin, gelatin, or proteins) or zwitterions (e.g., amino acids such as betaine, taurine, arginine, glycine, lysine, or histidine). In some embodiments, the pharmaceutical composition comprises a phosphate. In some embodiments, the pharmaceutical composition comprises propylene glycol and / or Tween 20. In some embodiments, the pharmaceutical composition comprises glycerol.

[0132] Parenteral administration may be performed by subcutaneous, intramuscular, intraperitoneal, or intravenous injection using a syringe, or possibly a pen-type syringe. Alternatively, parenteral administration may be performed using an infusion pump.

[0133] A pharmaceutical composition containing a fusion compound may be in several dosage forms, such as a solution, suspension, tablet, and capsule.

[0134] Accordingly, in a further aspect of the present invention, a process is provided for the preparation of the pharmaceutical composition / formulation defined above, the process comprising mixing the fusion compound defined above with one or more pharmaceutically acceptable excipients.

[0135] Accordingly, a fifth aspect of the present invention provides a pharmaceutical composition as defined in the fourth aspect of the present invention for use in treating or preventing a disease as defined herein, with reference to the third aspect of the present invention and all its embodiments.

[0136] Pharmaceutical compositions containing the fusion compound may be administered to patients in need of treatment or prevention at several sites, such as local sites (e.g., skin or mucous membranes), sites that bypass absorption (e.g., intra-artery, intra-vein, intra-cardiac), and sites involved in absorption (e.g., intracutaneous, subcutaneous, intramuscular, oral, or intra-abdominal).

[0137] Treatment using the fusion compound according to the present invention may also be combined with one or more additional pharmacologically active substances, such as an antifibrotic agent like an amylin analog or SGLT2, an FXR agonist like ACCi, or a THR beta-agonist.

[0138] Generation process The present invention also relates to a method for producing fusion compounds. The preparation of polypeptides such as GLP-1 analogs, FGF21 analogs, and spacers is well known in the art. Polypeptides incorporated into fusion compounds (or fragments thereof) can be prepared, for example, by classical recombination, i.e., by culturing host cells that contain a DNA sequence encoding the analog and are capable of expressing the polypeptide in a suitable nutrient medium under conditions that allow polypeptide expression. Non-limiting examples of host cells suitable for the expression of these polypeptides include Escherichia coli, Saccharomyces cerevisiae, and mammalian BHK or CHO cell lines. Similarly, or alternatively, polypeptides (or fragments thereof) introduced into fusion compounds can be prepared by classical solid peptide synthesis, e.g., solid-phase peptide synthesis using Boc or Fmoc chemistry or other well-established techniques; see, for example, Greene and Wuts, “Protective Groups in Organic Synthesis”, John Wiley & Sons, 1999; Florencio Zaragoza Dorwald, “Organic Synthesis on Solid Phase”, Wiley-VCH Verlag GmbH, 2000, and “Fmoc Solid Phase Peptide Synthesis”, Edited by WCChan and PDWhite, Oxford University Press 2000. Fusion compounds containing non-coding amino acids may be prepared as described in the art; see, for example, Hodgson et al: “The synthesis of peptides and proteins containing non-natural amino acids”, Chemical Society Reviews, vol.33, no.7(2004), pp. 422-430.

[0139] In some embodiments, the fusion compounds of the present invention are prepared stepwise by (i) recombinant preparation of the skeletal polypeptide and (ii) covalent bonding of substituents to the skeletal polypeptide (e.g., by alkylation). Specific examples of methods for preparing the fusion compounds are included in the Experiments section.

[0140] While this specification has illustrated and described certain features of the present invention, many modifications, substitutions, alterations, and equivalents will come to mind for those skilled in the art. It should therefore be understood that the appended claims are intended to cover all such modifications and alterations that fall within the true spirit of the invention.

[0141] Specific Embodiments The following are specific embodiments of the present invention. 1. A fusion protein comprising the polypeptide of chemical formula 1, wherein ABC, (wherein, A is the GLP-1 polypeptide, which is an analog of Sequence ID No. 1. B is a spacer consisting of 1 to 257 amino acids. C is the FGF21 polypeptide, an analogue of Sequence ID No. 2. or a pharmaceutically acceptable salt, amide, or ester thereof. 2. The fusion protein according to Embodiment 1, wherein the C-terminus of a GLP-1 polypeptide is fused to the N-terminus of a peptide spacer, and the C-terminus of the peptide spacer is fused to the N-terminus of an FGF21 analog. 3. A fusion protein according to Embodiment 1 or 2, wherein B consists of 5 to 257 amino acids. 4. A fusion protein according to any one of Embodiments 1 to 3, wherein B consists of 9 to 129 amino acids. 5. B is chemical formula 2(GAQP) x -C y -(GAQP) h -A jA fusion protein according to any one of Embodiments 1 to 4, comprising or consisting of (wherein x is an integer in the range of 0 to 64, y is an integer in the range of 0 to 1, h is an integer in the range of 1 to 64, and j is an integer in the range of 0 to 1). 6. The fusion protein according to any one of Embodiments 1 to 5, wherein B is selected from the list consisting of SEQ ID NOs: 13, SEQ ID NOs: 14, SEQ ID NOs: 15, SEQ ID NOs: 16, SEQ ID NOs: 17, and SEQ ID NOs: 18. 7. The fusion protein according to any one of Embodiments 1 to 6, wherein B is Sequence ID No. 13, Sequence ID No. 16, or Sequence ID No. 187. 8. The fusion protein according to any one of Embodiments 1 to 7, wherein A is at least 50%, preferably at least 55%, preferably at least 60%, preferably at least 65%, preferably at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, or most preferably at least 95% identical to SEQ ID NO: 1. 9. A fusion protein according to any one of Embodiments 1 to 8, wherein A comprises up to 15 amino acid changes, preferably up to 14 amino acid changes, preferably up to 13 amino acid changes, preferably up to 12 amino acid changes, preferably up to 11 amino acid changes, preferably up to 10 amino acid changes, preferably up to 9 amino acid changes, preferably up to 8 amino acid changes, preferably up to 7 amino acid changes, preferably up to 6 amino acid changes, preferably up to 5 amino acid changes, and most preferably up to 4 amino acid changes, compared to Sequence ID No. 1. 10. A fusion protein according to any one of Embodiments 1 to 9, wherein A contains or comprises amino acid sequence number 3. 11. A fusion protein according to any one of Embodiments 1 to 10, wherein A has 3 to 7 amino acid changes compared to SEQ ID NO: 1. 12. A fusion protein according to any one of Embodiments 1 to 11, wherein A contains a Cys residue at a position corresponding to position 26, 27, or 36 of GLP-1(7-37)(SEQ ID NO: 1), or optionally A contains an Arg residue at a position corresponding to position 26 of GLP-1(7-37)(SEQ ID NO: 1). 13. A fusion protein according to any one of Embodiments 1 to 12, wherein A has the following amino acid changes compared to GLP-1(7-37)(SEQ ID NO: 1): [8G, 22E, 26R], [8G, 22E, 26C, 36G], [8G, 22E, 27C, 36G], [8G, 22E, 26R, 27C, 36G], [8G, 22E, 26R, 36C], [8G, 22E, 36G], or [8G, 22E, 26R, 36G]. 14. A fusion protein according to any one of Embodiments 1 to 13, wherein A is selected from the list consisting of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 35. 15. The fusion protein according to any one of Embodiments 1 to 14, wherein C is identical to SEQ ID NO: 2 by at least 50%, preferably at least 55%, preferably at least 60%, preferably at least 65%, preferably at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, or most preferably at least 95%. 16. A fusion protein according to any one of Embodiments 1 to 15, wherein C comprises up to 10 amino acid changes, preferably up to 9 amino acid changes, preferably up to 8 amino acid changes, preferably up to 7 amino acid changes, preferably up to 6 amino acid changes, preferably up to 5 amino acid changes, preferably up to 4 amino acid changes, and most preferably up to 3 amino acid changes, compared to Sequence ID No. 2. 17. A fusion protein according to any one of Embodiments 1 to 16, wherein C has three or four amino acid changes compared to SEQ ID NO: 2. 18. A fusion protein according to any one of Embodiments 1 to 17, wherein C comprises 180C, 180E, or 180A. 19. A fusion protein according to any one of Embodiments 1 to 18, wherein C has the following amino acid changes compared to FGF21(1-181)(SEQ ID NO: 2): [121Q,168L,180C] or [121Q,168L,171G] or [121Q,168L,171G,180E]. 20. A fusion protein according to any one of embodiments 1 to 19, wherein C is selected from the list consisting of SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12. 21. A fusion protein according to any one of Embodiments 1 to 20, wherein C has FGF21 activity. 22. A fusion protein according to any one of Embodiments 1 to 21, wherein C can activate FGFR1c. 23. A fusion protein according to any one of Embodiments 1 to 22, wherein the components of Chemical Formula 1 are linked via amide bonds. 24. A fusion compound comprising a fusion protein according to any one of Embodiments 1 to 23, wherein the fusion compound comprises one or more substituents. 25. The fusion compound according to Embodiment 24, wherein the substituent includes an extended portion. 26. The fusion compound according to Embodiment 24 or Embodiment 25, wherein the extended portion is a functional group (FG) having a pKa of less than 7.0. 27. The fusion compound according to any one of embodiments 24 to 26, wherein the extended portion is lipophilic and / or negatively charged at physiological pH (7.4). 28. A fusion compound according to any one of embodiments 24 to 27, wherein the extended portion is a fatty acid. 29. The extended part is chemical formula 3: HOOC-(CH2) x -CO- * A fusion compound according to any one of embodiments 24 to 28, wherein (wherein x is an integer in the range of 10 to 20). 30. The fusion compound according to Embodiment 29, wherein x in chemical formula 3 is 12 to 20. 31. A fusion compound according to Embodiment 29 or Embodiment 30, wherein x in chemical formula 3 is 14 to 20. 32. A fusion compound according to any one of Embodiments 29 to 31, wherein x in chemical formula 3 is 14 to 18. 33. A fusion compound according to any one of embodiments 29 to 32, wherein x in chemical formula 3 is 16 to 20. 34. A fusion compound according to any one of embodiments 29 to 32, wherein x in chemical formula 3 is 16 to 18. 35. A fusion compound according to any one of embodiments 29 to 34, wherein x in chemical formula 3 is 16. 36. A fusion compound according to any one of embodiments 24 to 35, wherein the substituent includes a linker. 37. The fusion compound according to any one of embodiments 24 to 36, wherein the substituents are an extension portion and a linker. 38. The linker is chemical formula 4, chemical formula 5 and chemical formula 6 (In the formula, chemical formula 4 is, * -NH-CH(COOH)-(CH2)2-CO- * And, Chemical formula 5 is, * -NH-(CH2)2-[O-(CH2)2] k -O-[CH2] m -CO- * Here, k is an integer in the range of 1 to 5, and m is an integer in the range of 1 to 5. Chemical formula 6 is, * -NH-(CH2) n -NH-CO-CH2- * And n is an integer in the range of 1 to 5. Chemical formulas 4, 5, and 6 are interconnected via amide bonds, and in the shown arrangement, * - Protractor CO- at NH terminus * It is linked to the end (chemical formula 3), and its CH2- * A fusion compound according to any one of embodiments 36 to 37, comprising at least one of each of the following: (linked to an amino acid A, B, or C at the terminal). 39. The fusion compound according to Embodiment 38, wherein k in chemical formula 5 is 1. 40. The fusion compound according to Embodiment 38 or Embodiment 39, wherein m in chemical formula 5 is 1. 41. A fusion compound according to any one of embodiments 38 to 40, wherein n in chemical formula 6 is 2. 42. A fusion compound according to any one of embodiments 38 to 41, wherein the linker elements are linked via amide bonds. 43. A fusion compound according to any one of embodiments 38 to 42, wherein the linker comprises one element of chemical formula 4, two elements of chemical formula 5, and one element of chemical formula 6. 44. A fusion compound according to any one of embodiments 38 to 42, wherein the linker comprises one element of chemical formula 4, two elements of chemical formula 5, and one element of chemical formula 6. 45. The fusion compound according to any one of embodiments 38 to 43, wherein the extended portion is bonded to chemical formula 4 of the linker and chemical formula 5 of the linker is bonded to the polypeptide skeleton. 46. ​​A fusion compound according to any one of embodiments 38 to 44, wherein the substituent is of the following chemical formula 7. [ka] 47. A fusion compound according to any one of Embodiments 24 to 46, wherein the substituent is [2-[2-[[2-[[2-[[2-[[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoyl-amino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]-ethylamino]-2-oxoethyl]. 48. A fusion compound according to any one of Embodiments 24 to 47, wherein the fusion compound comprises a first and a second substituent. 49. The fusion compound according to any one of embodiments 24 to 48, wherein at least one substituent is bonded to one of the amino acid residues in A, B, or C. 50. A fusion compound according to any one of embodiments 24 to 49, wherein at least one substituent is bonded to an amino acid residue of A. 51. A fusion compound according to any one of embodiments 24 to 50, wherein at least one substituent is bonded to an amino acid residue of B. 52. A fusion compound according to any one of embodiments 24 to 51, wherein at least one substituent is bonded to an amino acid residue of C. 53. The fusion compound according to Embodiment 48 or Embodiment 49, wherein the first substituent is bonded to A or B and the second substituent is bonded to C. 54. A fusion compound according to any one of embodiments 24 to 53, wherein the substituent is bonded to a Cys residue. 55. A fusion compound according to any one of Embodiments 24 to 54, wherein the substituent is bonded to a Cys residue of A, B, or C. 56. A fusion compound according to any one of embodiments 24 to 55, wherein A comprises 26C, 27C, or 36C. 57. A fusion compound according to any one of Embodiments 24 to 55, wherein C contains 180C. 58. The fusion compound according to any one of embodiments 24 to 56, wherein the substituent is bonded to 26C, 27C, or 36C in A. 59. A fusion compound according to any one of embodiments 24 to 55 or 57, wherein the substituent is bonded to 180C at C. 60. The fusion compound, [ka] (Chemical formula 8), [ka] (Chemical formula 9), [ka] (Chemical formula 10), [ka] (Chemical formula 11), [ka] (Chemical formula 12), [ka] (Chemical formula 13), [ka] (Chemical formula 14), [ka] (Chemical formula 15), [ka] (Chemical formula 16), [ka] (Chemical formula 17), [ka] (Chemical formula 18), [ka] (Chemical formula 19), [ka] (Chemical formula 20), [ka] (Chemical formula 21), [ka] (Chemical formula 22), and [ka] A fusion compound according to any one of embodiments 24 to 59, selected from the list consisting of (chemical formula 23). 61. The fusion compound according to any one of embodiments 24 to 60, wherein the fusion compound is selected from the list consisting of chemical formulas 10, 11, 12, 14, 16, 17, 18, 21, and 23. 62. The fusion compound, [ka] (Chemical formula 12), [ka] (Chemical formula 14), [ka] (Chemical formula 16), and [ka] A fusion compound according to any one of embodiments 24 to 60, selected from the list consisting of (chemical formula 23). 63. The fusion compound, [ka] A fusion compound according to any one of embodiments 24 to 61, having chemical formula 23. 64. A fusion compound according to any one of embodiments 24 to 63, wherein the fusion compound has GLP-1 activity. 65. A fusion compound according to any one of embodiments 24 to 63, wherein the fusion compound can bind to the GLP-1 receptor. 66. A fusion compound according to any one of embodiments 24 to 63, wherein GLP-1 activity is measured in the absence of HSA. 67. A fusion compound according to any one of Embodiments 24 to 66, wherein the GLP-1 activity is measured as described in Example 2. 68. A fusion compound according to any one of Embodiments 24 to 66, wherein the GLP-1 activity is expressed as an EC50 value. 69. The fusion compound according to Embodiment 70, wherein the EC50 value, measured in the absence of HSA, is less than about 100 pM, preferably less than about 70 pM, preferably less than about 50 pM, preferably less than 20 pM. 70. A fusion compound according to any one of embodiments 24 to 66, wherein the fusion compound has FGF21 activity. 71. A fusion compound according to any one of Embodiments 24 to 70, wherein the fusion compound is measured in the absence of HSA. 72. A fusion compound according to any one of Embodiments 24 to 70, wherein the fusion compound is measured as described in Example 3. 73. A fusion compound according to any one of Embodiments 24 to 70, wherein the FGF21 activity is the ability to activate FGFR. 74. A fusion compound according to any one of Embodiments 24 to 70, wherein the FGF21 activity is the ability to activate FGFR1c. 75. A fusion compound according to any one of Embodiments 24 to 70, wherein FGF21 activity is measured in vitro using a whole-cell assay. 76. FGF21 activity is EC 50 It is expressed as a value, EC 50 However, the fusion compound according to any one of Embodiments 24 to 70, wherein the concentration is less than about 50 nM, preferably less than about 10 nM, preferably less than about 5 nM, and preferably less than about 2 nM. 77. A fusion compound according to any one of Embodiments 24 to 70, wherein the fusion compound has a desirable half-life. 78. A fusion compound according to any one of embodiments 24 to 77, wherein the fusion compound has a long half-life. 79. A fusion compound according to any one of Embodiments 24 to 77, wherein the fusion compound has a half-life suitable for once-weekly administration. 80. A fusion compound according to any one of embodiments 24 to 79, wherein the fusion compound is retarded. 81. A fusion compound according to any one of embodiments 24 to 77, wherein the fusion compound can lower plasma levels of triglycerides. 82. A pharmaceutical composition comprising a fusion compound described in any one of Embodiments 24 to 81, or a pharmaceutically acceptable salt, amide, or ester thereof, and one or more pharmaceutically acceptable excipients. 83. A fusion compound according to any one of Embodiments 24 to 81, or a pharmaceutical composition according to Embodiment 82, for use as a pharmaceutical. 84. A fusion compound according to any one of Embodiments 24 to 81, or a pharmaceutical composition according to Embodiment 82, for use as follows. (i) Prevention and / or treatment of all forms of diabetes, including hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, non-insulin-dependent diabetes, MODY (young-onset adult-onset diabetes), gestational diabetes, and / or reduction of HbA1c; (ii) Delaying or preventing the progression of diabetic disease, such as the progression of type 2 diabetes; delaying the progression from impaired glucose tolerance (IGT) to insulin-dependent type 2 diabetes; delaying or preventing insulin resistance; and / or delaying the progression from insulin-free type 2 diabetes to insulin-dependent type 2 diabetes; (iii) Improvement of β-cell function, such as a decrease in β-cell apoptosis, an increase in β-cell function and / or β-cell mass, and / or restoration of glucose sensitivity to β-cells; (iv) Prevention and / or treatment of cognitive impairment and / or neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, and / or multiple sclerosis; (v) For example, prevention and / or treatment of eating disorders such as obesity by reducing food intake, weight loss, appetite suppression, or satiety; treatment or prevention of obesity induced by bulimia nervosa, bulimia nervosa, and / or antipsychotic drugs or steroids; reduction of gastric motility, delay of gastric emptying, increase of physical mobility, and / or prevention and / or treatment of comorbidities of obesity such as osteoarthritis and / or urinary incontinence; (vi) Prevention and / or treatment of diabetic complications such as vascular disorders, neuropathy including peripheral neuropathy, nephropathy, and / or retinopathy; (vii) Improvement of lipid parameters, e.g., prevention and / or treatment of dyslipidemia in humans, reduction of total serum lipids, increase of HDL, reduction of small, high-density LDL, reduction of VLDL, reduction of triglycerides, reduction of cholesterol, reduction of plasma levels of lipoprotein a (Lp(a)), and / or suppression of apolipoprotein a (apo(a)) production in vitro and / or in vivo; (viii) Prevention and / or treatment of cardiovascular diseases (Syndrome X, arteriosclerosis, myocardial infarction, coronary heart disease, reperfusion injury, stroke, cerebral ischemia, early heart disease or early cardiovascular disease, left ventricular hypertrophy, coronary artery disease, hypertension, essential hypertension, acute hypertensive emergency, cardiomyopathy, heart failure, exercise intolerance, acute and / or chronic heart failure, arrhythmias, dysarthria, syncope, angina pectoris, cardiac bypass and / or stent reocclusion, intermittent claudication (obstructive arteriosclerosis), diastolic dysfunction, and / or systolic dysfunction; and / or prevention and / or treatment of hypotension (e.g., hypotension); (ix) Prevention and / or treatment of gastrointestinal disorders such as inflammatory bowel disease, short bowel syndrome, Crohn's disease, or colitis, indigestion, and / or gastric ulcers, and / or inflammation such as psoriasis, psoriatic arthritis, rheumatoid arthritis, and / or systemic lupus erythematosus; (x) Prevention and / or treatment of serious illnesses, e.g., treatment of critically ill patients, patients with multiple organ nephropathy (CIPNP) associated with serious illness, and / or patients with potential CIPNP; prevention of the development of serious illness or CIPNP; prevention, treatment, and / or cure of systemic inflammatory response syndrome (SIRS) in patients; prevention or reduction of the likelihood of hospitalized patients developing bacteremia, sepsis, and / or septic shock; and / or stabilization of blood glucose levels, insulin balance, and optionally metabolism in intensive care unit patients with acute illnesses; (xi) Prevention and / or treatment of polycystic ovary syndrome (PCOS); (xii) Prevention and / or treatment of brain diseases such as cerebral ischemia, cerebral hemorrhage, and / or traumatic brain injury; (xiii) Prevention and / or treatment of sleep apnea syndrome, and / or (xiv) Prevention and / or treatment of abuse such as alcohol abuse and / or drug abuse. 85. A fusion compound according to any one of Embodiments 24 to 81, or a pharmaceutical composition according to Embodiment 82, for use in the treatment and / or prevention of all forms of diabetes and related diseases, such as obesity, eating disorders, cardiovascular disease, and diabetic complications; and / or improvement of lipid parameters, improvement of β-cell function; and / or delay or prevention of the progression of diabetic disease; and / or treatment and / or prevention of hepatic steatosis and non-alcoholic fatty liver disease (NAFLD). 86. A fusion compound according to any one of embodiments 24 to 81, or a pharmaceutical composition according to embodiment 82, for use in the prevention and / or treatment of obesity. 87. A fusion compound according to any one of Embodiments 24 to 81, or a pharmaceutical composition according to Embodiment 82, for use in the treatment and / or prevention of fatty liver and non-alcoholic fatty liver disease (NAFLD). 88. A fusion compound according to any one of sections 24 to 81, or a pharmaceutical composition according to Embodiment 82, for use in the treatment and / or prevention of NASH. 89. A fusion compound according to any one of Embodiments 24 to 81, or a pharmaceutical composition according to Embodiment 82, for use in preventing or delaying an increase in relative liver weight, plasma alanine aminotransferase levels, liver triglyceride content, and / or liver cholesterol. 90. A fusion compound according to any one of Embodiments 24 to 81, or a pharmaceutical composition according to Embodiment 82, for use in reducing relative liver weight, plasma alanine aminotransferase levels, liver triglyceride content, and / or liver cholesterol. 91. A fusion compound according to any one of Embodiments 24 to 81, or a pharmaceutical composition according to Embodiment 82, for use in preventing, delaying, and / or reducing inflammation in the liver. 92. A fusion compound according to any one of Embodiments 24 to 81, or a pharmaceutical composition according to Embodiment 82, for use in preventing, delaying, and / or reducing fibrosis in the liver. 93. Use of a fusion compound according to any one of Embodiments 24 to 81, or a pharmaceutical composition according to Embodiment 82, in the manufacture of a pharmaceutical for the treatment and / or prevention of any form of diabetes and related diseases, such as obesity, eating disorders, cardiovascular disease, and diabetic complications; and / or improvement of lipid parameters, improvement of β-cell function; and / or delay or prevention of the progression of diabetic disease; and / or treatment and / or prevention of hepatic steatosis and non-alcoholic fatty liver disease (NAFLD). 94. Use of a fusion compound according to any one of embodiments 24 to 81, or a pharmaceutical composition according to embodiment 82, in the manufacture of a pharmaceutical for the treatment and / or prevention of NASH. 95. Use of a fusion compound according to any one of Embodiments 24 to 81, or a pharmaceutical composition according to Embodiment 82, in the manufacture of a pharmaceutical for the treatment of fatty liver and non-alcoholic fatty liver disease (NAFLD). 96. A method for treating and / or preventing any form of diabetes and related diseases, such as obesity, eating disorders, cardiovascular disease, and diabetic complications, by administering a pharmaceutically active amount of a fusion compound according to any one of embodiments 24 to 81, or a pharmaceutical composition according to embodiment 82; and / or improving lipid parameters, improving β-cell function; and / or delaying or preventing the progression of diabetic disease; and / or treating and / or preventing hepatic steatosis and non-alcoholic fatty liver disease (NAFLD). 97. A method for treating or preventing NASH by administering a pharmaceutically active amount of a fusion compound according to any one of embodiments 24 to 81, or a pharmaceutical composition according to embodiment 82. 98. A method for treating or preventing fatty liver and non-alcoholic fatty liver disease (NAFLD) by administering a pharmaceutically active amount of a compound described in any one of embodiments 24 to 81, or a pharmaceutical composition described in embodiment 82. 99. A method for producing a fusion compound according to any one of Embodiments 24 to 81, comprising the steps of (i) recombination preparation of a skeletal polypeptide, and (ii) covalent bonding of substituents to the skeletal polypeptide (e.g., by alkylation).

[0142] Further specific embodiments A. Formula Chemical Formula 1: ABC (wherein, A is the GLP-1 polypeptide, which is an analog of Sequence ID No. 1. B is a spacer consisting of 1 to 257 amino acids. C is the FGF21 polypeptide, an analog of SEQ ID NO: 2. ) Polypeptides of the polypeptide, and fusion compounds optionally containing one or more substituents, or a pharmaceutically acceptable salt, amide, or ester thereof. B. The fusion compound according to Embodiment A, wherein the C-terminus of a GLP-1 polypeptide is fused to the N-terminus of a peptide spacer, and the C-terminus of the peptide spacer is fused to the N-terminus of an FGF21 analog. C. A fusion compound according to Embodiment A or B, wherein B consists of 5 to 257 amino acids. D. A fusion compound according to any one of Embodiments A to C, wherein B consists of 9 to 129 amino acids. E. B is the chemical formula 2(GAQP) x -C y -(GAQP) h -A j A fusion compound according to any one of Embodiments A to D, comprising or consisting of (wherein x is an integer in the range of 0 to 64, y is an integer in the range of 0 to 1, h is an integer in the range of 1 to 64, and j is an integer in the range of 0 to 1). A fusion compound according to any one of Embodiments A to E, wherein F.B is selected from the list consisting of SEQ ID NOs: 13, 14, 15, 16, 17, and 18. A fusion compound according to any one of Embodiments A to F, wherein G.B is Sequence ID No. 13, Sequence ID No. 16, or Sequence ID No. 18. A fusion compound according to any one of Embodiments A to G, wherein H.A is identical to SEQ ID NO: 1 by at least 50%, preferably at least 55%, preferably at least 60%, preferably at least 65%, preferably at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, or most preferably at least 95%. I. A fusion compound according to any one of Embodiments A to H, wherein A comprises up to 15 amino acid changes, preferably up to 14 amino acid changes, preferably up to 13 amino acid changes, preferably up to 12 amino acid changes, preferably up to 11 amino acid changes, preferably up to 10 amino acid changes, preferably up to 9 amino acid changes, preferably up to 8 amino acid changes, preferably up to 7 amino acid changes, preferably up to 6 amino acid changes, preferably up to 5 amino acid changes, and most preferably up to 4 amino acid changes, compared to Sequence ID No. 1. A fusion compound according to any one of Embodiments A to I, wherein J.A contains or comprises amino acid sequence number 3. A fusion compound according to any one of Embodiments A to J, wherein K.A has 3 to 7 amino acid changes compared to SEQ ID NO: 1. A fusion compound according to any one of the preceding embodiments A to K, wherein L.A contains a Cys residue at a position corresponding to position 26, 27, or 36 of GLP-1(7-37)(SEQ ID NO: 1), or optionally A contains an Arg residue at a position corresponding to position 26 of GLP-1(7-37)(SEQ ID NO: 1). A fusion compound according to any one of Embodiments A to L, wherein M.A has the following amino acid changes compared to GLP-1(7-37)(SEQ ID NO: 1): [8G,22E,26R], [8G,22E,26C,36G], [8G,22E,27C,36G], [8G,22E,26R,27C,36G], [8G,22E,26R,36C], [8G,22E,36G], or [8G,22E,26R,36G]. A fusion compound according to any one of Embodiments A to M, wherein N.A is selected from the list consisting of SEQ ID NOs. 4, SEQ ID NOs. 5, SEQ ID NOs. 6, SEQ ID NOs. 7, SEQ ID NOs. 8, SEQ ID NOs. 9, and SEQ ID NOs. 35. A fusion compound according to any one of Embodiments A to N, wherein O.C is identical to SEQ ID NO: 2 by at least 50%, preferably at least 55%, preferably at least 60%, preferably at least 65%, preferably at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, or most preferably at least 95%. A fusion compound according to any one of Embodiments A to O, wherein P.C. contains up to 10 amino acid changes, preferably up to 9 amino acid changes, preferably up to 8 amino acid changes, preferably up to 7 amino acid changes, preferably up to 6 amino acid changes, preferably up to 5 amino acid changes, preferably up to 4 amino acid changes, and most preferably up to 3 amino acid changes, compared to Sequence ID No. 2. Q. A fusion compound according to any one of Embodiments A to P, wherein C has three or four amino acid changes compared to Sequence ID No. 2. A fusion compound according to any one of Embodiments A to Q, wherein R.C comprises 180C, 180E, or 180A. A fusion compound according to any one of Embodiments A to R, wherein S.C contains the following amino acid changes compared to FGF21(1-181)(SEQ ID NO: 2): [121Q,168L,180C] or [121Q,168L,171G] or [121Q,168L,171G,180E]. A fusion compound according to any one of Embodiments A to S, wherein T.C is selected from the list consisting of SEQ ID NOs: 10, SEQ ID NOs: 11, and SEQ ID NOs: 12. A fusion compound according to any one of embodiments A to T, wherein U.C. has FGF21 activity. A fusion compound according to any one of embodiments A to U, wherein V.C can activate FGFR1c. The fusion compound according to any one of Embodiments A to V, wherein the components of Chemical Formula 1 are linked via an amide bond.

[0143] List of Abbreviations Area Under the Curve (AUC) BEH Ethylene Bridged Hybrid Baby Hamster Kidney (BHK) β-Crot (BKL) Brain Natriuretic Peptide (BNP) Bovine Serum Albumin (BSA) Bis(p-sulfonatophenyl)phenylphosphine Dihydrate Dipotassium Salt (BSPP) Cholesterol Ester Transfer Protein (CETP) Chinese Hamster Ovary (CHO) Cleaning In Place (CIP) Cytomegalovirus (CMV) Column Volume (CV) Cardiovascular Disease (CVD) Dulbecco's Modified Eagle Medium (DMEM) Dipeptidyl Peptidase-IV (DPP-IV) Dithiothreitol (DTT) Escherichia coli (E. Coli) Epithelial Growth Factor-Like Domain A (EGF(A)) Enzyme-Linked Immunosorbent Assay (ELISA) Ethylenediaminetetraacetic Acid (EDTA) Extracellular Signal-Regulated Kinase (ERK) Fetal Bovine Serum (FBS) Fibroblast Growth Factor 21 (FGF21) Fibroblast Growth Factor Receptor (FGF-R) Glucagon-Like Peptide-1 (GLP-1) 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic Acid (HEPES) High-Density Lipoprotein Cholesterol (HDLc) Horseradish Peroxidase (HRP) Human Serum Albumin (HSA) Immobilized Metal Affinity Chromatography (IMAC) IPTG Isopropyl β-d-1-thiogalactopyranoside LB Luria-Bertani (Sambrook, J., EFFritsch, and T. Maniatis. (1989). Molecular cloning: a laboratory manual, 2nd edition. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.) LPa lipoprotein a LCMS (Liquid Chromatography Mass Spectrometry) LDL-C (low-density lipoprotein cholesterol) LDL-R low-density lipoprotein receptor LOCI (Luminescent Oxygen Channeling) Immunoassay HDL-C (High-Density Lipoprotein Cholesterol) MAPK Mitogen-Activated Protein Kinase MQ Milli-Q MSD Mass Selective Detector MWCO Molecular Weight Cutoff NAFLD (Non-Alcoholic Fatty Liver Disease) NEP Neutral Endopeptidase PBS (phosphate-buffered saline) PCA polyclonal antibody PCSK9 Proprotein Converter Subtilisin / Kexin Type 9 Pen / strep Penicillin-Streptomycin SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis) SGLT2 selective sodium-glucose cotransporter 2 TB Terrible Broth TFA (Trifluoroacetic Acid) TIC Total Ion Current Time-of-Flight (TOF) flight time type Tris (hydroxymethyl)aminomethane, or 2-amino-2-hydroxymethylpropane-1,3-diol UPLC Ultra-High-Speed ​​Liquid Chromatography VLDL-C (Very Low Density Lipoprotein Cholesterol)

[0144] Method for preparing fusion compounds The polypeptides prepared for the fusion compounds of the present invention can be commonly used in the art.

[0145] General method for polypeptide preparation in the E. coli host system

[0146] Cloning and expression A fusion protein backbone with a removable N-terminal extension was produced by recombination. This was done by expressing a DNA sequence encoding an amino acid sequence, subcloning it into a pET11d-derived vector, and subsequently transforming a suitable host cell, i.e., *Escherichia coli* BL21(DE3) or a derivative thereof. The DNA sequence was of synthetic origin and obtained from commercial providers (e.g., Thermofisher, Genescript).

[0147] E. coli transformation Transformation of E. coli was performed by Sambrook et al. (1989) [Sambrook J, Fritsch EF, Maniatis T.; Molecular Cloning: A Laboratory Manual, 2 nd Transformation was performed by electroporation using a Bio-Rad Gene Pulser set to 25 μF, 200 ohms, and 2.5 kV in a 2 mm cuvette, according to the standard method described in [edn; Cold Spring Harbor Laboratory Press: New York; 1989] or according to Dower et al. (1988) [Dower, WJ, Miller, JF, & Ragsdale, CW (1988) Nucleic Acids Res. 16, 6127-6145]. Transformed cells were selected on LB medium supplemented with appropriate selective antibiotics, namely ampicillin and / or kanamycin.

[0148] Cultivation of Escherichia coli Escherichia coli cells transformed with plasmid DNA were taken from a frozen stock or directly from fresh transformation on an LB plate (using the appropriate antibiotic). The cells were inoculated into a 500 ml Corning® disposable Erlenmeyer flask filled with 100 ml of LB medium and the appropriate antibiotic. The cells were grown overnight at 30 °C with shaking at 220 rpm. 40 ml of cells from the pre-culture were diluted into 2000 ml of TB medium filled in a 5-L Corning® Disposable Erlenmeyer Flask. The cells were cultured at 37 °C until the OD600 reached 2.0. Then, target protein expression was induced by the addition of 1 mM IPTG and further cultured at 37 °C. The expression samples were analyzed by SDS-PAGE. The inclusion body fraction was isolated and collected using sonication and centrifugation as described in the following paragraph.

[0149] Recovery of inclusion bodies The cell slurry in 20 mM histidine, 150 mM NaCl buffer pH 6.0 was lysed with a disruptor (900 bar, 4 passes) and pelleted by centrifugation (6000×g, 30 minutes). The inclusion bodies were washed twice with 20 mM histidine (aqueous solution), 1 M sodium acetate (aqueous solution), 0.1% Triton and once with water (MilliQ) and analyzed by SDS-PAGE.

[0150] SDS-PAGE SDS-PAGE was performed using NuPAGE™ Bis-Tris (Thermofisher) 4-12% gels according to the provided protocol to analyze the expression samples, which is standardly carried out in the art.

[0151] Refolding of the polypeptide The inclusion bodies were solubilized in 6M urea in 20 mM ethanolamine pH 9.0 and 20 mM cysteamine. The solution was diluted in refolding buffer (20 mM Tris, pH 8.0) to a final polypeptide concentration of 1 mg / ml. The refolding process was carried out at room temperature for at least 12 hours. Insoluble impurities were then removed by centrifugation (7000 × g, 45 minutes).

[0152] Purification of refolded polypeptides The refolded polypeptide solution was loaded onto Q Sepharose Big Beads resin (GE Healthcare) using anion exchange chromatography (20 mM Tris pH 8.0, 0–500 mM NaCl) as commonly described in Protein Purification. Principles and Practice Series: Springer Advanced Texts in Chemistry Scopes, Robert K. 3rd ed., 1994 (Chapters 6 and 8). The N-terminal extension was removed by enterokinase cleavage to obtain the specific N-terminus of the target polypeptide. The resulting polypeptide solution was applied to Capto Phenyl highsub (GE Healthcare) (10 mM Tris pH 8.0, 1.5–0 mM NaCl) to remove enterokinase and low molecular weight impurities. The resulting pool was packed into SOURSE30Q (GE Healthcare) (20 mM Tris pH 8.0, 0–250 mM NaCl) for polishing. The final pool of target polypeptides was concentrated to 5 mg / ml and frozen for storage. [Table 5] [Table 6]

[0153] General methods for introducing substituents into polypeptides and purifying fusion compounds The reagent (Chemical Formula 7) necessary for introducing substituents onto the polypeptide was prepared as described in International Publication No. 2016 / 102562. 17-{(S)-1-carboxy-3-[2-(2-{[2-(2-{[2-(2-bromoacetylamino)ethylcarbamoyl]methoxy}ethoxy)ethylcarbamoyl]methoxy}ethoxy)ethylcarbamoyl]propylcarbamoyl}heptadecanoic acid [ka] chemical formula 7

[0154] Alkylation The frozen polypeptide solution was thawed and then concentrated to over 1 mg / ml using a spin filter MWCO 10 kDa (30 minutes, 3000 rpm). The pH was adjusted to 8.5 with aqueous NaOH solution, and 5 equivalents of BSPP per capped cysteine ​​were added. After stirring for 2-3 hours, 4-5 equivalents of chemical formula 7 in 0.1 M NaHCO3 (aqueous solution) were added per free cysteine. The mixture was gently stirred in the dark for 1.5-16 hours. The reaction mixture was diluted with water before purification by anion exchange using an Akta system. [Table 7]

[0155] The pure fractions were pooled and buffer-exchanged using an Akta system to either buffer B1: 8 mM phosphate, 240 mM propylene glycol, 0.007% Tween 20, pH=8.2, or buffer B2: 10 mM phosphate, 2% (w / vol) glycerol, pH=8.2. [Table 8] If necessary, the pooled fractions were concentrated to 3-5 mg / ml using a spin filter (30 minutes, 3000 rpm).

[0156] Methods for general detection and methods for characterization Dilute the sample to approximately 1 mg / ml and inject it into the LC-MS system (e.g., 1 μl). Desalt the analog. The instrument should be calibrated, and if possible, calibrated using a lock mass spray. Generate an MS spectrum across the primary chromatographic peaks and reconstruct the intact mass using a deconvolution algorithm. [Table 9] [Table 10] [Table 11]

[0157] Examples 1.1-1.17 [Table 12-1] [Table 12-2] [Table 12-3] [Table 12-4]

[0158] Example 1.1 [8G,22E,26R]GLP-1(7-37)-(GAQP)x4,A- S{beta-180}-[2-[2-[[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoyl-amino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]-ethylamino]-2-oxoethyl]- [121Q,168L,180C]FGF21(1-181) [ka] Chemical formula 8; Purification using buffer B1. LC-MS method 1: Calculated mass: 25161.1; Measured mass: 25161.9.

[0159] Example 1.2 S{beta-26}-[2-[2-[[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoyl-amino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]-ethylamino]-2-oxoethyl]-[8G,22E,26C,36G]GLP-1(7-37)-(GAQP)x4,A-[121Q,168L,171G]FGF21(1-181) [ka] Chemical formula 9; Purification using buffer B1. LCMS36: Calculated mass: 24936.6; Measured mass: 24936.0

[0160] Example 1.3 S{beta-27}-[2-[2-[[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoyl-amino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]-ethylamino]-2-oxoethyl]-[8G,22E,27C,36G]GLP-1(7-37)-(GAQP)x4,A-[121Q,168L,171G,180E]FGF21(1-181) [ka] Chemical formula 10; Purification using buffer B1. LCMS47: Calculated mass: 24993.7; Measured mass: 24994.0

[0161] Example 1.4 S{beta-27}-[2-[2-[[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoyl-amino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]-ethylamino]-2-oxoethyl]-[8G,22E,27C,36G]GLP-1(7-37)-(GAQP)x8,A-[121Q,168L,171G,180E]FGF21(1-181) [ka] Chemical formula 11; Purification using buffer B1. LCMS47: Calculated mass: 26407.2; Measured mass: 26408.0

[0162] Example 1.5 S{beta-27}-[2-[2-[[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoyl-amino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]-ethylamino]-2-oxoethyl]-[8G,22E,27C,36G]GLP-1(7-37)-(GAQP)x16,A-[121Q,168L,171G,180E]FGF21(1-181) [ka] chemical formula 12, Purification using buffer B1 or B2. LCMS36: Calculated mass: 29234.2; Measured mass: 29234.0

[0163] Example 1.6 S{beta-36}-[2-[2-[[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoyl-amino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]-ethylamino]-2-oxoethyl]-[8G,22E,26R,36C]GLP-1(7-37)-(GAQP)x4,A-[121Q,168L,171G,180E]FGF21(1-181) [ka] chemical formula 13, Purification using buffer B1. LCMS36: Calculated mass: 25093.8; Measured mass: 25094.0

[0164] Example 1.7 S{beta-27}-[2-[2-[[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoyl-amino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]-ethylamino]-2-oxoethyl]- [8G,22E,27C,36G]GLP-1(7-37)-(GAQP)x4,A- S{beta-180}-[2-[2-[[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoyl-amino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]-ethylamino]-2-oxoethyl]-[121Q,168L,180C]FGF21 [ka] chemical formula 14, Purification using buffer B1 or B2. LCMS47: Calculated mass: 25823.8; Measured mass: 25824.0

[0165] Example 1.8 S{beta-26}-[2-[2-[[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoyl-amino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]-ethylamino]-2-oxoethyl]-[8G, 22E,26C,36G]GLP-1(7-37)-(GAQP)x4,A- S{beta-180}-[2-[2-[[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoyl-amino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]-ethylamino]-2-oxoethyl]-[121Q,168L,180C]FGF21 [ka] chemical formula 15, Purification using buffer B2. LCMS47: Calculated mass: 25824.7; Measured mass: 25826.0

[0166] Example 1.9 S{beta-27}-[2-[2-[[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoyl-amino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]-ethylamino]-2-oxoethyl]- [8G,22E,26R,27C,36G]GLP-1(7-37)-(GAQP)x4,A- S{beta-180}-[2-[2-[[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoyl-amino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]-ethylamino]-2-oxoethyl]-[121Q,168L,180C]FGF21 [ka] chemical formula 16, Purification using buffer B1 or B2. LCMS47: Calculated mass: 25851.8; Measured mass: 25854.0

[0167] Example 1.10 S{beta-27}-[2-[2-[[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoyl-amino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]-ethylamino]-2-oxoethyl]-[8G,22E,27C,36G]GLP-1(7-37)-(GAQP)x4,A-[121Q,168L,171G]FGF21(1-181) [ka] chemical formula 17, Purification using buffer B1. LCMS47: Calculated mass: 24935.7; Measured mass: 24937.0

[0168] Example 1.11 S{beta-27}-[2-[2-[[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoyl-amino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]-ethylamino]-2-oxoethyl]-[8G,22E,27C,36G]GLP-1(7-37)-(GAQP)x8,A-[121Q,168L,171G]FGF21(1-181) [ka] chemical formula 18 Purification using buffer B1. LCMS47: Calculated mass: 26351.2; Measured mass: 26351.0

[0169] Example 1.12 [8G,22E,26R,36G]GLP-1(7-37)-(GAQP)x2,S{beta}-[2-[2-[[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoyl-amino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]-ethylamino]-2-oxoethyl ]C,GAQPx2,AS{beta-180}-[2-[2-[[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoyl-amino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]-ethylamino]-2-oxoethyl]-[121Q,168L,180C]FGF21 [ka] chemical formula 19, Purification using buffer B1 or B2. LCMS47: Calculated mass: 25980.9; Measured mass: 25982.0

[0170] Example 1.13 [8G,22E,36G]GLP-1(7-37)-(GAQP)x2,S{beta}-[2-[2-[[2-[2-[[2-[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoyl-amino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]-ethylamino]-2-oxoethyl]C,GAQPx2,A[121Q,168L,171G]FGF21 [ka] chemical formula 20 Purification using buffer B1. LCMS47: Calculated mass: 25064.8; Measured mass: 25067.0

[0171] Example 1.14 S{beta-27}-[2-[2-[[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoyl-amino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]-ethylamino]-2-oxoethyl]-[8G,22E,26R,27C,36G]GLP-1(7-37)-(GAQP)x4,A-[121Q,168L,171G,180E]FGF21(1-181) [ka] chemical formula 21 Purification using buffer B1. LCMS36: Calculated mass: 25021.7; Measured mass: 25023.0

[0172] Example 1.15 [8G,22E,26R,36G]GLP-1(7-37)-(GAQP)x2,S{beta}-[2-[2-[[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoyl-amino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]-ethylamino]-2-oxoethyl]C,GAQPA,[121Q,168L,171G]FGF21 [ka] chemical formula 22 Purification using buffer B1. LCMS47: Calculated mass: 24739.4; Measured mass: 24741.0

[0173] Example 1.16 {S27-[(28S)-(28,46-Dicarboxy-2,7,16,25,30-Pentaoxo-9,12,18,21-Tetraoxa-3,6,15,24,29-Pentazahexatetracontan-1-yl)]-[Gly8,36,Glu22,Cys27]-GLP-1(Human)-(7-37)-Peptidyl}-(Gly-Ala-Gln-Prolinyl)32-Ala-[Gln121,Leu168,Gly171,Glu180]-FGF21(Human)-(1-181)-Peptide [ka] chemical formula 23 Purification using buffer B2. LCMS47: Calculated mass: 34888.2; Measured mass: 34888.0

[0174] Example 1.17 - Reference Compound N-epsilon26-[2-(2-[2-(2-[2-(2-[4-(17-carboxyheptadecanoylamino)-4(S)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Aib8,Arg34]GLP-1-(7-37)peptide [ka] Chemical formula 24, Sequence ID 37 This compound was prepared as described in International Publication No. 06097537.

[0175] Example 1.18 - Reference Compound 2 S{beta-180}-[2-[2-[[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]-ethylamino]-2-oxoethyl]-Ala[Gln121,Leu168,Cys180]FGF21(1-181) [ka] Chemical formula 25, Sequence ID 38 This compound was prepared as described in International Publication No. 1016102562.

[0176] Common methods for measuring GLP-1 activity To determine the GLP-1 receptor (GLP-1R) activity or potency of the GLP-1 moiety of a GLP-1 / FGF21 fusion compound and to assess how receptor activation may be affected by the presence of serum albumin, in vitro potency assays were performed in human GLP-1 receptor-expressing cells in the absence and presence of 1% (w / v) human serum albumin (HSA) as described below.

[0177] An increase in EC50 value (decreased potency) in the presence of serum albumin indicates binding to serum albumin and represents a method for predicting the long-term pharmacokinetic profile of a test substance in animal models.

[0178] The stronger the effect of a compound, the lower its EC50 value. A compound is considered a very potent GLP-1 receptor agonist if its EC50 is below approximately 50 pM, such as 20 pM, and EC50 is measured using the assay described herein without the addition of HSA. A compound is considered to have moderate potency if its EC50 value is between 50 and 250 pM. A compound is considered to have less potency if its EC50 value is between 250 and 1000 pM. A compound is considered ineffective if its EC50 value is above 1000 pM.

[0179] Assay principle Activation of the GLP-1 receptor leads to an increase in cellular concentrations of cyclic AMP (cAMP). Consequently, transcription is activated by a promoter containing multiple copies of the cAMP response element (CRE). Therefore, it is possible to measure GLP-1 receptor activity using a CRE luciferase reporter gene introduced into baby hamster kidney (BHK) cells co-expressing the GLP-1 receptor.

[0180] Cells and assay reagents Cell stocks were prepared by culturing stably transfected cell lines expressing the human GLP-1 receptor and CRE-responsive luciferase (CRE-Luc) reporter gene (BHK 467-12A KZ-10, prepared according to methods known to those skilled in the art) in growth medium consisting of 10% FBS (Gibco, 16140-071), 1% penicillin / streptomycin (Gibco, 15140-122), 1 mM Na-piruvate (Gibco, 11360-039), 1 mg / mL G418 (Gibco, 10131-027), and 240 nM MTX (Pfizer, 15936) in DMEM (Gibco, 61965-026). Cells at approximately 80-90% confluence were washed once with PBS and detached from the cell flask using Versene (Gibco, 15040-033). After centrifugation, the cell pellet was lysed and diluted to 1.5 × 10⁶ cells / mL in a medium consisting of 20% FBS (Gibco, 16140-071), 1% penicillin / streptomycin (Gibco, 15140-122), 1 mM Na-pirubate (Gibco, 11360-039), 1 mg / mL G418 (Gibco, 10131-027), 240 nM MTX (Pfizer, 15936), and DMEM (Gibco, 61965-026) supplemented with 10% DMSO (Sigma, D2650). The cells were divided equally and stored at -180°C until use.

[0181] The assay buffer consisted of DMEM supplemented with 1X GlutaMAX (Gibco, 35050-038), 10 mM HEPES (Gibco, 15630-056), 1% (w / v) ovalbumin (Sigma, A5503), and 0.1% (v / v) Pluronic F-68 (Gibco, 24040-032), without phenol red (Gibco, 11880-028).

[0182] procedure To perform the assay, serial dilutions (10-fold dilution per compound, 8 concentrations) of the reference compound and the GLP-1 / FGF21 fusion compound were performed in HSA-free assay buffer, often starting at approximately 100–200 nM in 96-well plates. Frozen stocks of human GLP-1R / CRE-Luc cells were thawed in a 37°C water bath, washed once in PBS, and diluted to 100,000 cells / mL in assay buffer with or without 2% (w / v) HSA (Sigma, A9511). For each dilution, a 50 μL aliquot of the reference compound or GLP-1 / FGF21 coagonist was transferred to two 96-well assay plates (ThermoFisher, 237105) with 50 μL of cell suspension (5,000 cells / well) added with or without 2% (w / v) HSA. The assay plate was incubated in 5% CO2 at 37°C for 3 hours, then left at room temperature for 5 minutes, after which 100 μL of SteadyLite Plus (PerkinElmer, 6066759) was added to each well. The plate was sealed and incubated at room temperature for 30 minutes with gentle shaking, protected from light. Luminescence was detected using a luminescence plate reader, e.g., Synergy 2 (BioTek). EC 50 The value [pM] was calculated by nonlinear curve fitting using a 4-parameter logistic model (hill slope = 1) with GraphPad Prism, or by using TIBCO Enterprise Runtime for R (TIBCO Software, Palo Alto, California, USA).

[0183] Example 2 The GLP-1 activity of exemplary compounds was investigated using standard methods. The results are shown in Table 6 (average of at least two distinct concentration-response curves). All exemplary compounds showed GLP-1 activity. [Table 13]

[0184] All fusion compounds exhibited activity against the GLP-1 receptor, which was comparable or higher in the presence of HSA. In vitro GLP-1R potency was dependent on the alkylation site. For both monoalkylated and bialkylated compounds, the highest potency was found for alkylation at 27C, followed by 36C, and then 26C. In the presence of albumin, the same pattern was observed, but the bialkylated compounds lost significantly higher potency compared to the monoalkylated compounds. For the monoalkylated 27C derivative, the decrease in potency in the presence of albumin increased with increasing spacer length, but 32xGAQP, the longest spacer in chemical formula 23, had comparable potency to chemical 24 in the presence of albumin. 26R did not appear to affect in vitro potency. Because chemical formula 24 has an extension factor at position 26, it is surprising that in the fusion context, the loss of potency was more than 10 times for the monoalkylated compound in the absence of HSA, and for the bialkylated version, a 40-fold loss was observed in the absence of HSA and a 26-fold loss in the presence of HSA.

[0185] General methods for investigating FGF21 activity The purpose of this example was to test the activation of the FGF21 receptor of the fusion compound in the example. The potency of the FGF21 receptor in vitro was measured by FGF receptor activation in a whole-cell assay.

[0186] The efficacy of the GLP-1 / FGF21 fusion compound in Example 2 was measured in HEK (human embryonic kidney cells) overexpressing human beta-clot (BKL) according to the method described below.

[0187] To verify the albumin binding of the GLP-1 / FGF21 fusion derivative, assays were performed under conditions without serum albumin and in the presence of human serum albumin (HSA) (final assay concentration 0.1%). The increase in EC50 value (decreased potency) of the FGF21 derivative in the presence of serum albumin suggests binding to serum albumin and serves as a method for predicting the sustained pharmacokinetic profile of the test substance in animal models. The results for the GLP-1 / FGF21 fusion compounds are shown in Table 7. Chemical formula 25 is included for reference purposes.

[0188] Assay principle HEK293 cells endogenously express several FGF receptors, including FGFR1c, FGFR3c, and FGFR4. These cells do not respond to FGF21 until transfected with the co-receptor beta-cloto (BKL). Activation of the FGF receptor / BKL complex leads to activation of the MAPK / ERK signaling pathway and phosphorylation of ERK. The level of phosphorylated ERK (pERK) at a given time point increases with increasing FGF21 concentration. As described below, pERK levels were measured after 12 minutes of stimulation at various test compound concentrations.

[0189] Assay description 1) Day 1: HEK293 / beta-clotocytes were seeded at a rate of 5,000 cells / well in 96-well plates containing 4500 mg / l glucose (Gibco #31966-021) supplemented with 10% HI FBS (Gibco #16140-71), 1% penicillin / streptomycin (Gibco #15140-122), and 100 μg / ml hygromycin B (Gibco #10687-010). 2) Day 2: Two hours before adding the test compound, the cell medium was replaced with 100 μl of basal medium (DMEM containing 4500 mg / l glucose (Gibco #31966-021)). 3) The test compound was diluted in 2× assay medium (DMEM containing 4500 mg / l glucose (Gibco #31966-021) supplemented with 0.01% Tween20), warmed to 37°C, added to cells in basal medium (100 μl), and incubated at 37°C for 12 minutes. The test compound was tested in and without 0.1% HSA (Sigma-A1887). 4) All culture medium was rapidly removed, and 50 μl of lysis buffer was added per well. The plate was shaken for 10 minutes, and the lysate was prepared for pERK measurement. 5) pERK is measured in a 384-well plate using the AlphaScreen SureFire kit (PerkinElmer # TGRESB10K). This kit is based on ERK and pERK-specific antibodies bound to donor beads and acceptor beads. The presence of pERK brings the acceptor beads and donor beads closer together and generates a signal that can be read on the EnVision plate reader.

[0190] The data was analyzed using GraphPad Prism software. 50 The values ​​were calculated using nonlinear regression via software and reported in nM.

[0191] Example 3 The FGF21 activity of the exemplary compounds was investigated using standard methods. The results are shown in Table 7 (average of at least two independent experiments). All exemplary compounds showed FGF21 activity. [Table 14]

[0192] As can be seen from Table 7, all fusion compounds showed higher FGF21 receptor potency compared to the FGF21 moiety alone (including the protractor substituent). Generally, FGF21 potency was improved compared to chemical formula 25. In the case of monoalkylated compounds, the potency in the presence of HSA had only a slight effect, which is most likely due to the absence of an albumin-binding agent in the FGF21 moiety of the fusion. The bialkylated compounds showed improved potency compared to chemical formula 25 in the absence of HSA, but in the presence of HSA, the potency was equivalent to chemical formula 25, and in the presence of albumin, they showed a larger shift compared to chemical formula 25.

[0193] Example 4: A general method for investigating mean residence time (pharmacokinetics) The objective of this study was to determine the average residence time of compounds in different species. Mice, miniature pigs, and cynomolgus monkeys were selected for this purpose.

[0194] Mean residence time (MRT) MRT was performed because an early, rapid decrease in plasma concentration was observed. 1 / 2 It was decided to use this instead, which may indicate that the distribution phase of the PK profile is fast and the terminal disappearance phase is very slow. To avoid overemphasizing the slow terminal disappearance phase, and because this phase has few data points and a very low AUC, it is likely to be less effective, so it was decided to adopt MRT, a more robust parameter based on the total AUC of the PK profile that encompasses all the data in the profile. The results are shown in Tables 8, 11 and 12.

[0195] Example 4.1: Tests in mice Administration and Sampling: 7-8 week old mice (male C57BL / 6J (Mus musculus), Janvier Labs, Le Genest-Saint-Isle, France) were used in the study. The study used a sparse sampling method with n=15 mice per test compound, and blood was collected two or three times from each mouse, resulting in n=3 at each time point. The animals were intravenously administered a dose of 5 nmol / kg of the test compound (in a buffer solution of 8 mM phosphate, 240 mM propylene glycol, 0.007% polysorbate 20, pH=8.2) at a concentration of 1 nmol / ml. Approximately 100 μl of blood was collected from the sublingual plexus of conscious mice according to the following regimen: 5 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 18 hours, 24 hours, 30 hours, 48 ​​hours, and 72 hours after administration. Blood was collected in tubes coated with EDTA (Sarstedt microvette 1293) and placed on ice until centrifuged at 6000G for 5 minutes at 4°C. Plasma (approximately 50 μl) was transferred to a 0.7 ml Micronic tube and stored at -20°C until analysis.

[0196] Bioassay: Since there are no immuno-based assays for detecting GLP-1 / FGF21 compounds in plasma, we decided to use assays to detect GLP1 and FGF21 separately, using combinations of antibodies that react to the GLP1 and FGF21 moieties. The selected GLP1 assay was able to detect GLP1 with an intact N-terminus, and the selected FGF21 assay was able to detect the intact C-terminus of FGF21.

[0197] FGF21 assay Sample Analysis: Samples were analyzed using luminescent oxygen channeling immunoassay (LOCI or AlphaLISA). Donor beads were coated with streptavidin (lot number 2298681, PerkinElmer, USA) according to a standard protocol, while acceptor beads were conjugated with an in-house produced monoclonal antibody (lot number 2356893, PerkinElmer, USA) according to a standard procedure specific to the C-terminal epitope of the FGF21 polypeptide of the test compound (exemplified by Lu et al (Journal of Biomedical Science (2020) 27:1)). A secondary antibody, a commercially available polyclonal antibody against human FGF21 (lot BAF2539, R&D systems, USA), was biotinylated according to a standard procedure. The three reactants were combined with their respective analytes to form two-site immune complexes. Irradiation of the complexes released a single oxygen atom from the donor beads. These were guided to acceptor beads, which induced a chemiluminescent response, which was measured using an EnVision plate reader (PerkinElmer, USA). The amount of light was proportional to the concentration of the test compound.

[0198] The protocol was as follows: 5 μL of plasma sample and assay accuracy control calibrators (high, medium, and low concentrations within the calibrator range) were applied to appropriate wells of a white 384-well plate, followed by a 15 μL mixture of the aforementioned in-house monoclonal antibody (0.5 μg / well) and acceptor beads coated with biotinylated PCA human FGF21 (4.5 nM / well). The plate was incubated at room temperature for 1 hour. Then, 30 μL of streptavidin-coated donor beads (2 μg / well) were added to each well and incubated at room temperature for 30 minutes. The plate was read at room temperature using an Envision plate reader (Perkin Elmer) with excitation using a filter with a bandwidth of 520–645 nm and a 680 nm laser. The total measurement time per well was 210 ms, including a 70 ms excitation time.

[0199] Data Analysis and Reporting: Plasma concentration-time data after a single IV dose were subjected to non-compartmental analysis using Phoenix® and WinNonlin® (Pharsight®, St. Louis, Missouri, USA). The area under the plasma concentration-time profile was calculated using the log-linear trapezoidal method. The area under the first moment curve was also calculated using the log-linear trapezoidal method. The mean residence time was calculated as the ratio of the area under the first moment curve to the area under the plasma concentration-time curve. The terminal phase half-life was calculated using "individual best fits" of log-linear regression of concentration-time and reported as the harmonic mean and pseudo-standard deviation.

[0200] GLP-1 assay The assays and calculations were performed in the same manner as described above for the FGF21 assay, specifically using a proprietary monoclonal antibody with biotin labeling against the N-terminus of GLP-1 and a proprietary monoclonal antibody against the GLP-1 intermediate conjugated to receptor beads. [Table 15]

[0201] The mean residence times observed using the GLP-1 assay and the FGF21 assay were similar, ranging from 2.5 hours to 21 hours (2.5 hours to 17 hours using the GLP-1 assay and 2.4 hours to 21.1 hours using the FGF21 assay). The MRT of monoalkylated compounds increased as the spacer length increased from 2.5 hours (GLP-1 assay, chemical formula 21) to 9.8 hours (GLP-1 assay, chemical formula 23). Bialkylated compounds showed significantly longer MRTs, with compound chemical 19 having the longest MRTs of 17.0 hours and 21.1 hours, respectively, using the GLP-1 assay and the FGF21 assay.

[0202] Example 4.2: Tests in miniature pigs The overall objective is to confirm that compound chemical formulas 16, 19, and 23 have clearance and terminal phase half-life / MRT consistent with once-weekly dosing in humans.

[0203] Administration The study was conducted on 18 female Gottingen minipigs (18 animals) from Ellegaard Gottingen Minipigs A / S, Soro Landevej 302, DK-4261 Dalmose. At the start of the acclimatization period, the pigs weighed approximately 15-20 kg and were 7-9 months old. The animals were administered intravenously or subcutaneously according to the following schedule:

[0204] Dosage schedule [Table 16] Intravenous administration: The compound was administered intravenously (IV) via an ear vein catheter. After administration, 10 mL of sterile saline solution was flushed through the catheter. Subcutaneous administration: The compound was administered using a 1 mL or 2 mL syringe and a 25 G x 5 / 8 (orange needle) needle (with a stopper on the needle) to a depth of 5 mm, or a 21 G x 3 / 4 butterfly needle (with a stopper on the needle) to a depth of 5 mm. Subcutaneous administration was performed on the side of the neck of miniature pigs. The needle was held in the skin for approximately 10 seconds after administration.

[0205] Blood sample Complete plasma concentration-time profiles were obtained from each animal. Blood samples were collected via ear vein catheters according to the following schedule.

[0206] Blood (1.3 ml) was collected in an EDTA tube (a 1.3 ml tube containing K3EDTA to obtain 1.6 mg of K3EDTA / ml blood (Sarstedt, Germany)). After each blood sample collection, the catheter was washed with 10 ml of sterile 0.9% NaCl solution and 10 IE / ml heparin.

[0207] The samples were held on moist ice for up to 30 minutes before centrifugation (10 minutes, 4°C, 2000×g), and then transferred to Micronic tubes for exposure measurement. [Table 17]

[0208] Bioanalysis GLP-1 / FGF21 fusion compounds were assayed in porcine plasma by immunocapsulation, protease digestion, and liquid chromatography-mass spectrometry (LC-MS). The GLP-1 / FGF21 fusion compounds were quantified using surrogate peptides covering the N-terminal and C-terminal portions of the compounds.

[0209] In short, calibrators were prepared by spiking blank plasma with relevant fusion compounds in the range of 1–200 nM. 25 μL of calibrator, blank plasma, or test sample was mixed with 25 μL of blank porcine plasma (containing a 50 nM internal standard), 195 μL of PBS buffer, and 5.5 μL of biotinylated monoclonal antibody (in-house manufactured and specific to the central region of FGF21 (PGQKSPHRDPAPRGP)). The mixture was incubated at 37°C for 2 hours. After incubation, 25 μL of Dynabeads MyOne Streptavidin T1 magnetic beads (10 mg / ml, ThermoFisher Scientific) were added, and the mixture was incubated at room temperature for 1 hour. After incubation, the beads were washed three times with PBS buffer, and the GLP-1 / FGF21 fusion compound was eluted from the beads with 100 μL of elution buffer containing 10% acetonitrile, 1% formic acid, and 0.005% Tween20 in Milli-Q water. After elution, 50 μL of trypsin or LysC (0.02 μL in 1M TRIS buffer (pH 9)) was added to the elution buffer. Digestion was completed by adding 4.5 μL of formic acid after 18 hours at 37°C. The mixture was centrifuged, and the supernatant was transferred to a microtiter plate (coated with BSA). The mixture was analyzed by LC-MS using either a Thermo Accucore 150-C4 column (100 × 2.1 mm inner diameter; 2.6 μm) operated at 60°C, or a Waters Acquity UPLC Peptide BEH C18 column (300 angstroms, 1.7 μm, 2.1 × 100 mm) operated at 80°C. Gradient elution was performed using a Nexera UHPLC system (Shimadzu Corporation) with mobile phase A (Milli-Q water containing 0.1% formic acid and 5% acetonitrile) and mobile phase B (acetonitrile containing 0.1% formic acid and 5% Milli-Q water). The flow rate was 0.6 ml / min. A TripleTOF 5600 mass spectrometer (Sciex) was used as the detector and operated in cationization mode. The concentration in the plasma sample was calculated using a calibration curve. Including quality control samples, the deviation between nominal concentration and calculated concentration was less than 20% (25% for LLOQ).

[0210] Pharmacokinetic analysis Plasma concentration-time data were analyzed by non-compartmental pharmacokinetics using Phoenix 8 (Certara, Princeton, New Jersey 08540, USA).

[0211] Calculations were performed using the individual concentration-time values from each animal at each time point. The following pharmacokinetic parameters were calculated at each time point: AUC, AUC / dose, AUC %Extrapol , C0, C max , λ z , t max , t 1 / 2 , CL, CL / f, V z , V z / f, V ss , MRT and f.

[0212] Treatment of concentration values below the lower limit of quantification (LLOQ) Plasma concentrations below the LLOQ were treated as follows: Plasma concentrations before dosing (0 hours) below the limit of detection were displayed as "<LLOQ" in the data file. The values were automatically taken as zero by Phoenix.

[0213] If the concentration value after one dose was quantifiable and another value at the same dose and time was below the LLOQ, the latter was set to 1 / 2 LOD for the calculation of the average concentration. If the resulting average value was below the LLOQ, this value was replaced with "<LLOQ" in the data file and was not included in the calculations as a result.

[0214] If the (average) concentration value after one dose was below the LLOQ and subsequent (average) values were quantifiable, the former was set to 1 / 2 LLOQ in the calculation of pharmacokinetic parameters.

Table 18

[0215] The results indicate that the same MRT(h) was obtained for both the N-terminal and C-terminal peptides after intravenous administration. After subcutaneous administration, the MRT of the N-terminal portion of the monoalkylated compound (chemical formula 23) was longer than that of the C-terminal portion. The opposite was true for the bialkylated compound, which had one protractor within the spacer. In general, the bialkylated compounds exhibited the longest MRTs. All compounds were evaluated to have an MRT in pigs of sufficient length to support low doses in humans.

[0216] Example 4.3: Tests in cynomolgus monkeys Administration and Sampling: Non-naive (but not pre-administered with FGF21 or GLP-1 products) female wine-eating monkeys (Macaca fascicularis, Nafovanny / KHI Group, Vietnam) that were at least 2 years old and weighed 2.0–3.0 kg at the start of treatment were used in the study. Each group consisted of n=3 monkeys. The test compound was administered at a dose of 5 nmol / kg in an aqueous solution containing 1 mg / ml (10 mM phosphate, 2% (w / vol) glycol, pH=8.15) intravenously to one group and subcutaneously to the other. Approximately 600 μl of blood was collected from the femoral vein according to the following regimen: before administration, and at 5, 30, 1, 2, 4, 8, 24, 48, 96, 168, 240, 336, 408, and 504 hours after administration. Blood was collected in a Teklab K3EDTA tube containing 1.75 mg of EDTA / mL blood (part number 3K200PP) and placed on ice for up to 30 minutes before centrifugation at 2000 g for 10 minutes at 2–8°C. Plasma (at least 100 μl) was transferred to a Micronic tube and stored at -30 to -10°C until analysis.

[0217] In vivo and pharmacokinetic analyses were performed, as in the miniature pig study. Pharmacokinetic parameters are shown in Table 12. [Table 19]

[0218] The average residence time of the C-terminal portion of chemical formula 23 in monkeys was 56 hours after subcutaneous administration, which was deemed long enough to support low-dose administration in humans.

[0219] Example 5: Evaluation of the GLP-1 PD effect of the fusion compound The purpose of this study was to investigate the GLP-1 effect of GLP-1 / FGF21 fusion compounds by examining their GLP-1 effect on acute food intake after single-dose intravenous administration in lean mice.

[0220] FGF21 has no effect on food intake (FI) 24 hours after a single injection. Conversely, GLP-1 is known to reduce FI. Therefore, we used a model of acute FI to investigate the pharmacodynamic effective dose and in vivo efficacy of the GLP-1 portion of the GLP-1 / FGF21 bifunctional molecule.

[0221] method: Acute FI was measured in single-container mice using 56 eight-week-old male C57BL6J mice in a BioDaq system (New Brunswick, New Jersey, USA), ad libitum feeding of solid diet Altromin 1324 in a reverse light / dark cycle (dark 11 a.m. to 11 p.m.). Mice were acclimatized to BioDaq cages for 14 days and randomly divided into seven groups of eight mice / group based on body weight. Baseline FI measurements were collected over 24 hours prior to administration. Mice were intravenously injected into the tail vein one hour before dark with 10 nmol / kg of either chemical formula 23 or control compounds chemical formulas 24, 12, 14, 16, 19, or a vehicle. FI data was collected 24 hours post-administration. One animal in the chemical formula 16 group was excluded due to technical issues. Statistical analysis was performed using GraphPad Prism 8.0.2 with Dunnett-corrected one-way ANOVA, p<0.05. ***=p<0.001, ****=p<0.0001, ns=no significant difference

[0222] result There were no significant differences in food intake between the groups before administration of the compounds. After administration of the active compounds, food intake decreased significantly in all groups compared to the vehicle. Chemical formula 23 reduced FI by 58%, followed by chemical formula 12 (-52%), chemical formula 14 (-41%), chemical formula 16 (-39%), and chemical formula 19 (-27%). The positive control, chemical formula 24, showed the greatest decrease over 24 hours (-70%) in Figure 1.

[0223] All tested dual GLP-1 / FGF21 compounds, with chemical formulas 23 and 12, as well as chemical formula 24, significantly reduced food intake and demonstrated in vivo efficacy against the GLP-1 receptor.

[0224] Example 6: Evaluation of the effect of the fusion compound on body weight

[0225] Evaluation of GLP-1 / FGF21 fusion compounds in the efficacy of weight reduction in low-density lipoprotein receptor-deficient mouse models (LDLr- / -). The objective of the following study was to evaluate selected GLP-1 / FGF21 fusion compounds and body weight (BW) in a mouse model of proatherosclerosis and LDLr- / - with severe dyslipidemia and increased body weight (BW).

[0226] Test design Eighty male LDLr- / - mice (JAX, USA, STOCK:2207) were fed a high-fat and cholesterol diet (WD;D12049B, Research Diets, USA) for at least 10 weeks prior to entering the study protocol. Two days before the start of the experiment, the mice were randomly divided into eight groups based on their morning bedweight. The animals were administered the test substance or vehicle subcutaneously daily at noon for 21 days. The dose of the test substance was adjusted daily based on bedweight. For group 3 (Chemical Formula 24), the dose was escalated over 5 days, starting at 1.0 ml / kg, equivalent to 2 nmol / kg, and ending at 10 nmol / kg.

[0227] Weight data Baseline adjusted body weight for one day is shown in Figure 2. All compounds showed a significant reduction in body weight (BW). Analogues 12 and 23 showed the most efficient reduction in BW, reaching a reduction of over 20% from baseline after 7 days of administration. This BW reduction by 12 and 23 was deemed very potent, and for ethical reasons, the dose was reduced in all groups. With the 23 analogue, the very efficient reduction in BW persisted, so the dose was further reduced to 2 nmol / kg. However, this dose was deemed too low, and the dose was adjusted again to 4 nmol / kg and continued throughout the study. Chemical formulas 25 (FGF21) and 24, in particular, showed similarly significant BW reductions.

[0228] Example 7: Chemical stability. IsoAsp formation. FGF21 contains several aspartic acid molecules that readily form isoaspartic acid (isoAsp) (D5, D24, D25, D38, and D102). Stability tests were conducted to evaluate the degree of isoAsp formation.

[0229] Procedure stability testing Chemical formulas 12, 14, 16, 19, and 23 (20 mg / ml) were prepared in 10 mM phosphate buffer, 2% glycerol, pH 8.2, filtered, dispensed into dust-free sterile HPLC vials, and stored at static temperature (5C, 25C, 37C). Samples were taken weekly for 4 weeks to ensure chemical and physical stability.

[0230] Peptide mapping for data analysis The formation of IsoAsp at the aforementioned locations was determined by peptide mapping and LC-MS. The samples were digested with trypsin (E:S 1:20 w / w; pH 7.5; 2 hours at 37°C, barocycler: 72 cycles 90 sec / 10 sec) and reduced before LC-UV215-MS / MSMS analysis (0.25 M DTT, pH 7.5, 0.5 hours at 37°C). The amount of isobaric trypsin peptide containing isoaspartic acid (%) was calculated as a percentage of the unmodified trypsin peptide based on the area of ​​the extracted ion chromatogram (XIC). The following IsoAsp sites were directly identified within the isobaric trypsin peptide using electron transfer dissociation (ETD) MS / MS: namely, 25IsoAsp, 38IsoAsp, and 102IsoAsp. For isotopic peptides containing IsoAsp24 and IsoAsp25 (amino acid position numbering according to Sequence ID No. 2), the ions reported in ETD were not observed. [Table 20]

[0231] result IsoAsp formation was confirmed at positions D5, D24, D25, D38, and D102 (amino acid position numbering is based on Sequence ID 2) in all analyzed analogs, including the reference compound chemistry formula 25. While isoAsp content was comparable at time zero, compounds with two side chains (chemistry formulas 14, 16, and 19) had higher content of all isoAsp derivatives compared to compounds with one side chain (chemistry formulas 12 and 23). Table 13 shows the formation of isoAsp at position 102. The formation of the 102 IsoAsp in compounds chemistry formulas 12 and 23 was comparable to the formation of the isoAsp in the reference compound chemistry formula 25 after 33 days at 37°C.

Claims

1. Chemical formula 1: ABC (In the formula, (i) A is a GLP-1 polypeptide which is an analog of GLP-1(7-37) (SEQ ID NO: 1), (i) B is a spacer consisting of 1 to 257 amino acids, (i) C is an FGF21 polypeptide, which is an analog of FGF21(1-181) (SEQ ID NO: 2). A fusion protein containing the polypeptide, or a pharmaceutically acceptable salt, amide, or ester thereof.

2. The fusion protein according to claim 1, wherein B contains or comprises 5 to 257 amino acids, preferably 9 to 129 amino acids.

3. The fusion protein according to claim 1 or 2, wherein B is SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 15, or SEQ ID NO: 17, preferably SEQ ID NO:

16.

4. The fusion protein according to any one of claims 1 to 3, wherein A has 3 to 7 amino acid changes compared to SEQ ID NO:

1.

5. A is a fusion protein according to any one of claims 1 to 4, wherein A comprises 26C, 27C, 36C, or 26R.

6. The fusion protein according to any one of claims 1 to 5, wherein A is selected from the list consisting of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, preferably SEQ ID NO:

35.

7. A fusion protein according to any one of claims 1 to 6, wherein C comprises 180C, 180E, or 180A.

8. The fusion protein according to any one of claims 1 to 7, wherein C is selected from the list consisting of SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, preferably SEQ ID NO:

12.

9. A fusion compound comprising a fusion protein according to any one of claims 1 to 8 and one or more substituents.

10. The substituent includes or consists of an extension and / or optionally a linker, wherein the extension is of chemical formula 3: HOOC-(CH ２ ) ｘ -CO-*, where x is an integer in the range of 10 to 20, and the linker includes at least one of chemical formulas 4, 5, and 6. Chemical formula 4 is, ＊ -NH-CH(COOH)-(CH ２ ) ２ -CO- ＊ And, Chemical formula 5 is ＊ -NH-(CH ２ ) ２ -[O-(CH ２ ) ２ ｋ -O-[CH ２ ｍ -CO- ＊ where k is an integer in the range of 1 to 5, and m is an integer in the range of 1 to 5.​​ Chemical formula 6 is, ＊ -NH-(CH ２ ) ｎ -NH-CO-CH ２ - ＊ And n is an integer in the range of 1 to 5. Chemical formulas 4, 5, and 6 are interconnected via amide bonds, in the order shown, ＊ - The NH terminus is linked to the CO-* terminus of the protractor (chemical formula 3), and its CH ２ - ＊ The fusion compound according to claim 9, wherein the fusion compound is linked to amino acids A, B, or C at its terminal end.

11. The fusion compound according to claim 9 or 10, wherein the substituent is the following chemical formula 7. 【Chemistry 1】

12. The fusion compound according to any one of claims 9 to 11, wherein the compound is selected from a list consisting of chemical formulas 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, and 23, and preferably the compound is chemical formula 23.

13. A fusion compound having the structure shown in chemical formula 23 below. 【Chemistry 2】

14. A fusion compound according to any one of claims 9 to 13, for use as a pharmaceutical.

15. A fusion compound according to any one of claims 9 to 13 for use in the treatment and / or prevention of one or more conditions selected from the group consisting of type 1 diabetes, type 2 diabetes, metabolic syndromes, prediabetes, obesity, insulin resistance, diabetic retinopathy, diabetic neuropathy, diabetic nephropathy, chronic kidney disease, diabetic kidney disease, diabetic dyslipidemia, liver diseases such as NAFDL, MAFLD, MASH, NASH, ALD, and MetALD, ASH, and HCC, and atherosclerosis.

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