Fusion compounds and uses thereof

Abstract

The present invention relates to a fusion protein comprising a GLP-1 polypeptide and a FGF21 polypeptide separated by a spacer. The invention also relates to a fusion compound comprising the fusion protein and at least one substituent. The invention also relates to the pharmaceutical use of the fusion compound.

Description

Technical Field

[0001] This invention relates to fusion compounds comprising a GLP-1 peptide and an FGF21 peptide. Furthermore, this invention also relates to pharmaceutical compositions comprising such fusion compounds.

[0002] sequence list This application is filed together with the electronic sequence list, the entire contents of which are incorporated herein by reference. Background Technology

[0003] Fusion proteins are generated by genetic engineering by linking two or more genes that originally encode individual proteins. The result is a single polypeptide with the functional properties of both parent proteins, i.e., a bifunctional fusion protein. Combining unrelated proteins is challenging because they may prove difficult to prepare due to incompatibility of properties. For example, this can lead to aggregation or misfolding in one domain, conditions that are perfect for the other. Additionally, the two or more proteins (or polypeptides) fused together may have conflicting stability requirements, making formulation challenging. Furthermore, controlling and adjusting the relative amounts of each component can be difficult, complicating dosing for optimal efficacy and safety. Moreover, different polypeptide moieties in the fusion protein may interact, resulting in undesirable reduced biological activity. Direct fusion of polypeptide moieties without linkers can lead to a number of adverse consequences, including misfolding of the fusion protein, low protein yield, or impaired biological activity.

[0004] GLP-1 is an intestinal hypoglycemic hormone produced by intestinal endocrine cells after food intake. GLP-1 is a regulator of glucose metabolism and a regulator of insulin secretion from pancreatic β-cells in the pancreas. GLP-1 also induces insulin secretion in diabetic states. Furthermore, GLP-1 reduces food intake by enhancing satiety, thereby limiting weight gain and potentially even leading to weight loss. Taken together, these effects give GLP-1 unique properties, making it considered a highly desirable antidiabetic agent, especially because its glucose-dependent antihyperglycemic effect minimizes any risk of severe hypoglycemia. However, its pharmacokinetic / pharmacodynamic profile makes natural GLP-1 unavailable for therapeutic use. GLP-1 is highly sensitive to enzymatic degradation in vivo, and cleavage by dipeptidyl peptidase IV (DPP-IV) is likely the most relevant, as this occurs rapidly and generates non-insulin-stimulating metabolites. Therefore, methods to prolong the half-life of GLP-1 in vivo have attracted considerable attention. Various approaches have been employed to modify the structure of glucagon-like peptide-1 (GLP-1) compounds to provide a longer duration of action in vivo, leading to the development of once-weekly products such as abiglutide, dulaglutide, and smegglutide.

[0005] FGF21 belongs to the FGF19 subfamily of atypical fibroblast growth factors (FGFs) and has metabolic rather than mitogenic effects. FGF21 binds to and activates FGF receptors (FGFR1c, FGFR2c, and FGFR3c), but this activation only occurs in the presence of the non-signaling co-receptor β-klotho (BKL). Tissue-specific expression of BKL determines the metabolic activity of FGF21. FGF21 transgenic mice are resistant to diet-induced obesity and have extended lifespans. FGF21 is a metabolic regulator of energy expenditure, glucose, and lipid metabolism. FGF21 may have the potential to reverse weight, hyperglycemia, and dyslipidemia in obese patients with diabetes and dyslipidemia.

[0006] Due to proteolytic activity, FGF21 is unstable in vivo, with up to half of endogenous circulating human FGF21 being inactive. This loss of activity is due to C-terminal degradation, with most of these metabolites terminating at P171 rather than S181. Therefore, for therapeutic FGF21 molecules, it is necessary to protect the C-terminal region from metabolic degradation. Several methods have been reported to attempt to prolong the in vivo half-life of recombinant FGF21 proteins. One such example is PEGylation. However, PEGylation at position 179 of [-1M, 179C]FGF21 leads to a sharp decrease in in vitro activity (J. Xu et al., Bioconjugate Chemistry (2013)). 24 Fc fusion technology has also been used. However, the potency of Fc fusion proteins resulting from attaching Fc to the C-terminus of FGF21 is far lower than that of natural FGF21 and the N-terminal Fc fusion of FGF21 (Hecht et al., PLoS One 2012, 7(11), e49345). The molecular weight of the Fc moiety is approximately 50 kDa. Therefore, incorporating Fc into the fusion protein increases its molecular weight by at least 50 kDa. However, an increase in molecular weight is generally not desirable because it makes the corresponding drug formulation more challenging, for example, due to increased viscosity. In addition, the incorporation of the Fc domain may lead to steric hindrance between peptide moieties, which can result in reduced biological activity and altered biodistribution and metabolism of the protein moieties due to interference between domains.

[0007] The combined administration of GLP-1 and FGF21 has been reported (see WO 2010 / 142665). Combined 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 of administration, but are inconvenient for patients due to compliance and pain concerns. Co-formulations also offer some dosage flexibility, but finding formulation conditions that ensure chemical and physical stability for both compositions is often very challenging or impossible due to the different molecular properties of the two products.

[0008] Therefore, there is a need for fusion compounds containing GLP-1 and FGF21 peptides that have improved bioavailability, extended half-life, and / or increased potency. Summary of the Invention

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

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

[0011] Figure 1 The cumulative food intake (g) of lean mice after a single intravenous administration of the carrier or active compound is shown.

[0012] Figure 2 Baseline corrected body weight data of LDLr- / - mice treated for 21 days with formulas 24, 25, 14, 16, 19, 12, and 23 are shown. Data: mean + / - SEM (n=9-10 / group) Detailed Implementation

[0013] In the following text, "a (species)" as used herein may mean "one or more (species)" or "at least one (species)". Greek letters may be represented by their symbols or corresponding written names, for example: α = alpha; β = beta; ε = epsilon; γ = gamma; ω = omega; etc. In addition, the Greek letter μ may also be represented by "u", for example μl = ul or μM = uM.

[0014] The asterisk in the chemical formula ( () indicates a connection point. In the following text, unless otherwise stated in the specification, terms presented in the singular also include plural cases; for example, when referring to "fusion compound," it should be understood that this includes all individual variations falling within the broad definition of said fusion compound.

[0015] As used herein, the term “about” or “approximately” when used with a numerical value (e.g., 5, 10%, 1 / 3) refers to a range of values ​​that may be less than or greater than that number. For example, “about 5” refers to a range of values ​​that are 10%, 5%, 2%, or 1% smaller or greater than 5, such as 4.5 to 5.5, or 4.75 to 5.25, or 4.9 to 5.1, or 4.95 to 5.05. In some cases, “about 5” refers to a range of values ​​that are 2% or 1% smaller or greater than 5, such as 4.9 to 5.1 or 4.95 to 5.05. In some embodiments, the term “about” as used herein refers to the mentioned value ±10% and includes that value.

[0016] Unless the context indicates otherwise, all scopes mentioned herein should be interpreted as including their endpoints, and open scopes should be interpreted as including only commercially viable values. Similarly, all lists of values ​​should be considered to include intermediate values ​​unless the context indicates otherwise.

[0017] In a first aspect, the present invention relates to a fusion protein comprising a polypeptide of formula 1: ABC, wherein: A is a GLP-1 polypeptide, which is an analogue of SEQ ID NO: 1; B is a spacer composed of 1-257 amino acids; C is an FGF21 polypeptide, which is an analogue of SEQ ID NO: 2; Or its pharmaceutically acceptable salt, amide, or ester.

[0018] Alternatively or concurrently, the present invention relates to a fusion compound comprising... Polypeptide of chemical formula 1: ABC, wherein: A is a GLP-1 polypeptide, which is an analogue of SEQ ID NO: 1; B is a spacer composed of 1-257 amino acids; and C is an FGF21 polypeptide, which is an analogue of SEQ ID NO: 2; And substituents; or pharmaceutically acceptable salts, amides or esters thereof.

[0019] Alternatively or concurrently, the present invention relates to a fusion compound capable of activating the human GLP-1 receptor and / or capable of activating the FGFR complex.

[0020] 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 invention, including all embodiments and specific features thereof) and optionally one or more pharmaceutically acceptable excipients.

[0021] In a third aspect, the present invention relates to fusion proteins or fusion compounds (i.e., fusion proteins or fusion compounds as defined in the first aspect of the invention, including all embodiments and specific features thereof) or pharmaceutical compositions (i.e., compositions as defined in the second aspect of the invention, including all embodiments and specific features thereof), which are used as pharmaceuticals or for medical purposes.

[0022] In a fourth aspect, the present invention relates to fusion proteins or fusion compounds (i.e., fusion proteins or fusion compounds as defined in the first aspect of the invention, including all embodiments and specific features thereof) or pharmaceutical compositions (i.e., compositions as defined in the second aspect of the invention, including all embodiments and specific features thereof) for (i) prevention and / or treatment of all forms of diabetes; (ii) delaying or preventing the progression of diabetes, and / or delaying the progression of insulin-free type 2 diabetes to insulin-requiring type 2 diabetes; (iii) improving β-cell function; (iv) preventing and / or treating cognitive impairment and / or neurodegenerative diseases; (v) preventing and / or treating eating disorders; and / or preventing and / or treating comorbid obesity; (vi) preventing and / or treating diabetic complications; (vii) improving lipid parameters; and (viii) preventing and / or treating cardiovascular diseases. (ix) Prevention and / or treatment of gastrointestinal diseases; (x) prevention and / or treatment of critical illness; prevention or reduction of the likelihood of patients developing bacteremia, sepsis and / or septic shock during hospitalization; and / or stabilization of blood glucose, insulin balance and optional 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 brain diseases, such as cerebral ischemia, cerebral hemorrhage and / or traumatic brain injury; (xiii) prevention and / or treatment of sleep apnea; (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) non-alcoholic fatty liver disease (NAFLD) and / or acute and chronic pancreatitis.

[0023] In a fifth aspect, the present invention relates to a method for preparing fusion proteins or fusion compounds (i.e., fusion proteins or fusion compounds as defined in the first aspect of the invention, including all embodiments and specific features thereof).

[0024] General definition As used herein, the term "compound" refers to a molecular entity; therefore, a "compound" may have different structural elements in addition to having the minimum elements defined for each compound or group of compounds. Fusion compounds may be referred to as "compounds," and the term "compound" is also intended to cover its pharmaceutically relevant form, namely, the invention relates to compounds as defined herein or their pharmaceutically acceptable salts, amides, or esters.

[0025] As used herein, the term "polypeptide" or "polypeptide sequence" refers to a compound comprising a series of amino acids linked together by amide (or peptide) bonds. The term polypeptide is used interchangeably with the terms "peptide" and "protein".

[0026] The term "part" refers to a fragment or portion of a molecule, such as a fusion compound or fusion protein.

[0027] The term "peptide moiety" refers to a fragment or portion of a polypeptide molecule, such as a fusion compound or fusion protein. For example, "GLP-1 polypeptide moiety" refers to A in Formula 1, while "FGF21 polypeptide moiety" refers to C in Formula 1. Similarly, the term "spacer moiety" refers to B in Formula 1. In other words, the term "GLP-1 polypeptide moiety" refers to a fragment or portion of the "GLP-1 polypeptide" of Formula 1.

[0028] As used herein, the term "analyte" generally refers to a polypeptide whose sequence has one or more amino acid alterations compared to a reference amino acid sequence. Such amino acid alterations may include amino acid additions, deletions, and / or substitutions. An amino acid "substitution" may also be referred to as a "mutation." In a particular embodiment, the analog "comprises" the specified alteration. In other particular embodiments, the analog "composes of" or "has" the specified alteration. When the terms "comprises" or "contains" are used in connection with amino acid alterations in an analog, it should be understood that the analog may have further amino acid alterations compared to its reference sequence. When the terms "composes of" or "has" are used in connection with amino acid alterations in an analog, it should be understood that the specified amino acid mutation is the only amino acid alteration in the analog compared to the reference sequence. In the context of this application, the term "analyte" means analogues of human glucagon-like peptide-1 GLP-1(7-37) (SEQ ID NO: 1), analogues of human endogenous FGF21 (FGF21(1-181) (SEQ ID NO: 2)) and / or analogues of fusion compounds.

[0029] The term "derivative" generally refers to a polypeptide that can be prepared from a natural polypeptide or its analogues through chemical modification, particularly through the covalent linkage of one or more substituents. Derivatives may also be referred to as alkylated analogues. For example, fusion compounds as defined herein are derivatives of fusion proteins derived herein.

[0030] As used herein, the term "amino acid" refers to any amino acid, both proteinogenic and non-proteinogenic. As used herein, "proteinogenic amino acid" refers to the 20 standard amino acids encoded by the human genetic code. As used herein, "non-proteinogenic amino acid" refers to all amino acids that do not qualify as proteinogenic amino acids. Generally, amino acid residues as used herein (e.g., in the context of a polypeptide sequence) can be represented by their full name, their single-letter code, and / or their three-letter code. These three methods are completely equivalent and interchangeable. In the following text, each amino acid of the present invention's peptides, unless its optical isomer is specified, should be understood to refer to the L-isomer (unless otherwise stated).

[0031] As used herein, the terms “fusion” and “fused” refer to compounds comprising two or more individually defined polypeptides covalently linked by peptide bonds or by spacers. The term “spacer” as used herein refers to a molecular portion that separates two individually defined polypeptides.

[0032] The term "bifunctional" refers to bifunctional activity. For example, in a bifunctional fusion compound, both peptides of the bifunctional fusion protein—such as GLP-1 and FGF21—are active.

[0033] As used herein, the term "sequence identity" refers to the degree to which two amino acid sequences (e.g., polypeptides) have identical residues at the same positions in an alignment. This can also be simply referred to as "identity." Sequence identity is conveniently expressed as a percentage, i.e., if 85 amino acids are identical across 100 alignment positions between two sequences, the degree of identity is 85%. For the purposes of this invention, sequence identity between two amino acid sequences is determined using simple handwriting and visual inspection, and / or standard protein or peptide alignment procedures, such as "align" based on the Needleman-Wunsch algorithm. This algorithm is described in Needleman, SB and Wunsch, CD, (1970), Journal of Molecular Biology, 48: 443-453, while the align procedure is described by Myers and W. Miller in "Optimal Alignments in Linear Space," CABIOS (computer applications in the biosciences) (1988) 4:11-17. For alignment, the default scoring matrix BLOSUM62 and the default identity matrix can be used, and the penalty for the first residue in the vacancy can be set to -12, or preferably -10, while the penalty for the other residues in the vacancy can be set to -2, or preferably -0.

[0034] As used in this article, the term "FGFR complex" refers to the FGF receptor β-klotho (FGFR-BKL) complex, such as FGFR1c, FGFR3, and FGFR2.

[0035] GLP-1 peptide As used herein, the term "GLP-1 polypeptide" refers to an analogue (or variant) of human glucagon-like peptide-1 (GLP-1(7-37)), the sequence of which is included in the sequence listing as SEQ ID NO: 1HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG. 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 alterations compared to natural human GLP-1, wherein said amino acid alterations may be in the form of amino acid addition, amino acid deletion, and / or amino acid substitution. The numbering of amino acid residues in the GLP-1 polypeptide of the fusion compound or fusion protein of the present invention follows established practice in the art for natural human GLP-1, i.e., the first (N-terminal) amino acid residue is numbered or corresponds to position 7, and subsequent downstream amino acid residues toward the C-terminus are numbered 8, 9, 10, etc., until the last (C-terminal) amino acid residue. In natural human GLP-1, the C-terminal amino acid residue is Gly, numbered 37. The numbering scheme differs in the sequence listing, where the first amino acid residue (His) of SEQ ID NO: 1 is designated as number 1, and the last (Gly) is designated as number 31. However, as mentioned above, we follow the established numbering practice in the art here, namely, the first (N-terminal) amino acid residue in SEQ ID 1 is assigned number 7. 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 alteration and ii) the position of the altered native amino acid residue. Thus, an amino acid alteration in the form of substitution can be referred to as "Xaa", where aa is the amino acid introduced at the substitution position, and where X is a number corresponding to the position of the substituted amino acid residue in SEQ ID NO: 1.

[0036] For example, the GLP-1 polypeptide portion of the fusion compound or fusion protein of the present invention may be referred to by reference to amino acid changes relative to GLP-1(7-37) (SEQ ID NO: 1), for example, as follows: '[8G, 22E, 27C, 36G]GLP-1(7-37)'. In this example, the GLP-1 polypeptide is an analog of GLP-1(7-37) (SEQ ID NO: 1), wherein the analog is Gly at position 8 corresponding to position 8 of GLP-1(7-37) (SEQ ID NO: 1), Glu at position 22 corresponding to position 22 of GLP-1(7-37), Cys at position 27 corresponding to position 27 of GLP-1(7-37), and Gly at position 36 corresponding to position 36 of GLP-1(1-37). If the amino acid changes in the GLP-1 peptide are presented relative to GLP-1(7-37) (SEQ ID NO: 1), it should be understood that this refers only to the changes in the GLP-1 peptide portion and does not involve any other portion of the fusion compound or fusion protein, such as the spacer or FGF21 peptide portion.

[0037] “GLP-1 peptide” is used interchangeably with “GLP-1 analog” and “GLP-1 variant”. Compared with GLP-1 (7-37) (SEQ ID NO: 1), a GLP-1 peptide “containing” certain specified modifications may contain further modifications.

[0038] In some embodiments, the GLP-1 polypeptide moiety of the fusion protein or fusion compound is an analog of SEQ ID NO: 1. In some embodiments, the GLP-1 polypeptide moiety 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 moiety has 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, preferably up to 4 amino acid changes, or preferably up to 3 amino acid changes compared to SEQ ID NO: 1. In some embodiments, the GLP-1 polypeptide moiety has 3-7 changes compared to SEQ ID NO: 1.

[0039] In some embodiments, the GLP-1 peptide comprises the amino acid sequence H-Xaa8-EGTFTSDVSSYLE-Xaa22 -QAA-Xaa 26 -Xaa 27 -FIAWLVKG-Xaa 36 -G (SEQ ID NO: 3) or composed of this amino acid sequence, wherein Xaa8 is G; Xaa 22 for E; Xaa 26 For R, C, or K; Xaa 27 For C or E; Xaa 36 It can be G or C.

[0040] In some implementations, the GLP-1 peptide can undergo Cys-alkylation.

[0041] Table 1 provides non-limiting examples of GLP-1 peptides.

[0042] Table 1. Non-limiting examples of the GLP-1 polypeptide moiety of the fusion protein or fusion compound of the present invention.

[0043] FGF21 peptide The terms “human FGF21,” “natural FGF21,” “wild-type FGF21,” “human endogenous FGF21,” and “FGF21(1-181)” are used interchangeably to refer to a polypeptide consisting of the sequence SEQ ID NO: 2. HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS. In this formula, the amino acid residues are numbered according to FGF21(1-181) (SEQ ID NO: 2), where the first (N-terminal) amino acid residue (H) is numbered as position 1, and subsequent amino acid residues toward the C-terminus are numbered 2, 3, 4, etc., until the last (C-terminal) amino acid residue (S), which is position 181 in FGF21(1-181). In the sequence listing, the first amino acid residue (H) of SEQ ID NO: 2 is designated as number 1, and the last residue (S) is designated as number 181; therefore, the numbering in this document is the same as the numbering in the sequence listing. This also applies to other FGF21 polypeptide sequences.

[0044] The term “FGF21 polypeptide” is used interchangeably with “FGF21 analog” and “FGF21 variant”.

[0045] As used herein, the term "FGF21 polypeptide" refers to a polypeptide capable of activating the human FGF21 receptor. In some embodiments, the FGF21 polypeptide moiety of the fusion compound or fusion protein is capable of activating 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 moiety of the fusion compound is an analog of FGF21(1-181). In some embodiments, the FGF21 polypeptide moiety of the 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), wherein the analogue is Gln at position 121 corresponding to position 121 of FGF21(1-181), Leu at position 168 corresponding to position 168 of FGF21(1-181), and Cys at position 180 corresponding to position 180 of FGF21(1-181). The N-terminal Ala addition may also be referred to as "-1A" or "-1Ala" because it corresponds to position -1 of FGF21(1-181). If the amino acid changes in the FGF21 polypeptide are presented relative to FGF21(1-181), it should be understood that this refers only to changes in the FGF21 polypeptide moiety and does not involve any other moiety in the fusion compound, such as the spacer or GLP-1 polypeptide moiety.

[0046] If the amino acid changes in the FGF21 peptide are presented relative to FGF21(1-181) (SEQ ID NO: 2), it should be understood that this refers only to the changes in the FGF21 peptide portion and does not involve any other portion of the fusion compound, such as the spacer or GLP-1 peptide portion.

[0047] In some embodiments, the FGF21 polypeptide moiety of the fusion compound or fusion protein is an analog 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, compared to SEQ ID NO: 2, the FGF21 polypeptide contains 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, preferably up to 5 amino acid changes, preferably up to 4 amino acid changes, or preferably up to 3 amino acid changes. In some embodiments, the FGF21 polypeptide has 4 amino acid changes compared to SEQ ID NO: 2. In some embodiments, the FGF21 peptide comprises 180C. In some embodiments, the FGF21 peptide, compared to SEQ ID NO: 2, comprises or has the following amino acid changes: [121Q, 168L, 180C] or [121Q, 168L, 171G] or [121Q, 168L, 171G, 180E]. In some embodiments, the FGF21 peptide moiety 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 peptide has FGF21 activity. In some embodiments, FGF21 is capable of activating FGFR1c. In some embodiments, FGF21 is capable of activating FGFR3c.

[0048] In some implementations, the FGF21 peptide can undergo Cys-alkylation.

[0049] Table 2 provides non-limiting examples of FGF21 peptides.

[0050] Table 2: Non-limiting examples of the FGF21 polypeptide moiety of the fusion protein or fusion compound of the present invention.

[0051] spacer Fusion compounds or fusion proteins typically contain spacers that separate the biologically active portions of the compound to ensure that any function present in the biologically active portion is not interfered with by the proximity of other biologically active portions.

[0052] As used herein, the term "spacer" refers to an element that covalently links the bioactive portion of the fusion compound or fusion protein of the present invention. A spacer may also be referred to as a "spacer polypeptide," a "spacer portion," or a "spacer element." In some embodiments, the spacer comprises an amino acid sequence in which its N-terminus is linked to the C-terminus of the GLP-1 polypeptide portion via an amide bond, and its C-terminus is linked to the N-terminus of the FGF21 polypeptide portion via an amide bond. In some embodiments, the spacer comprises chemical formula 2 (GAQP). x -C y -(GAQP) h -A j The repeating elements are defined as follows: x is an integer in the range of 0-64, y is an integer in the range of 0-1, h is an integer in the range of 1-64, and j is an integer in the range of 0-1. An example of the spacer nomenclature used in this paper is '[GAQP]8-A' or '(GAQP)x8, A', both of which are abbreviations for a polypeptide sequence consisting of eight GAQP segments followed by an A segment. The full sequence is written as GAQPGAQPGAQPGAQPGAQPGAQPGAQPA.

[0053] Spacers can affect the pharmacokinetic properties of fusion compounds, for example, by prolonging the half-life of the fusion compound. In some embodiments, spacers may be able to improve the half-life of the fusion compound. The statement "improves the half-life of the fusion compound" may mean that it prolongs the plasma half-life of the fusion compound to make it suitable for once-daily or twice-weekly injection, preferably once-weekly injection.

[0054] In some embodiments, the spacer may consist of 1 to 257, such as 5 to 257, such as 9 to 129 amino acids. In some embodiments, the spacer may contain at least two GAQP segments. In some embodiments, the spacer may contain (GAQP). 32 -A. In some embodiments, the spacer is selected from the list consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18. In some embodiments, the spacer comprises or consists of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18.

[0055] In some implementations, the spacer can undergo Cys-alkylation.

[0056] Table 3 provides non-restrictive examples of spacers.

[0057] Table 3: Non-restrictive examples of spacers.

[0058] Substituents As used herein, the term "substituent" refers to a portion covalently linked to the GLP-1 polypeptide or FGF21 polypeptide or spacer of the compound of the present invention. The terms "substituent" and "side chain" are used interchangeably. Substituents are capable of forming non-covalent bonds with albumin, thereby promoting the circulation of the derivative in the bloodstream, and also have the effect of prolonging the duration of action of the fusion compound because the association of the fusion compound with albumin is only slowly dissociated to release the derivative in its free form. Therefore, the substituent as a whole may also be referred to as the "albumin-binding portion". The substituent contains a portion particularly associated with albumin binding, and thus with the prolongation of action, which may be referred to as the "prolongation portion" or "prolongation body". The prolongation portion may be close to, preferably at, the end (or distal, or free end) of the substituent relative to its connection point with the peptide. The substituent may contain a portion located between the prolongation portion and the peptide connection point, which may be referred to as the "linker body". The "substituent" may be lipophilic and / or negatively charged at physiological pH (7.4). The "elongation" or "substituent" can be covalently linked to the thiol group of a cysteine ​​residue in a GLP-1 peptide, FGF21 analog, or spacer via alkylation. The substituent can be synthesized as a haloacetamide group and activated therein, reacting with the thiol group of the cysteine ​​residue under conditions of covalent sulfur-carbon bond formation (a process known as Cys-alkylation), also called a thioether bond. Therefore, no halogen atom is present in the derivative; the substituent is linked by a sulfur atom. When the thiol group is mentioned in relation to the derivative, it must be understood as the sulfur atom that is part of the cysteine ​​thiol group prior to Cys-alkylation. Alternatively, the substituent can be activated with a maleimide group, which reacts with the thiol group of the cysteine ​​residue under conditions of covalent sulfur-carbon bond formation. The substituent can function as an albumin binder or albumin-binding moiety.

[0059] Elongation body: The elongator can be located at or near the distal end of the side chain, relative to its protein binding site. In one aspect, each elongator comprises or consists of an elongator of formula 3: HOOC-(CH2)x-CO- The carbon chain length, defined by x, can vary from 8 to 18, such as 14-18 or 14-16.

[0060] The naming follows the conventions of the field; for example, in the above formula, -CO- It refers to carbonyl ( -C(=O)- For example, in any general formula (R-CO-) in this paper (where R is as defined in each general formula) In R-CO- It refers to RC(=O)- .

[0061] Connector The linker may include at least one of the following linker elements: chemical formula 4, chemical formula 5, and chemical formula 6. Elements of chemical formula 4 and chemical formula 5 each have -NH- and CO- ends, respectively, thereby allowing them to be linked to each other via amide bonds and to the -CO- or -NH- of chemical formula 3 or chemical formula 6.

[0062] Formula 6 has an -NH- terminus (capable of forming an amide bond with Formula 3, Formula 4, or Formula 5) and an -NH-CO-CH2- terminus. In its unreacted form, it is a haloacetamide that can react with the thiol groups of cysteine ​​residues incorporated in the GLP-1 polypeptide moiety, the spacer moiety, or the FGF21 polypeptide moiety of the fusion protein or fusion compound of the present invention.

[0063] The chemical formulas 4, 5, and 6 of the connecting elements are shown below: Chemical formula 4 is -NH-CH(COOH)-(CH2)2-CO- ; Chemical formula 5 is -NH-(CH2)2-[O-(CH2)2] k -O-[CH2] m -CO- ,in k is an integer in the range of 1 to 5, where m is an integer in the range of 1 to 5; Chemical formula 6 is -NH-(CH2) n -NH-CO-CH2- , where n is an integer in the range of 1 to 5.

[0064] Formulas 4, 5, and 6 can be interconnected via amide bonds. -NH terminal connected to CO- according to the extension of chemical formula 3 end, and in its CH2- Amino acids such as cysteine ​​that are terminally linked to A, B, or C.

[0065] Chemical formula 4 can also be referred to here as gGlu, gamma Glu, or γGlu.

[0066] Chemical Formula 4 Chemical formula 5 can also be referred to as "Ado" here.

[0067] Chemical formula 5 Chemical Formula 6 In some implementations, the substituent is of chemical formula 7: In one embodiment, the substituent is [2-[2-[[2-[2-[2-[2-[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoyl-amino)butyryl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]-ethylamino]-2-oxoethyl] (Formula 7).

[0068] In some embodiments, the fusion compound comprises one or more substituents, such as first and second substituents. In some embodiments, the fusion compound comprises at least one substituent, such as one, two, three, or seven substituents. Substituents may be attached to any amino acid residue in the fusion compound. In some embodiments, substituents are attached to Cys residues incorporated in the fusion compound. In some embodiments, substituents are attached to Cys residues of the GLP-1 peptide moiety of the fusion compound. In some embodiments, substituents are attached to Cys residues of the FGF21 peptide moiety of the fusion compound. In some embodiments, substituents are attached to Cys residues of the spacer portion of the fusion compound. In some embodiments, a first substituent is attached to Cys residues of the GLP-1 peptide moiety of the fusion compound, while a second substituent is attached to Cys residues of the FGF21 peptide moiety of the fusion compound. In some embodiments, a first substituent is attached to Cys in the spacer portion, while a second substituent is attached to Cys in the FGF21 peptide. In some embodiments, substituents are attached to amino acid residues of the peptide backbone of the fusion compound. In some embodiments, the substituent is attached to the spacer portion or the FGF21 polypeptide portion of the fusion compound. In some embodiments, the substituent is attached to a Cys residue of the GLP-1 polypeptide portion of the fusion compound, wherein the Cys residue is located at position 27 or 26. In some embodiments, the substituent is attached to a Cys residue of the FGF21 polypeptide portion of the fusion compound, wherein the Cys residue is located at position 180.

[0069] Fusion protein Fusion proteins are proteins created by linking together two or more genes that originally encode individual proteins. Translation of the fusion gene produces a single polypeptide with functional properties derived from each of the original proteins. Fusion proteins may contain two or more biologically active moieties, which exert their biological activity primarily through interactions with two distinct sites. Thus, the GLP-1 / FGF21 fusion protein comprises a GLP-1 polypeptide and an FGF21 polypeptide. The GLP-1 / FGF21 fusion protein may further comprise a peptide spacer. Therefore, the GLP-1 / FGF21 fusion protein comprises a GLP-1 polypeptide moiety, an FGF21 polypeptide moiety, and an optional spacer moiety. The fusion protein containing the GLP-1 polypeptide and the FGF21 polypeptide can 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. The GLP-1 peptide exerts its biological activity through GLP-1R, which exerts its biological activity primarily by activating the FGFR complex.

[0070] The complete linear amino acid string that forms a bioactive polypeptide (e.g., GLP-1 and FGF21 polypeptides) and any spacers separating the bioactive polypeptide may be referred to herein as a “polypeptide backbone.” The terms “fusion protein” and “polypeptide backbone” are used interchangeably herein. Therefore, the polypeptide backbone of a fusion protein does not include substituents. If substituents are present, the compound is referred to herein as a “fusion compound” rather than a “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 proteins of this invention may be incorporated into the fusion compounds of this invention.

[0071] In some embodiments, the fusion protein is a bifunctional fusion protein. In some embodiments, the fusion protein is a fusion protein according to ABC (Chemical Formula 1), wherein A is a GLP-1 polypeptide, an analog of SEQ ID NO: 1; B is a peptide spacer composed of 1-257 amino acids; and C is an FGF21 analog, an analog of SEQ ID NO: 2. In some embodiments, A is a protein containing the amino acid sequence H-Xaa8-EGTFTSDVSSYLE-Xaa 22 -QAA-Xaa 26 -Xaa 27 -FIAWLVKG-Xaa 36 -G (SEQ ID NO: 3) or a GLP-1 polypeptide consisting of this amino acid sequence, wherein Xaa8 is G; Xaa 22 for E; Xaa 26 For R, C, or K; Xaa 27 For C or E; Xaa36 The value is G or C. In some embodiments, B is a peptide spacer, wherein the length of the peptide spacer is 5 to 257 amino acids. In some embodiments, the C-terminus of the GLP-1 peptide 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 NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, and SEQ ID NO: 34. In some embodiments, the fusion protein is selected from the list consisting of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 32, and SEQ ID NO: 33. In some embodiments, the fusion protein is SEQ ID NO: 27.

[0072] In some embodiments, the fusion protein according to ABC (Chemical Formula 1) is incorporated into the fusion compound of the present invention.

[0073] Table 4 provides non-limiting examples of fusion proteins.

[0074] Table 4: Non-limiting examples of fusion proteins that can be incorporated into the fusion compounds of the present invention.

[0075] Fusion Compound The term "fusion compound" refers to a derivative of a fusion protein. In some embodiments, the fusion compound comprises a fusion protein and substituents. "GLP-1 / FGF21 fusion compound" is a derivative of the GLP-1 / FGF21 fusion protein. In some embodiments, the compound of the present invention is a GLP-1 / FGF21 fusion compound. For example, the fusion compound may be referred to with reference to GLP-1 peptide, spacer, FGF21 peptide, and substituents. An example of the nomenclature for fusion compounds used in this paper is: S{Beta-27}-[2-[2-[2-[2-[2-[2-[2-[2-[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butyryl]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 embodiment, the fusion compound consists of a GLP-1 polypeptide of formula [8G, 22E, 27C, 36G]GLP-1 (7-37) and an FGF21 polypeptide of formula [121Q, 168L, 171G, 180E]FGF21 (1-181), wherein the GLP-1 polypeptide and the FGF21 polypeptide are separated by a spacer of formula (GAQP)x32, A, and wherein a substituent of formula S{Beta-27}-[2-[2-[2-[2-[2-[2-[2-[2-[2-[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butyryl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]-ethylamino]-2-oxoethyl] is attached to the 27th Cys residue of the GLP-1 polypeptide.

[0076] In some embodiments, the fusion compound comprises a fusion protein according to ABC (Chemical Formula 1), wherein A is a GLP-1 polypeptide analogue of GLP-1 (7-37) (SEQ ID NO: 1); B is a peptide spacer comprising 1-257 amino acids or composed of 1-257 amino acids; and C is an FGF21 polypeptide analogue of FGF21 (1-180) (SEQ ID NO: 2); and at least one substituent.

[0077] In some implementations, the fusion compound has an improved half-life.

[0078] In some embodiments, the fusion compound is a bifunctional fusion compound. In some embodiments, the fusion compound contains substituents. In some embodiments, the fusion compound contains one or more substituents, such as two or four substituents. In some embodiments, the fusion compound contains a substituent attached to a Cys residue at position 27 corresponding to GLP-1(7-37) (SEQ ID NO: 1). In some embodiments, the fusion compound contains a substituent attached to a Cys residue at position 26 corresponding to GLP-1(7-37) (SEQ ID NO: 1). In some embodiments, the fusion compound contains a substituent attached to a Cys residue at position 36 corresponding to GLP-1(7-37) (SEQ ID NO: 1). In some embodiments, the fusion compound contains a substituent attached to a Cys residue of a spacer. In some embodiments, the fusion compound contains a substituent attached to a Cys residue at position 180 corresponding to FGF21(1-180) (SEQ ID NO: 2). In some embodiments, the fusion compound comprises a first substituent and a second substituent, the first substituent being attached to a Cys residue of the GLP-1 polypeptide moiety of the fusion compound, for example, to a Cys residue at a position corresponding to position 26 of GLP-1 (7-37) (SEQ ID NO: 1) or, for example, to a Cys residue at a position corresponding to position 27 of GLP-1 (7-37) (SEQ ID NO: 1), and the second substituent being attached 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 comprises a first substituent attached to a Cys residue of the spacer body moiety of the fusion compound and a second substituent attached to a Cys residue of the FGF21 polypeptide moiety of the fusion compound, wherein the Cys residue is located at a position corresponding to position 180 of FGF21 (1-180) (SEQ ID NO: 2).

[0079] 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.

[0080] In some implementations, the fusion compound is selected from chemical formula 23. Chemical formula 12 Chemical formula 14 and chemical formula 16 A list of components.

[0081] Pharmaceutically acceptable salts, amides or esters Fusion compounds can be in the form of pharmaceutically acceptable salts, amides, or esters.

[0082] For example, salts are formed through a chemical reaction between a base and an acid, such as: 2NH3 + H2SO4 → (NH4)2SO4.

[0083] The salt can be a basic salt, an acidic salt, or neither (i.e., a neutral salt). In water, basic salts produce hydroxide ions while acidic salts produce hydrated hydrogen ions.

[0084] Salts of fusion compounds can be formed by adding cations or anions, respectively, between anionic or cationic groups. These groups can be located in the polypeptide backbone and / or substituents of the fusion compound.

[0085] Non-limiting examples of anionic groups in the fusion compounds of this invention include free carboxyl groups in substituents (if any) and in GLP-1 and / or FGF21 peptides. The amino acid sequence typically contains a free carboxylic acid group at the C-terminus, and may also contain a free carboxyl group at internal acidic amino acid residues such as Asp and Glu.

[0086] Non-limiting examples of cationic groups in fusion compounds include a free amino group at the N-terminus (if present), and any free amino group of internal basic amino acid residues such as His, Arg, and Lys. The amino group at the N-terminus of the fusion compounds of the present invention can be free or acetylated.

[0087] Esters of the derivatives of the present invention can be formed, for example, by reacting a free carboxylic acid group with an alcohol or phenol, such reaction resulting in the substitution of at least one hydroxyl group with an alkoxy or aryloxy group.

[0088] The formation of esters may involve a free carboxyl group at the C-terminus of the polypeptide backbone, and / or any free carboxyl group in the substituents.

[0089] The amides of the derivatives of the present invention can 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.

[0090] The formation of amides may involve a free carboxyl group at the C-terminus of the polypeptide backbone, any free carboxyl group in the substituent, an amino group at the N-terminus of the polypeptide backbone, and / or any amino group in the substituent.

[0091] 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.

[0092] GLP-1 activity In terms of functionality, the fusion compound possesses 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 referred to as "potency".

[0093] In some embodiments, the term refers to in vivo agonistic activity / potency. In some embodiments, activation of the GLP-1 receptor is determined by measuring the cAMP response of cells stably expressing the GLP-1 receptor in vitro after exposure to an agonist. In some embodiments, the cells are according to Example 2. In some embodiments, the GLP-1 receptor is the human GLP-1 receptor. GLP-1 activity can be expressed as EC50. 50 Value or relative to a reference compound with GLP-1 activity, such as EC of formula 24. 50 Value. GLP-1 activity can be determined in the presence of HSA. Preferably, GLP-1 activity is determined as described in Example 2.

[0094] In some embodiments, GLP-1 activity is measured in young hamster kidney (BHK) cells stably expressing the human GLP-1 receptor and the CRE-luciferase reporter gene, thereby enabling indirect measurement of cAMP formation induced by adenylate 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 Determining GLP-1 Activity" / Example 2, and is expressed as EC. 50 Value, of which EC 50 Below 100 pM, preferably below 70 pM, preferably below 50 pM, and preferably below 20 pM.

[0095] FGF21 activity In terms of functionality, the fusion compound possesses FGF21 activity. As used herein, the term FGF21 activity refers to the ability to activate the FGFR complex; this activation may also be referred to as “potency.” For example, activity can be determined in vitro using HEK293 cells endogenously expressing several FGF receptors, including FGFR1c, FGFR3c, and BKL. For example, the response of human FGFR can be measured using HEK (human embryonic kidney cells) overexpressing human β-klotho (BKL). FGF21 activity can be expressed as EC 50 The FGF21 activity can be determined in the presence of HSA. Preferably, FGF21 activity is determined as described in Example 3.

[0096] In some embodiments, FGF21 activity is measured in HEK293 cell lines overexpressing FGFR1c and human β-klotho receptor (BKL). In some embodiments, FGF21 activity is measured in the presence of 0.1% HSA. In some embodiments, FGF21 activity is measured as activation of the FGFR complex in the absence of HSA, as described in Example 2, and is expressed as EC. 50 Value, of which EC 50 Below 50 nM, preferably below 10 nM, preferably below 5 nM, and preferably below 2 nM.

[0097] Pharmacokinetics In some embodiments, the fusion compounds of the present invention may have an extended mean residence time “MRT” compared to natural FGF21 and natural GLP-1, respectively.

[0098] In some embodiments, MRT can be measured in vivo using mice. The MRT measured in vivo using mice is preferably determined as described in Example 4.1. In some embodiments, MRT can 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 can be measured in vivo using cynomolgus monkeys. The MRT measured in cynomolgus monkeys is preferably determined as described in Example 4.3.

[0099] In some embodiments, the half-life of the fusion compound is calculated using an individual best-fit of a log-linear regression of concentration-time. In some embodiments, the MRT is calculated as MRT = AUMC / AUC, where AUC is the AUMC calculated using a trapezoidal method. In some embodiments, the MRT of the fusion compound is calculated based on non-compartmental analysis. In some embodiments, when measured in mice according to Example 4.1, the MRT of the fusion compound 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, preferably at least 9.4 hours. In some embodiments, when measured in miniature pigs according to Example 4.2, the MRT of the fusion compound is at least 50 hours, preferably at least 60 hours, preferably at least 70 hours, preferably at least 80 hours, preferably at least 90 hours. In some embodiments, when measured in cynomolgus monkeys according to Example 4.3, the MRT of the fusion compound is at least 30 hours, preferably at least 40 hours, preferably at least 50 hours, and preferably at least 52 hours.

[0100] Pharmacodynamics FGF21 is a human hormone synthesized in the liver and involved in glucose, lipid, and energy homeostasis. Treatment with FGF21 effectively reduces triglycerides, LDL-C, and VLDL-C while increasing HDL-C. Therefore, those skilled in the art will expect that compounds with FGF21 activity can reduce plasma levels of triglycerides, LDL-C, and VLDL-C. Treatment of obese rodents with FGF21 effectively reduces body weight, which is associated with IGF-1, hepatic triglycerides, and liver enzymes such as ALT and AST. Therefore, the PD effect of FGF21 can be studied in obese animal models.

[0101] GLP-1 is an intestinal glucose-lowering hormone secreted in the intestines, involved in glucose metabolism and satiety. Treatment with GLP-1 strongly and effectively reduces food intake and leads to weight loss.

[0102] In one embodiment, as measured in mice, the fusion compound reduces acute food intake. In one embodiment, as measured in mice, the fusion compound effectively reduces body weight. In one embodiment, as measured in DIO mice or a DIO-NASH model (GAN diet), the fusion compound reduces hepatic triglycerides. In one embodiment, as measured in DIO mice or a DIO-NASH model (GAN diet), the fusion compound reduces liver enzymes such as ALT.

[0103] In terms of functionality, the fusion compound may be able to reduce plasma concentrations of low-density lipoprotein cholesterol (LDL-C). Alternatively or additionally, the fusion compound of the present invention may be able to reduce plasma levels of triglycerides. Alternatively or additionally, the fusion compound of the present invention may be able to reduce plasma levels of total cholesterol.

[0104] Drug indications / medical uses This invention also relates to fusion compounds used as pharmaceuticals. As used herein, the term "treatment" refers to medical treatment for any human subject in need. This treatment can be preventative, precipitatory, palliative, symptomatic, and / or curative. The timing and purpose of the treatment may vary from individual to individual, depending on the subject's health condition.

[0105] According to a second aspect of the invention, a fusion compound as defined above (i.e., the compound as defined in the first aspect of the invention, including all its embodiments and specific features) is provided for use as a medicament (or for medical purposes).

[0106] For the avoidance of doubt, references to the compounds defined in the first aspect of the invention will include fusion proteins of Formula 1 (including all embodiments thereof) and their pharmaceutically acceptable salts, esters and amides.

[0107] In some implementations, the fusion compound is specifically used to treat and / or prevent eating disorders, cardiovascular disease, and diabetic complications; and / or to improve lipid parameters, such as preventing and / or treating dyslipidemia, reducing total serum lipids; increasing HDL; reducing small dense LDL; reducing VLDL; reducing triglycerides; reducing cholesterol; reducing human lipoprotein a (Lp(a)) plasma levels; increasing human plasma adiponectin; inhibiting apolipoprotein A (apo(A)) production and improving β-cell function; and / or to delay or prevent the progression of diabetes; and / or to treat and / or prevent hepatic steatosis, nonalcoholic fatty liver disease (NAFLD), metabolic dysfunction-associated steatosis (MAFLD), alcoholic liver disease (ALD), MetALD, and nonalcoholic steatohepatitis (NASH), and metabolic dysfunction-associated steatohepatitis (MASH).

[0108] It is proposed to use MASH as an alternative term to describe NASH, which is the same diagnostic entity (ME Rinella et al., j.aohep.2023.101133). As used herein, the terms “NASH” and MASH have the same meaning and are used interchangeably.

[0109] It is proposed that MAFLD be used as an alternative term to describe NAFLD, which is the same diagnostic entity (ME Rinella et al., j.aohep.2023.101133). As used herein, the terms “NAFDL” and “MAFLD” have the same meaning and are used interchangeably.

[0110] In a third aspect of the invention, compounds of the invention as defined above are provided for the treatment and / or prevention of diseases. These diseases may be selected from diabetes and related diseases, such as eating disorders, cardiovascular diseases, and diabetic complications; and / or for improving lipid parameters, improving β-cell function, for example, preventing and / or treating dyslipidemia, reducing total serum lipids; increasing HDL; reducing small dense LDL; reducing VLDL; reducing triglycerides; reducing cholesterol; reducing human lipoprotein a (Lp(a)) plasma levels; inhibiting the production of apolipoprotein A (apo(A)); and / or for delaying or preventing the progression of diabetes; and / or for treating and / or preventing hepatic steatosis, NAFDL, and NASH.

[0111] In an alternative third aspect of the invention, a method is provided for treating and / or preventing diseases such as diabetes and related diseases, such as eating disorders, cardiovascular diseases, and diabetic complications; and / or for improving lipid parameters, such as preventing and / or treating dyslipidemia, reducing total serum lipids; increasing HDL; reducing small dense LDL; reducing VLDL; reducing triglycerides; reducing cholesterol; reducing human lipoprotein a (Lp(a)) plasma levels; inhibiting the production of apolipoprotein A (apo(A)) and improving β-cell function; and / or for delaying or preventing the progression of diabetes; and / or for treating and / or preventing hepatic steatosis, NAFDL, and NASH, the method comprising administering a therapeutically effective amount of the compound of the invention as defined above to a patient in need.

[0112] In a further alternative third aspect of the invention, the use of the compounds of the invention as defined above in the preparation of medicaments for the treatment or prevention of diseases such as eating disorders, cardiovascular diseases, and diabetic complications; and / or for improving lipid parameters, such as preventing and / or treating dyslipidemia, reducing total serum lipids; increasing HDL; reducing small dense LDL; reducing VLDL; reducing triglycerides; reducing cholesterol; reducing human lipoprotein a (Lp(a)) plasma levels; inhibiting the production of apolipoprotein A (apo(A)) and improving β-cell function; and / or for delaying or preventing the progression of diabetes; and / or for the treatment and / or prevention of hepatic steatosis, NAFDL, and NASH.

[0113] In some embodiments, the compounds of the present invention can be used for the following pharmaceutical treatments: (i) Prevention and / or treatment of all forms of diabetes, such as hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, non-insulin-dependent diabetes, MODY (mature-onset diabetes mellitus), gestational diabetes, and / or for reducing HbA1C. (ii) Delay or prevent the progression of diabetes, such as the progression of type 2 diabetes, delay the progression of impaired glucose tolerance (IGT) to insulin-requiring type 2 diabetes, delay or prevent insulin resistance, and / or delay the progression of insulin-free type 2 diabetes to insulin-requiring type 2 diabetes. (iii) For example, by reducing food intake, losing weight, suppressing appetite, inducing satiety to prevent and / or treat eating disorders such as obesity; treat or prevent bulimia, bulimia nervosa and / or obesity induced by antipsychotic drugs or steroid administration; reduce gastric motility; delay gastric emptying; increase physical activity; and / or prevent and / or treat comorbidities of obesity such as osteoarthritis and / or urinary incontinence; (iv) Weight maintenance after successful weight loss (whether induced by medication or by diet and exercise) – that is, preventing weight gain after successful weight loss. (v) Prevention and / or treatment of liver conditions such as hepatic steatosis, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), liver inflammation, or fatty liver.

[0114] In some embodiments, the present invention relates to compounds of the present invention for the prevention and / or treatment of acute and / or chronic pancreatitis.

[0115] In some embodiments, the present invention relates to compounds for the prevention and / or treatment of diseases selected from type 2 diabetes, metabolic syndrome, obesity, insulin resistance, as well as prediabetes, diabetic retinopathy, diabetic neuropathy, diabetic nephropathy, chronic kidney disease, diabetic nephropathy, diabetic dyslipidemia, fatty liver disease including non-alcoholic fatty liver disease (NASH), and atherosclerosis.

[0116] In some embodiments, the present invention relates to compounds of the present invention for the prevention and / or treatment of hepatocellular carcinoma (HCC).

[0117] In some embodiments, the present invention relates to compounds of the present invention for the prevention and / or treatment of alcoholic steatohepatitis (ASH) or alcoholic fatty liver disease (AFDL).

[0118] In some embodiments, the present invention relates to compounds of the invention for the prevention and / or treatment of NASH, wherein said 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 the percentage of liver weight to total body weight. In some embodiments, said use reduces relative liver weight, plasma alanine aminotransferase levels, liver triglyceride content, and / or liver cholesterol.

[0119] In some embodiments, the present invention relates to compounds of the present invention for the prevention and / or treatment of NASH, wherein said use prevents, delays and / or reduces any histopathological indications of fatty degeneration.

[0120] In some embodiments, the present invention relates to compounds of the present invention for the prevention and / or treatment of NASH, wherein the use prevents, delays and / or reduces inflammation in the liver.

[0121] NASH is the most extreme form of NAFLD. NAFLD is a type of fatty liver that occurs when fat deposits in the liver due to causes other than alcohol consumption. Fat deposition is also known as steatosis. Patients with NASH often have obesity, type 2 diabetes, dyslipidemia, and / or metabolic syndrome. Symptoms of NASH include fatigue, malaise, or discomfort in the right upper quadrant of the abdomen.

[0122] In some embodiments, the present invention relates to compounds of the present invention for the prevention and / or treatment of NASH, wherein the use prevents, delays, and / or reduces fibrosis in the liver. In some embodiments, the present invention relates to compounds for the prevention and / or treatment of NASH, wherein the compounds are administered in the form of a pharmaceutical composition comprising 1-50 mg / ml of the compound, such as 5-40 mg / ml, such as 10-30 mg / ml of the compound.

[0123] In some embodiments, the present invention relates to compounds of the present invention for the prevention and / or treatment of NASH, wherein the compounds are administered subcutaneously once a week.

[0124] In some embodiments, the present invention relates to compounds of the invention for the prevention and / or treatment of NASH, wherein the compounds are administered subcutaneously daily, every other day, every three days, every four days, every five days, or every six days. In some embodiments, the compounds are administered for at least 12 months.

[0125] In some embodiments, the present invention relates to compounds of the invention for the prevention and / or treatment of NASH, wherein said compounds are administered to subjects in need in a therapeutically effective amount.

[0126] In some embodiments, the subject is obese and / or has diabetes. In some embodiments, the subject is overweight, obese, has hyperglycemia, type 2 diabetes, impaired glucose tolerance, and / or type 1 diabetes.

[0127] In some embodiments, the indication may be type 2 diabetes and / or dyslipidemia and / or obesity. In some embodiments, the present invention relates to a weight management method. 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.

[0128] Typically, all subjects suffering from obesity are also considered 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 invention for treating or preventing obesity. In some embodiments, the subjects suffering from obesity are persons, such as adults or pediatric patients (including infants, children, and adolescents). Body Mass Index (BMI) is a measure of body fat based on height and weight. The calculation formula is BMI = weight in kilograms / height in meters. 2 Human subjects suffering from obesity may have a BMI >30; such subjects may also be referred to as obese. In some embodiments, human subjects suffering from obesity may have a BMI >35 or a BMI in the range of >30 to <40. In some embodiments, the obesity is severe obesity or morbid obesity, wherein human subjects may have a BMI >40.

[0129] In some embodiments, the present invention relates to methods for treating or preventing overweight 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 in the presence of at least one weight-related comorbidity. In some embodiments, the overweight subject is a person, such as an adult or a pediatric patient (including infants, children, and adolescents). In some embodiments, the overweight human subject may have a BMI >25, such as a BMI >27. In some embodiments, the overweight human subject has a BMI in the range of 25 to <30 or in the range of 27 to <30. In some embodiments, the weight-related comorbidity is selected from hypertension, diabetes (such as type 2 diabetes), dyslipidemia, high cholesterol, and obstructive sleep apnea.

[0130] In some embodiments, the present invention relates to methods 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 to undergo weight loss according to the present invention may have a BMI >25, such as a BMI >27 or a BMI >30. In some embodiments, a person to undergo weight loss according to the present invention may have a BMI >35 or a BMI >40. The term "weight loss" may include the treatment or prevention of obesity and / or overweight.

[0131] Those skilled in the art will understand that references to treatment of a specific condition (or similarly, references to treating the condition) will have their normal meaning in the medical field. In particular, the term may refer to the achievement of a reduction in the severity and / or frequency of one or more clinical symptoms associated with the condition, as determined by the attending physician of a patient who has or is susceptible to such symptoms. For example, in the case of NASH, the term may refer to increased liver enzymes, increased plasma lipids, increased liver stiffness, increased hepatic steatosis, loss of liver function, and increased hepatotoxicity.

[0132] As used herein, reference to a patient (or multiple patients) refers to a living subject receiving treatment, including mammalian (e.g., human) patients. In particular, reference to a patient refers to a human patient.

[0133] For the avoidance of doubt, those skilled in the art will understand that such treatment [or prevention] will be administered to patients (or subjects) in need. Those skilled in the art can use conventional techniques to assess a patient's (or subject's) need for such treatment [or prevention]. As used herein, the terms disease and symptom (and similarly, the terms condition, ailment, medical problem, etc.) are used interchangeably. As used herein, the term effective amount refers to the amount of compound that produces a therapeutic effect on the treated patient. The effect can be observed in an objective manner (i.e., measured by certain tests or markers) or subjectively (i.e., indicated and / or felt by the subject). In particular, the effect can be observed (e.g., measured) objectively using appropriate tests known to those skilled in the art.

[0134] Pharmaceutical Composition This invention also relates to pharmaceutical compositions comprising a fusion compound. In one embodiment, the pharmaceutical composition comprising the fusion compound includes at least one pharmaceutically acceptable excipient. The pharmaceutical compositions / formulations described herein can be prepared according to standards and / or recognized pharmaceutical practices.

[0135] The term "excipient" broadly refers to any component other than the active therapeutic ingredient. Excipients can be inert, inactive, and / or pharmaceutically inactive. Excipients can be used for a variety of purposes, such as as carriers, loading agents, diluents, tablet excipients, and / or to improve administration and / or absorption of the active ingredient. Formulation of pharmaceutically active ingredients with various excipients is known in the art; see, for example, Remington: The Science and Practice of Pharmacy (e.g., 19th edition (1995) and any subsequent editions). Other optional components of a pharmaceutical composition include, for example, wetting agents, emulsifiers, antioxidants, fillers, metal ions, oily loading agents, and proteins. Non-limiting examples of excipients include solvents, diluents, buffers, preservatives, tension modifiers, chelating agents, surfactants, and stabilizers.

[0136] In a fourth aspect of the invention, a pharmaceutical composition is provided comprising a fusion compound as defined above and optionally one or more pharmaceutically acceptable excipients. The injectable composition can be prepared using conventional techniques of the pharmaceutical industry, which include appropriately dissolving and mixing the components to obtain the desired end product. Thus, according to a procedure, the fusion compound is dissolved in a suitable buffer solution at an appropriate pH to minimize or avoid precipitation. In embodiments, the pharmaceutical composition may comprise a wetting agent, emulsifier, antioxidant, filler, tonicotinic agent, chelating agent, metal ion, oily carrier, protein (e.g., human serum albumin, gelatin, or protein) or zwitterionic (e.g., amino acids such as betaine, taurine, arginine, glycine, lysine, and 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.

[0137] Parenteral administration can be performed via subcutaneous, intramuscular, intraperitoneal, or intravenous injection using a syringe, optionally a pen syringe. Alternatively, parenteral administration can be performed using an infusion pump.

[0138] Pharmaceutical compositions containing fusion compounds can be in several dosage forms, such as solutions, suspensions, tablets, and capsules.

[0139] Therefore, in another aspect of the invention, a method for preparing a pharmaceutical composition / formulation as defined above is provided, the method comprising mixing a fusion compound as defined above with one or more pharmaceutically acceptable excipients.

[0140] Therefore, in a fifth aspect of the invention, a pharmaceutical composition as defined in the fourth aspect of the invention is provided for treating or preventing diseases as defined herein, with reference to the third aspect of the invention and all embodiments thereof.

[0141] Pharmaceutical compositions containing fusion compounds can be applied to several sites in patients in need, such as local sites, like the skin or mucous membranes; sites of bypass absorption, like the arteries, veins, or heart; and sites involving absorption, such as the skin, subcutaneous tissue, muscle, or orally or in the abdomen.

[0142] Treatment using the fusion compounds of this invention can also be combined with one or more other pharmacologically active substances, such as amylin analogs or antifibrotic agents, such as SGLT2, FXR agonists, ACCI, or THRβ agonists.

[0143] Production process This invention also relates to a method for producing fusion compounds. The preparation of peptides such as GLP-1 analogs, FGF21 analogs, and spacers is well known in the art. For example, peptides incorporated into a fusion compound (or a fragment thereof) can be prepared by classical recombinant methods, i.e., by culturing host cells containing DNA sequences encoding analogs and capable of expressing the peptides in a suitable nutrient medium under conditions allowing expression of the peptides. Non-limiting examples of host cells suitable for expressing these peptides are: *Escherichia coli* (…). Escherichia coli ), brewer's yeast ( Saccharomyces cerevisiaeAnd mammalian BHK or CHO cell lines. Alternatively or concurrently, peptides incorporated into fusion compounds (or fragments thereof) can be prepared by classical solid-phase peptide synthesis, such as solid-phase peptide synthesis using Boc or Fmoc chemistry, or other established techniques, see, for example, Greene and Wuts, “Protective Groups in Organic Synthesis”, John Wiley & Sons, 1999; Florencio Zaragoza Dörwald, “Organic Synthesis on solid Phase”, Wiley-VCH Verlag GmbH, 2000; and “Fmoc SolidPhase Peptide Synthesis”, edited by WC Chan and PD White, Oxford University Press, 2000. Fusion compounds containing non-coding amino acids can be prepared as described in the art, 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.

[0144] In some embodiments, the fusion compounds of the present invention are prepared in a stepwise manner: (i) recombinant preparation of the backbone polypeptide, and (ii) covalent linkage of substituents to the backbone polypeptide (e.g., by alkylation). Specific examples of methods for preparing the fusion compounds are included in the experimental section.

[0145] While certain features of the invention have been set forth and described herein, many modifications, substitutions, alterations, and equivalents will now occur to those skilled in the art. Therefore, it should be understood that all such modifications and alterations falling within the true scope of the invention are intended to be covered by the appended claims.

[0146] Specific implementation plan The following are specific embodiments of the present invention: 1. A fusion protein comprising a polypeptide of formula 1: ABC, wherein: A is a GLP-1 polypeptide, which is an analogue of SEQ ID NO: 1; B is a spacer composed of 1-257 amino acids; C is an FGF21 polypeptide, which is an analogue of SEQ ID NO: 2; Or its pharmaceutically acceptable salt, amide, or ester.

[0147] 2. The fusion protein according to embodiment 1, wherein the C-terminus of the GLP-1 peptide 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.

[0148] 3. The fusion protein according to one of the aforementioned embodiments, wherein B consists of 5 to 257 amino acids.

[0149] 4. The fusion protein according to one of the aforementioned embodiments, wherein B consists of 9 to 129 amino acids.

[0150] 5. The fusion protein according to one of the foregoing embodiments, wherein B comprises chemical formula 2 (GAQP). x -C y -(GAQP) h -A j Or derived from chemical formula 2 (GAQP) x -C y -(GAQP) h -A j The expression is composed of x, which is an integer in the range of 0-64, y, which is an integer in the range of 0-1, h, which is an integer in the range of 1-64, and j, which is an integer in the range of 0-1.

[0151] 6. The fusion protein according to one of the foregoing embodiments, wherein B is selected from the list consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18.

[0152] 7. The fusion protein according to one of the foregoing embodiments, wherein B is SEQ ID NO: 13, SEQ ID NO: 16 or SEQ ID NO: 17.

[0153] 8. The fusion protein according to one of the foregoing embodiments, 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.

[0154] 9. The fusion protein according to one of the foregoing embodiments, wherein, compared with SEQ ID NO: 1, A contains a maximum of 15 amino acid changes, preferably a maximum of 14 amino acid changes, preferably a maximum of 13 amino acid changes, preferably 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, and most preferably a maximum of 4 amino acid changes.

[0155] 10. The fusion protein according to any of the foregoing embodiments, wherein A comprises or consists of the amino acid sequence SEQ ID NO: 3.

[0156] 11. The fusion protein according to any of the foregoing embodiments, wherein A has 3-7 amino acid changes compared to SEQ ID NO: 1.

[0157] 12. The fusion protein according to any of the foregoing embodiments, 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 wherein A contains an Arg residue at a position corresponding to position 26 of GLP-1 (7-37) (SEQ ID NO: 1).

[0158] 13. The fusion protein according to any of the foregoing embodiments, wherein A comprises 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].

[0159] 14. The fusion protein according to any of the foregoing embodiments, 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.

[0160] 15. The fusion protein according to any of the foregoing embodiments, wherein C 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% of SEQ ID NO: 2.

[0161] 16. The fusion protein according to any of the foregoing embodiments, wherein C, compared with SEQ ID NO: 2, 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.

[0162] 17. The fusion protein according to any of the foregoing embodiments, wherein C has 3 or 4 amino acid changes compared to SEQ ID NO: 2.

[0163] 18. The fusion protein according to any of the foregoing embodiments, wherein C comprises 180C, 180E, or 180A.

[0164] 19. The fusion protein according to any of the foregoing embodiments, wherein C comprises 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].

[0165] 20. The fusion protein according to any of the foregoing embodiments, wherein C is selected from the list consisting of SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12.

[0166] 21. The fusion protein according to any of the foregoing embodiments, wherein C has FGF21 activity.

[0167] 22. The fusion protein according to any of the foregoing embodiments, wherein C is capable of activating FGFR1c.

[0168] 23. The fusion protein according to any of the foregoing embodiments, wherein the components of Formula 1 are linked by amide bonds.

[0169] 24. A fusion compound comprising a fusion protein according to any one of embodiments 1-23, wherein the fusion compound comprises one or more substituents.

[0170] 25. The fusion compound according to embodiment 24, wherein the substituent comprises an elongated portion.

[0171] 26. The fusion compound according to embodiment 24 or embodiment 25, wherein the extended portion is a functional group (FG) with pKa < 7.0.

[0172] 27. The fusion compound according to any one of embodiments 24-26, wherein the extended portion is lipophilic and / or negatively charged at physiological pH (7.4).

[0173] 28. The fusion compound according to any one of embodiments 24-27, wherein the extended portion is a fatty acid.

[0174] 29. The fusion compound according to any one of embodiments 24-28, wherein the extended portion is of chemical formula 3: HOOC-(CH2) x -CO- , where x is an integer in the range of 10-20.

[0175] 30. The fusion compound according to embodiment 29, wherein x in chemical formula 3 is 12-20.

[0176] 31. The fusion compound according to embodiment 29 or embodiment 30, wherein x in chemical formula 3 is 14-20.

[0177] 32. The fusion compound according to any one of embodiments 29-31, wherein x in chemical formula 3 is 14-18.

[0178] 33. The fusion compound according to any one of embodiments 29-32, wherein x in chemical formula 3 is 16-20.

[0179] 34. The fusion compound according to any one of embodiments 29-32, wherein x in chemical formula 3 is 16-18.

[0180] 35. The fusion compound according to any one of embodiments 29-34, wherein x in chemical formula 3 is 16.

[0181] 36. The fusion compound according to any one of embodiments 24-35, wherein the substituent comprises a linker.

[0182] 37. The fusion compound according to any one of embodiments 24-36, wherein the substituent comprises an elongation portion and a linker.

[0183] 38. The fusion compound according to any one of embodiments 36-37, wherein the linker comprises at least one of chemical formula 4, chemical formula 5 and chemical formula 6; Chemical formula 4 is -NH-CH(COOH)-(CH2)2-CO- , Chemical formula 5 is -NH-(CH2)2-[O-(CH2)2] k -O-[CH2] 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-(CH2) n -NH-CO-CH2- Where n is an integer in the range of 1 to 5. Formulas 4, 5, and 6 are interconnected by amide bonds and, in the order shown, are... The -NH end is connected to the CO- of the extended body (Chemical Formula 3). end, and in its CH2- An amino acid whose end is attached to A, B, or C.

[0184] 39. The fusion compound according to embodiment 38, wherein k of chemical formula 5 is 1.

[0185] 40. The fusion compound according to embodiment 38 or embodiment 39, wherein m of chemical formula 5 is 1.

[0186] 41. The fusion compound according to any one of embodiments 38-40, wherein n of chemical formula 6 is 2.

[0187] 42. The fusion compound according to any one of embodiments 38-41, wherein the linker elements are connected by amide bonds.

[0188] 43. The fusion compound according to any one of embodiments 38-42, wherein the linker comprises a chemical formula 4 element, two chemical formula 5 elements and a chemical formula 6 element.

[0189] 44. The fusion compound according to any one of embodiments 38-42, wherein the linker comprises a chemical formula 4 element, two chemical formula 5 elements and a chemical formula 6 element.

[0190] 45. The fusion compound according to any one of embodiments 38-43, wherein the extended portion is attached to chemical formula 4 of the linker, and wherein chemical formula 5 of the linker is attached to the polypeptide backbone.

[0191] 46. ​​The fusion compound according to any one of embodiments 38-44, wherein the substituent is of chemical formula 7: .

[0192] 47. The fusion compound according to any one of embodiments 24-46, wherein the substituent is [2-[2-[[2-[2-[2-[2-[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoyl-amino)butyryl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]-ethylamino]-2-oxoethyl].

[0193] 48. The fusion compound according to any one of embodiments 24 to 47, wherein the fusion compound comprises first and second substituents.

[0194] 49. The fusion compound according to any one of embodiments 24 to 48, wherein at least one substituent is attached to any one of the amino acid residues A, B or C.

[0195] 50. The fusion compound according to any one of embodiments 24 to 49, wherein at least one substituent is attached to an amino acid residue of A.

[0196] 51. The fusion compound according to any one of embodiments 24 to 50, wherein at least one substituent is attached to an amino acid residue of B.

[0197] 52. The fusion compound according to any one of embodiments 24 to 51, wherein at least one substituent is attached to an amino acid residue of C.

[0198] 53. The fusion compound according to any one of embodiments 48 or 49, wherein the first substituent is attached to A or B and the second substituent is attached to C.

[0199] 54. The fusion compound according to any one of embodiments 24-53, wherein the substituent is attached to a Cys residue.

[0200] 55. The fusion compound according to any one of embodiments 24-54, wherein the substituent is attached to a Cys residue in A, B, or C.

[0201] 56. The fusion compound according to any one of embodiments 24-55, wherein A comprises 26C, 27C, or 36C.

[0202] 57. The fusion compound according to any one of embodiments 24-55, wherein C comprises 180C.

[0203] 58. The fusion compound according to any one of embodiments 24-56, wherein the substituent is attached to 26C, 27C, or 36C in A.

[0204] 59. The fusion compound according to any one of embodiments 24-55 or 57, wherein the substituent is attached to 180C in C.

[0205] 60. The fusion compound according to any one of embodiments 24-59, wherein the fusion compound is selected from... (Chemical Formula 8) (Chemical Formula 9) (Chemical Formula 10) (Chemical Formula 11) (Chemical Formula 12) (Chemical Formula 13) (Chemical Formula 14) (Chemical Formula 15) (Chemical Formula 16) (Chemical Formula 17) (Chemical Formula 18) (Chemical Formula 19) (Chemical Formula 20) (Chemical Formula 21) (Chemical Formula 22) and List of (chemical formula 23).

[0206] 61. The fusion compound according to any one of embodiments 24-60, wherein the fusion compound is selected from the list consisting of chemical formula 10, chemical formula 11, chemical formula 12, chemical formula 14, chemical formula 16, chemical formula 17, chemical formula 18, chemical formula 21 and chemical formula 23.

[0207] 62. The fusion compound according to any one of embodiments 24-60, wherein the fusion compound is selected from... (Chemical Formula 12) (Chemical Formula 14) (Chemical Formula 16) and List of (chemical formula 23).

[0208] 63. The fusion compound according to any one of embodiments 24-61, wherein the fusion compound is (Chemical formula 23).

[0209] 64. The fusion compound according to any one of embodiments 24-63, wherein the fusion compound has GLP-1 activity.

[0210] 65. The fusion compound according to any one of embodiments 24-63, wherein the fusion compound is capable of binding to the GLP-1 receptor.

[0211] 66. The fusion compound according to any one of embodiments 24-63, wherein the GLP-1 activity is measured in the absence of HSA.

[0212] 67. The fusion compound according to any one of embodiments 24-66, wherein the GLP-1 activity is measured as described in Example 2.

[0213] 68. The fusion compound according to any one of embodiments 24-66, wherein the GLP-1 activity is expressed as an EC50 value.

[0214] 69. The fusion compound according to embodiment 70, wherein the EC50 value is measured in the absence of HSA and is below about 100 pM, preferably below about 70 pM, preferably below about 50 pM, and preferably below about 20 pM.

[0215] 70. The fusion compound according to any one of embodiments 24-66, wherein the fusion compound has FGF21 activity.

[0216] 71. The fusion compound according to any one of embodiments 24-70, wherein the fusion compound is measured in the absence of HSA.

[0217] 72. The fusion compound according to any one of embodiments 24-70, wherein the fusion compound is measured as described in Example 3.

[0218] 73. The fusion compound according to any one of embodiments 24-70, wherein the FGF21 activity is the ability to activate FGFR.

[0219] 74. The fusion compound according to any one of embodiments 24-70, wherein the FGF21 activity is the ability to activate FGFR1c.

[0220] 75. The fusion compound according to any one of embodiments 24-70, wherein the FGF21 activity is measured in vitro using a whole-cell assay.

[0221] 76. The fusion compound according to any one of embodiments 24-70, wherein the FGF21 activity is expressed as EC 50 The value, and in which the EC 50 Below about 50 nM, preferably below about 10 nM, preferably below about 5 nM, and preferably below about 2 nM.

[0222] 77. The fusion compound according to any one of embodiments 24-70, wherein the fusion compound has a desired half-life.

[0223] 78. The fusion compound according to any one of embodiments 24-77, wherein the fusion compound has a long half-life.

[0224] 79. The fusion compound according to any one of embodiments 24-77, wherein the fusion compound has a half-life suitable for once-weekly administration.

[0225] 80. The fusion compound according to any one of embodiments 24-79, wherein the fusion compound is extended.

[0226] 81. The fusion compound according to any one of embodiments 24-77, wherein the fusion compound is capable of reducing plasma triglyceride levels.

[0227] 82. A pharmaceutical composition comprising a fusion compound according to any one of embodiments 24-81 or a pharmaceutically acceptable salt, amide or ester thereof, and one or more pharmaceutically acceptable excipients.

[0228] 83. The fusion compound according to any one of embodiments 24-81, or the pharmaceutical composition according to embodiment 82, used as a drug.

[0229] 84. The fusion compound according to any one of embodiments 24-81, or the pharmaceutical composition according to embodiment 82, for use in... (i) Prevention and / or treatment of all forms of diabetes, such as hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, non-insulin-dependent diabetes, MODY (mature-onset diabetes mellitus), gestational diabetes, and / or for reducing HbA1C. (ii) Delay or prevent the progression of diabetes, such as the progression of type 2 diabetes, delay the progression of impaired glucose tolerance (IGT) to insulin-requiring type 2 diabetes, delay or prevent insulin resistance, and / or delay the progression of insulin-free type 2 diabetes to insulin-requiring type 2 diabetes. (iii) Improve β-cell function, such as reducing β-cell apoptosis, improving β-cell function and / or β-cell quality, and / or restoring β-cell glucose sensitivity; (iv) Prevention and / or treatment of cognitive impairment and / or neurodegenerative conditions, such as Alzheimer's disease, Parkinson's disease and / or multiple sclerosis; (v) For example, by reducing food intake, losing weight, suppressing appetite, inducing satiety to prevent and / or treat eating disorders such as obesity; treat or prevent bulimia, bulimia nervosa and / or obesity induced by antipsychotic drugs or steroid administration; reduce gastric motility; delay gastric emptying; increase physical activity; and / or prevent and / or treat comorbidities of obesity such as osteoarthritis and / or urinary incontinence; (vi) Prevention and / or treatment of diabetic complications, such as vascular disease; neuropathy, including peripheral neuropathy; nephropathy; and / or retinopathy; (vii) Improve lipid parameters, such as prevent and / or treat dyslipidemia, reduce total serum lipids; increase HDL; reduce small dense LDL; reduce VLDL; reduce triglycerides; reduce cholesterol; reduce human lipoprotein a (Lp(a)) plasma levels; inhibit the production of apolipoprotein a (apo(a)) in vitro and / or in vivo; (viii) Prevention and / or treatment of cardiovascular diseases, such as syndrome X, atherosclerosis, myocardial infarction, coronary artery 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, arrhythmia, syncope, angina pectoris, coronary artery bypass grafting and / or stent re-occlusion, intermittent claudication (occlusive arteriosclerosis), diastolic dysfunction and / or systolic dysfunction; and / or lowering blood pressure, such as lowering systolic blood pressure; (ix) Prevention and / or treatment of gastrointestinal diseases, 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 critical illness, such as treatment of critically ill patients, patients with critically ill multiple nephropathy (CIPNP) and / or potential CIPNP patients; prevention of the progression of critical illness or CIPNP; prevention, treatment and / or cure of systemic inflammatory response syndrome (SIRS) in patients; prevention or reduction of the likelihood of patients developing bacteremia, sepsis and / or septic shock during hospitalization; and / or stabilization of blood glucose, insulin balance and optional 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 brain diseases, such as cerebral ischemia, cerebral hemorrhage and / or traumatic brain injury; (xiii) Prevention and / or treatment of sleep apnea; and / or (xiv) Prevention and / or treatment of abuse, such as alcohol abuse and / or drug abuse.

[0230] 85. The fusion compound according to any one of embodiments 24-81, or the pharmaceutical composition according to embodiment 82, for the treatment and / or prevention of any of all forms of diabetes and related diseases, such as obesity, eating disorders, cardiovascular disease, and diabetic complications; and / or for improving lipid parameters, improving β-cell function; and / or for delaying or preventing the progression of diabetes; and / or for the treatment and / or prevention of hepatic steatosis and non-alcoholic fatty liver disease (NAFLD).

[0231] 86. The fusion compound according to any one of embodiments 24-81, or the pharmaceutical composition according to embodiment 82, for the prevention and / or treatment of obesity.

[0232] 87. The fusion compound according to any one of embodiments 24-81, or the pharmaceutical composition according to embodiment 82, for the treatment and / or prevention of hepatic steatosis and non-alcoholic fatty liver disease (NAFLD).

[0233] 88. The fusion compound according to any one of embodiments 24-81, or the pharmaceutical composition according to embodiment 82, for the treatment and / or prevention of NASH.

[0234] 89. The fusion compound according to any one of embodiments 24-81, or the pharmaceutical composition according to embodiment 82, for preventing or delaying increases in relative liver weight, plasma alanine aminotransferase levels, liver triglyceride content, and / or liver cholesterol.

[0235] 90. The fusion compound according to any one of embodiments 24-81, or the pharmaceutical composition according to embodiment 82, for reducing relative liver weight, plasma alanine aminotransferase levels, liver triglyceride content, and / or liver cholesterol.

[0236] 91. The fusion compound according to any one of embodiments 24-81, or the pharmaceutical composition according to embodiment 82, for the prevention, delay and / or reduction of inflammation in the liver.

[0237] 92. The fusion compound according to any one of embodiments 24-81, or the pharmaceutical composition according to embodiment 82, for preventing, delaying and / or reducing fibrosis in the liver.

[0238] 93. Use of the fusion compound according to any one of embodiments 24-81 or the pharmaceutical composition according to embodiment 82 in the preparation of a medicament for 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 for improving lipid parameters and β-cell function; and / or for delaying or preventing the progression of diabetes; and / or for the treatment and / or prevention of hepatic steatosis and non-alcoholic fatty liver disease (NAFLD).

[0239] 94. Use of the fusion compound according to any one of embodiments 24-81 or the pharmaceutical composition according to embodiment 82 in the preparation of a medicament for the treatment and / or prevention of NASH.

[0240] 95. Use of the fusion compound according to any one of embodiments 24-81 or the pharmaceutical composition according to embodiment 82 in the preparation of a medicament for treating hepatic steatosis and non-alcoholic fatty liver disease (NAFLD).

[0241] 96. A method for treating or preventing all forms 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 24-81 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 diabetes; and / or treating and / or preventing hepatic steatosis and non-alcoholic fatty liver disease (NAFLD).

[0242] 97. A method for treating or preventing NASH by administering a pharmaceutically active amount of a fusion compound according to any one of embodiments 24-81 or a pharmaceutical composition according to embodiment 82.

[0243] 98. A method for treating or preventing hepatic steatosis and nonalcoholic fatty liver disease (NAFLD) by administering a pharmaceutically active amount of a fusion compound according to any one of embodiments 24-81 or a pharmaceutical composition according to embodiment 82.

[0244] 99. A method for producing a fusion compound according to any one of embodiments 24-81, the method comprising the steps of: (i) recombinantly preparing a backbone polypeptide, and (ii) covalently attaching (e.g., by alkylation) a substituent to said backbone polypeptide.

[0245] Further specific implementation plans A. A fusion compound comprising a polypeptide of formula 1: ABC, and optionally one or more substituents, wherein: A is a GLP-1 polypeptide, which is an analogue of SEQ ID NO: 1; B is a spacer composed of 1-257 amino acids; C is an FGF21 polypeptide, which is an analogue of SEQ ID NO: 2; Or its pharmaceutically acceptable salt, amide, or ester.

[0246] B. The fusion compound according to any one of the foregoing embodiments, wherein the C-terminus of the GLP-1 peptide 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.

[0247] C. The fusion compound according to one of the aforementioned embodiments, wherein B consists of 5 to 257 amino acids.

[0248] D. The fusion compound according to one of the aforementioned embodiments, wherein B consists of 9 to 129 amino acids.

[0249] E. The fusion compound according to one of the foregoing embodiments, wherein B comprises chemical formula 2 (GAQP). x -C y -(GAQP) h -A j Or derived from chemical formula 2 (GAQP) x -C y -(GAQP) h -A j The expression is composed of x, which is an integer in the range of 0-64, y, which is an integer in the range of 0-1, h, which is an integer in the range of 1-64, and j, which is an integer in the range of 0-1.

[0250] F. The fusion compound according to one of the foregoing embodiments, wherein B is selected from the list consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18.

[0251] G. The fusion compound according to one of the foregoing embodiments, wherein B is SEQ ID NO: 13, SEQ ID NO: 16 or SEQ ID NO: 18.

[0252] H. The fusion compound according to one of the foregoing embodiments, 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.

[0253] I. The fusion compound according to one of the foregoing embodiments, wherein, compared with SEQ ID NO: 1, A contains 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.

[0254] J. The fusion compound according to any of the foregoing embodiments, wherein A comprises or consists of the amino acid sequence SEQ ID NO:3.

[0255] K. The fusion compound according to any of the foregoing embodiments, wherein A has 3-7 amino acid changes compared to SEQ ID NO: 1.

[0256] L. The fusion compound according to any of the foregoing embodiments, 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 wherein A contains an Arg residue at a position corresponding to position 26 of GLP-1 (7-37) (SEQ ID NO: 1).

[0257] M. The fusion compound according to any of the foregoing embodiments, wherein A comprises 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].

[0258] N. The fusion compound according to any of the foregoing embodiments, 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.

[0259] O. The fusion compound according to any of the foregoing embodiments, wherein C 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% of SEQ ID NO: 2.

[0260] P. The fusion compound according to any of the foregoing embodiments, wherein, compared with SEQ ID NO: 2, 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.

[0261] Q. The fusion compound according to any of the foregoing embodiments, wherein C has 3 or 4 amino acid changes compared to SEQ ID NO: 2.

[0262] R. The fusion compound according to any of the foregoing embodiments, wherein C comprises 180C, 180E, or 180A.

[0263] S. The fusion compound according to any of the foregoing embodiments, wherein C comprises 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].

[0264] T. The fusion compound according to any of the foregoing embodiments, wherein C is selected from the list consisting of SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12.

[0265] U. The fusion compound according to any of the foregoing embodiments, wherein C has FGF21 activity.

[0266] V. The fusion compound according to any of the foregoing embodiments, wherein C is capable of activating FGFR1c.

[0267] W. The fusion compound according to any of the foregoing embodiments, wherein the components of Formula 1 are linked by amide bonds.

[0268] List of abbreviations Area under the AUC curve BEH Ethylene-bridged hybrid BHK baby hamster kidney BKL β klotho BNP (Brain Natriuretic Peptide) BSA (Bovine Serum Albumin) BSPP (Dipotassium salt of bis(p-sulfonylphenyl)phenylphosphine dihydrate) CETP (cholesterol ester transfer protein) CHO Chinese hamster ovary CIP in situ cleanup CMV (Cytomegalovirus) CV column volume CVD (Cardiovascular Disease) DMEM Dubelocco Modified Eagle Medium DPP-IV dipeptidyl peptidase-IV DTT dithiothreitol E. coli EGF(A) epidermal growth factor-like domain A ELISA (Enzyme-Linked Immunosorbent Assay) EDTA (ethylenediaminetetraacetic acid) ERK extracellular signal-regulated kinase FBS Fetal Bovine Serum FGF21 fibroblast growth factor 21 FGF-R fibroblast growth factor receptor GLP-1 glucagon-like peptide-1 HEPES 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid HDLc (High-density lipoprotein cholesterol) HRP (Hordeum peroxidase) HSA human serum albumin IMAC Immobilized Metal Affinity Chromatography IPTG isopropyl β-d-1-thiogalactopyranoside LB Luria-Bertani (Sambrook, J., EF Fritsch and T. Maniatis. (1989). Molecular cloning: a laboratory manual, 2nd ed. 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 channel immunoassay HDL-C (High-density lipoprotein cholesterol) MAPK mitogen-activated protein kinase MQ Milli-Q MSD quality selective detector MWCO molecular weight cutoff value NAFLD (Non-alcoholic fatty liver disease) NEP neutral endopeptidase PBS (Phosphate Buffered Saline) PCA polyclonal antibody PCSK9 preprotein convertase, subtilisin / Kexin type 9 Pen / step Penicillin-Streptomycin SDS-PAGE (Sodium Lauryl Sulfate-Polyacrylamide Gel Electrophoresis) SGLT2 Selective sodium-glucose cotransporter 2 TB Terrif Broth TFA (trifluoroacetic acid) TIC Total Ion Current Time of Flight (TOF) Tris tris(hydroxymethyl)aminomethane or 2-amino-2-hydroxymethylpropane-1,3-diol UPLC (Ultra-High Performance Liquid Chromatography) VLDL-C (Very Low Density Lipoprotein Cholesterol) Preparation method of fusion compound The polypeptides prepared for the fusion compounds of the present invention can be prepared using conventional methods in the art.

[0269] General methods for preparing peptides in Escherichia coli host systems Cloning and expression Recombination produces a fusion protein backbone with a removable N-terminal extension. This is achieved by expressing a DNA sequence encoding an amino acid sequence and subcloning it into a pET11d-derived vector, followed by transformation into a suitable host cell, E. coli BL21(DE3) or a derivative thereof. The DNA sequence is of synthetic origin and is obtained from commercial suppliers such as Thermofisher and Genescript.

[0270] E. coli transformation Transformation of *E. coli* was performed according to the standard method of Sambrook et al. (1989) [Sambrook J, Fritsch EF, Maniatis T.; Molecular Cloning: A Laboratory Manual, 2nd ed.; Cold Spring Harbor Laboratory Press: New York; 1989], or by electroporation in 2-mm cuvettes using a Bio-Rad Gene Pulser set to 25 µF, 200 ohm, and 2.5 kV, as described by Dower et al. (1988) [Dower, WJ, Miller, JF, and Ragsdale, CW (1988) Nucleic Acids Res. 16, 6127-6145]. Transformed cells were selected on LB medium supplemented with an appropriate selective antibiotic, either ampicillin or kanamycin.

[0271] E. coli culture E. coli cells transformed with plasmid DNA were obtained from frozen stock solution or freshly transformed cells directly from LB agar plates (containing appropriate antibiotics). Cells were seeded into 500 ml Corning® disposable Erlenmeyer flasks containing 100 ml LB medium with appropriate antibiotics. Cells were allowed to grow overnight at 30°C and shaking at 220 rpm. 40 ml of cells from the pre-culture was diluted in 2000 ml TB medium filled into 5-L Corning® disposable Erlenmeyer flasks. Cells were cultured at 37°C until an OD600 of 2.0 was reached. Target protein expression was then induced by adding 1 mM IPTG and cultured further at 37°C. Expression samples were analyzed by SDS-PAGE. Inclusion body fractions were separated and collected by sonication and centrifugation, as described in the following paragraph.

[0272] Inclusion body recycling Cell slurries in 20 mM histidine, 150 mM NaCl buffer (pH 6.0) were lysed using a disruptor (900 bar, four times) and precipitated by centrifugation (6000 x g, 30 min). Inclusion bodies were washed twice with 20 mM histidine (aqueous solution), 1 M sodium acetate (aqueous solution), and 0.1% Triton, and once with water (MilliQ), and analyzed by SDS-PAGE.

[0273] SDS-PAGE SDS-PAGE was performed using NuPAGE™ 4-12% gel Bis-Tris (Thermofisher) according to the standards of the art to analyze the expressed samples in accordance with the provided protocol.

[0274] Peptide refolding Inclusion bodies were dissolved in 6M urea in 20 mM ethanolamine (pH 9.0) and 20 mM cysteine. The solution was diluted in refolding buffer (20 mM Tris, pH 8.0) to a final peptide 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 xg, 45 min).

[0275] Purification of refolded peptides like Protein Purification As outlined in *Principles and Practice Series: Springer Advanced Texts in Chemistry Scopes*, Robert K., 3rd edition, 1994 (Chapters 6 and 8), a solution of refolded peptides was loaded onto an anion exchange chromatography (20 mM Tris, pH 8.0, 0–500 mM NaCl) Q Sepharose Big Beads resin (GE Healthcare). The N-terminal extension was removed by enterokinase cleavage to obtain the specific N-terminus of the target peptide. The resulting peptide solution was then added 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 conjugate was then loaded into SOURSE30Q (GE Healthcare) (20 mM Tris pH 8.0, 0–250 mM NaCl) for purification. The final conjugate of the target peptide was concentrated to 5 mg / ml and cryopreserved.

[0276] Capto Phenyl highsub (GE Healthcare)

[0277] Refined SOURSE30Q (GE Healthcare)

[0278] General methods for introducing substituents onto peptides and purifying fusion compounds The reagent (formula 7) required for introducing substituents onto the polypeptide is prepared as described in WO2016 / 102562: 17-{(S)-1-carboxy-3-[2-(2-{[2-(2-{[2-(2-bromoacetyl-amino)ethylcarbamoyl]methoxy}ethoxy)ethylcarbamoyl]methoxy}ethoxy)ethyl-carbamoyl]propylcarbamoyl}heptadecanoic acid Chemical Formula 7 Alkylation Thaw the frozen peptide solution and concentrate it to above 1 mg / ml using a rotary filter (MWCO 10 kDa, 30 min, 3000 rpm) at 30 min. Adjust the pH to 8.5 with aqueous NaOH solution and add 5 equivalents of BSPP for each capped cysteine. After stirring for 2–3 hours, add 4–5 equivalents of Formula 7 for each free cysteine ​​in 0.1 M NaHCO3 (aqueous solution). Gently stir the mixture in the dark for 1.5–16 hours. Dilute the reaction mixture with water and then purify it by anion exchange using the Äkta system.

[0279] Combine the pure fractions and exchange the buffers using the Äkta 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.

[0280] If necessary, concentrate the combined fractions to 3-5 mg / ml using a rotary filter (30 min, 3000 rpm).

[0281] General methods for detection and characterization Dilute the sample to approximately 1 mg / ml and inject it into the LC-MS system (e.g., 1 μl). Desalt the analogue. Calibrate the instrument, if possible, using lock-in mass spray. Generate the MS spectrum on the main peak and reconstruct the full mass using a deconvolution algorithm.

[0282] LC-MS Method 1 (LCMS 29)

[0283] LC-MS Method 2 (LCMS 36)

[0284] LC-MS Method 3 (LCMS 47)

[0285] Examples 1.1-1.17 Table 5. Chemical Formulas 8-23 of the Fusion Compounds

[0286] Example 1.1 [8G, 22E, 26R]GLP-1(7-37)-(GAQP)x4, A-S{Beta-180}-[2-[2-[2-[2-[2-[2-[2-[2-[2-[(4S)-4-carboxy-4-(17-carboxyheptadecanoyl-amino)butyryl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]-ethylamino]-2-oxoethyl]- [121Q, 168L,180C]FGF21(1-181) Chemical formula 8; Purification was performed using buffer B1. LCMS Method 1: Calculated mass: 25161.1; Actual mass: 25161.9.

[0287] Example 1.2 S{Beta-26}-[2-[2-[2-[2-[2-[2-[2-[2-[2-[(4S)-4-carboxy-4-(17-carboxyheptadecanoyl-amino)butyryl]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) Chemical formula 9; Purification was performed using buffer B1. LCMS36: Calculated mass: 24936.6; Measured mass: 24936.0 Example 1.3 S{Beta-27}-[2-[2-[2-[2-[2-[2-[2-[2-[2-[(4S)-4-carboxy-4-(17-carboxyheptadecanoyl-amino)butyryl]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) Chemical formula 10; Purified using buffer B1. LCMS47: Calculated mass: 24993.7; Measured mass: 24994.0 Example 1.4 S{Beta-27}-[2-[2-[2-[2-[2-[2-[2-[2-[2-[(4S)-4-carboxy-4-(17-carboxyheptadecanoyl-amino)butyryl]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) Chemical formula 11; Purification was performed using buffer B1. LCMS47: Calculated mass: 26407.2; Measured mass: 26408.0 Example 1.5 S{Beta-27}-[2-[2-[2-[2-[2-[2-[2-[2-[2-[(4S)-4-carboxy-4-(17-carboxyheptadecanoyl-amino)butyryl]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) Chemical formula 12, Purification was performed using buffer B1 or B2. LCMS36: Calculated mass: 29234.2; Measured mass: 29234.0 Example 1.6 S{Beta-36}-[2-[2-[[2-[2-[2-[2-[2-[2-[2-[(4S)-4-carboxy-4-(17-carboxyheptadecanoyl-amino)butyryl]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) Chemical formula 13, Purification was performed using buffer B1. LCMS36: Calculated mass: 25093.8; Measured mass: 25094.0 Example 1.7 S{Beta-27}-[2-[2-[2-[2-[2-[2-[2-[2-[2-[(4S)-4-carboxy-4-(17-carboxyheptadecanoyl-amino)butyryl]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-[2-[(4S)-4-carboxy-4-(17-carboxyheptadecanoyl-amino)butyryl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]-ethylamino]-2-oxoethyl]- [121Q, 168L, 180C]FGF21 Chemical formula 14, Purification was performed using buffer B1 or B2. LCMS47: Calculated mass: 25823.8; Measured mass: 25824.0 Example 1.8 S{Beta-26}-[2-[2-[2-[2-[2-[2-[2-[2-[2-[(4S)-4-carboxy-4-(17-carboxyheptadecanoyl-amino)butyryl]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-[2-[(4S)-4-carboxy-4-(17-carboxyheptadecanoyl-amino)butyryl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]-ethylamino]-2-oxoethyl]- [121Q, 168L, 180C]FGF21 Chemical formula 15, Purified using buffer B2. LCMS47: Calculated mass: 25824.7; Measured mass: 25826.0 Example 1.9 S{Beta-27}-[2-[2-[2-[2-[2-[2-[2-[2-[2-[(4S)-4-carboxy-4-(17-carboxyheptadecanoyl-amino)butyryl]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-[2-[2-[(4S)-4-carboxy-4-(17-carboxyheptadecanoyl-amino)butyryl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]-ethylamino]-2-oxoethyl]- [121Q, 168L, 180C]FGF21 Chemical formula 16, Purification was performed using buffer B1 or B2. LCMS47: Calculated mass: 25851.8; Measured mass: 25854.0 Example 1.10 S{Beta-27}-[2-[2-[2-[2-[2-[2-[2-[2-[2-[(4S)-4-carboxy-4-(17-carboxyheptadecanoyl-amino)butyryl]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) Chemical formula 17, Purified using buffer B1. LCMS47: Calculated mass: 24935.7; Measured mass: 24937.0 Example 1.11 S{Beta-27}-[2-[2-[2-[2-[2-[2-[2-[2-[(4S)-4-carboxy-4-(17-carboxyheptadecanoyl-amino)butyryl]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) Chemical Formula 18 Purification was performed using buffer B1. LCMS47: Calculated mass: 26351.2; Measured mass: 26351.0 Example 1.12 [8G, 22E, 26R, 36G]GLP-1(7-37)-(GAQP)x2, S{Beta}-[2-[2-[2-[2-[2-[2-[2-[2-[2-[(4S)-4-carboxy-4-(17-carboxyheptadecanoyl-amino)butyryl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]-ethylamino]-2-oxoethyl]C, GAQPx2, A- S{Beta-180}-[2-[2-[[2-[2-[2-[2-[2-[2-[2-[(4S)-4-carboxy-4-(17-carboxyheptadecanoyl-amino)butyryl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]-ethylamino]-2-oxoethyl]- [121Q, 168L, 180C]FGF21 Chemical formula 19, Purification was performed using buffer B1 or B2. LCMS47: Calculated mass: 25980.9; Measured mass: 25982.0 Example 1.13 [8G, 22E, 36G]GLP-1(7-37)-(GAQP)x2, S{Beta}-[2-[2-[2-[2-[2-[2-[2-[2-[2-[(4S)-4-carboxy-4-(17-carboxyheptadecanoyl-amino)butyryl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]-ethylamino]-2-oxoethyl]C, GAQPx2,A [121Q, 168L,171G]FGF21 Chemical formula 20 Purified using buffer B1. LCMS47: Calculated mass: 25064.8; Measured mass: 25067.0 Example 1.14 S{Beta-27}-[2-[2-[2-[2-[2-[2-[2-[2-[2-[(4S)-4-carboxy-4-(17-carboxyheptadecanoyl-amino)butyryl]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) Chemical Formula 21 Purification was performed using buffer B1. LCMS36: Calculated mass: 25021.7; Actual mass: 25023.0 Example 1.15 [8G, 22E, 26R, 36G]GLP-1(7-37)-(GAQP)x2, S{Beta}-[2-[2-[2-[2-[2-[2-[2-[2-[2-[(4S)-4-carboxy-4-(17-carboxyheptadecanoyl-amino)butyryl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]-ethylamino]-2-oxoethyl]C, GAQPA, [121Q,168L, 171G]FGF21 Chemical formula 22 Purified using buffer B1. LCMS47: Calculated mass: 24739.4; Measured mass: 24741.0 Example 1.16 {S27-[(28S)-(28,46-dicarboxy-2,7,16,25,30-pentoxo-9,12,18,21-tetraoxa-3,6,15,24,29-pentazahexadecane-1-yl)]-[Gly8,36,Glu22,Cys27]-GLP-1(human)-(7-37)-peptide}-(Gly-Ala-Gln-prolyl)32-Ala-[Gln121,Leu168,Gly171,Glu180]-FGF21(human)-(1-181)-peptide Chemical formula 23 Purified using buffer B2. LCMS47: Calculated mass: 34888.2; Measured mass: 34888.0 Example 1.17 – Reference Compound 1 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 Chemical formula 24, SEQ ID NO: 37 The compound was prepared as described in WO06097537.

[0288] Example 1.18 – Reference Compound 2 S{Beta-180}-[2-[2-[[2-[2-[2-[2-[2-[2-[(4S)-4-carboxy-4-(17-carboxyheptadecanoyl-amino)butyryl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]-ethylamino]-2-oxoethyl]-Ala[Gln121,Leu168,Cys180]FGF21(1-181) Chemical formula 25, SEQ ID NO: 38 The compound was prepared as described in WO1016102562.

[0289] General methods for determining GLP-1 activity To determine the GLP-1 receptor (GLP-1R) activity or potency of the GLP-1 moiety of the GLP-1 / FGF21 fusion compound and to assess how the presence of serum albumin may potentially affect receptor activation, in vitro potency assays were performed in cells expressing the human GLP-1 receptor in the absence and in the presence of 1% (w / v) human serum albumin (HSA), as described below.

[0290] An increase in EC50 value (reduced potency) in the presence of serum albumin indicates binding to serum albumin and represents a method for predicting prolonged pharmacokinetic profiles of test substances in animal models.

[0291] The more potent a compound, the lower its EC50 value. When a compound's EC50 is below approximately 50 pM, such as 20 pM, it is considered a very potent GLP-1 receptor agonist, where the EC50 is measured using the assay described herein without the addition of HSA. When a compound's EC50 value is 50–250 pM, it is considered to have moderate potency. When a compound's EC50 value is 250–1000 pM, it is considered to have poor potency. When a compound's EC50 value is above 1000 pM, it is considered ineffective.

[0292] Measurement principle Activation of the GLP-1 receptor leads to an increase in cellular concentration of cyclic AMP (cAMP). Transcription is thus activated by a promoter containing multiple copies of the cAMP response element (CRE). Therefore, GLP-1 receptor activity can be measured using a CRE-luciferase reporter gene introduced into young hamster kidney (BHK) cells co-expressing the GLP-1 receptor.

[0293] Cells and assay reagents Cell stock solutions were prepared by culturing a stably transfected cell line (BHK 467-12A KZ-10, prepared according to methods known to those skilled in the art) expressing the human GLP-1 receptor and CRE-responsive luciferase (CRE-Luc) reporter gene in growth medium. This growth medium consisted of DMEM (Gibco, 61965-026) supplemented with 10% FBS (Gibco, 16140-071), 1% penicillin / streptomycin (Gibco, 15140-122), 1 mM sodium pyruvate (Gibco, 11360-039), 1 mg / mL G418 (Gibco, 10131-027), and 240 nM MTX (Pfizer, 15936). Approximately 80-90% confluenced cells were washed once in PBS and released from the cell flasks with Versene (Gibco, 15040-033). After centrifugation, the cell pellet was dissolved and diluted in culture medium to a concentration of 1.5 x 10⁶ cells / mL. This medium consisted of DMEM (Gibco, 61965-026) supplemented with 20% FBS (Gibco, 16140-071), 1% penicillin / streptomycin (Gibco, 15140-122), 1 mM sodium pyruvate (Gibco, 11360-039), 1 mg / mL G418 (Gibco, 10131-027), 240 nM MTX (Pfizer, 15936), and 10% DMSO (Sigma, D2650). Cells were aliquoted and stored at -180°C until use.

[0294] The assay buffer consisted of phenol red-free DMEM (Gibco, 11880-028) 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).

[0295] program To perform this assay, serial dilutions (10-fold dilution, 8 concentrations per compound) of the reference compound and the GLP-1 / FGF21 fusion compound were performed in HSA-free assay buffer, typically starting at approximately 100–200 nM in 96-well plates. Human GLP-1R / CRE-Luc cells were thawed at 37°C, 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, 50 µL aliquots of the reference compound or GLP-1 / FGF21 co-agonist were transferred to two 96-well assay plates (ThermoFisher, 237105), to which 50 µL of cell suspension with or without 2% (w / v) HSA (5,000 cells / well) was added. The assay plate was incubated at 37°C and 5% CO2 for 3 hours, then left at room temperature for 5 minutes. Afterward, 100 µL of SteadyLite Plus (PerkinElmer, 6066759) was added to each well. The plate was sealed and incubated at room temperature with gentle shaking for 30 minutes while protected from light. Emissions were detected using a luminescence reader such as Synergy 2 (BioTek). EC was calculated by nonlinear curve fitting using a four-parameter logistic model (slope = 1) applied with GraphPadPrism or by means of TIBCO Enterprise Runtime for R (TIBCO Software, Palo Alto, CA, USA). 50 Value [pM].

[0296] Example 2 The GLP-1 activity of the example compounds was investigated using the general method described above. The results are presented in Table 6 (average of at least two individual concentration response curves). All example compounds exhibited GLP-1 activity.

[0297] Table 6. GLP-1 potency of GLP-1 / FGF21 fusion compounds.

[0298] All fusion compounds exhibited activity against the GLP-1 receptor, with comparable or higher activity in the presence of HSA. In vitro GLP-1R potency depended on the alkylation position. For both monoalkylated and dialkylated compounds, the highest potency was observed with alkylation at position 27C, followed by position 36C, spacer C, and 26C. The same pattern was observed in the presence of albumin; however, dialkylated compounds showed a significantly greater loss of potency compared to monoalkylated compounds. For the monoalkylated 27C derivative, the decrease in potency in the presence of albumin increased with increasing spacer length, although the longest spacer in Formula 23, 32xGAQP, exhibited potency comparable to Formula 24 in the presence of albumin. 26R did not appear to affect in vitro potency. Surprisingly, given that Formula 24 has an elongated spacer at position 26, in the fusion case, for monoalkylated compounds, a potency loss exceeding 10-fold was observed in the absence of HSA, while for the dialkylated form, a 40-fold potency loss was observed in the absence of HSA and a 26-fold potency loss was observed in the presence of HSA.

[0299] General methods for studying FGF21 activity The purpose of this embodiment is to test the activation of the FGF21 receptor by the fusion compound of the embodiment. The in vitro FGF21 receptor potency of the activated FGF receptor was measured in whole-cell assays.

[0300] As further described below, the potency of the GLP-1 / FGF21 fusion compound of Example 2 was determined in HEK (human embryonic kidney cells) overexpressing human β-klotho (BKL).

[0301] To test the binding of the GLP-1 / FGF21 fusion derivative to albumin, the assay was performed both in the absence of serum albumin and in the presence of human serum albumin (HSA) (0.1% final assay concentration). The increased EC50 value (decreased potency) of the FGF21 derivative in the presence of serum albumin indicates binding to serum albumin and represents a method for predicting prolonged pharmacokinetic profiles of test substances in animal models.

[0302] The results for the GLP-1 / FGF21 fusion compound are shown in Table 7. Chemical formula 25 is included for reference. Measurement principle HEK293 cells endogenously express several FGF receptors, including FGFR1c, FGFR3c, and FGFR4. These cells are unresponsive to FGF21 until transfected with the co-receptor β-klotho (BKL). Activation of the FGF receptor / BKL complex leads to activation of the MAPK / ERK signaling pathway and phosphorylation of ERK. Phosphorylated ERK (pERK) levels at a given time point increase with increasing FGF21 concentration. As described below, pERK levels were measured 12 minutes after stimulation with a series of test compound concentrations.

[0304] Measurement Description 1) Day 1: HEK293 / β-klotho cells were seeded at 50,000 cells / well in 96-well plates in DMEM (Gibco #31966-021) containing 4500 mg / L glucose, supplemented with 10% HI FBS (Gibco #16140-71), 1% penicillin / streptomycin (Gibco #15140-122), and 100 µg / ml hygromycin B (Gibco, #10687-010).

[0305] 2) Day 2: Two hours before adding the test compound, the cell culture medium was replaced with 100 µl of basal medium (DMEM containing 4500 mg / l glucose (Gibco #31966-021)).

[0306] 3) Dilute the test compound in 2x assay medium (DMEM (Gibco #31966-021) containing 4500 mg / L glucose, supplemented with 0.01% Tween 20), incubate at 37°C, add 100 µL of basal medium to the cells, and incubate at 37°C for 12 minutes. Test the compound in the presence and absence of 0.1% HSA (Sigma – A1887).

[0307] 4) Quickly remove all culture medium and add 50 µl of lysis buffer to each well. Shake the plate for 10 minutes; the lysate is ready for pERK measurement.

[0308] 5) pERK was measured in 384-well plates using the AlphaScreen SureFire kit (PerkinElmer # TGRESB10K). This kit is based on ERK and pERK-specific antibodies conjugated to donor and recipient beads. The presence of pERK will bring the recipient and donor beads into close proximity, generating a signal that can be read on an EnVision plate reader. The data was analyzed using GraphPad Prism software. EC was calculated using nonlinear regression with this software. 50 The value is reported in nM.

[0310] Example 3 The FGF21 activity of the example compounds was investigated using the general methods described above. Results are presented (average of at least two independent experiments). All example compounds exhibited FGF21 activity.

[0311] Table 7. FGF21 potency of the GLP-1 / FGF21 fusion compound in HEK293 / BKL cells.

[0312] As shown in Table 7, all fusion compounds exhibited higher FGF21 acceptor potency than the individual FGF21 moiety (including the elongated substituents). Overall, FGF21 potency was improved compared to Formula 25. For monoalkylated compounds, the potency was only slightly affected in the presence of HSA, likely due to the lack of albumin binding in the FGF21 moiety of the fusion. In the absence of HSA, dialkylated compounds showed improved potency compared to Formula 25; however, in the presence of HSA, their potency was similar to that of Formula 25, and they exhibited greater variation in the presence of albumin compared to Formula 25.

[0313] Example 4: General methods for studying mean residence time (pharmacokinetics) The aim of this study was to determine the average residence time of compounds in different species. For this purpose, mice, miniature pigs, and cynomolgus monkeys were selected.

[0314] Average stay (MRT) Because of the observed initial rapid decrease in plasma concentration, it was decided to use MRT instead of T. 1 / 2 This may indicate the rapid distribution phase and the extremely slow terminal elimination phase of the PK curve. To avoid overemphasizing the slow terminal elimination phase (which represents only a few data points and an extremely low AUC, and therefore likely low power), we decided to use the MRT, a more robust parameter based on the total AUC of the PK spectrum involving all data points in the spectrum. The results are shown in Tables 8, 11, and 12.

[0315] Example 4.1: Study in mice Drug administration and sampling This study used 7- to 8-week-old mice (male C57BL / 6J mice). Mus musculus(Janvier Labs, Le Genest-Saint-Isle, France). This study used n=15 mice for each test compound, employing a sparse sampling method, collecting two or three blood samples from each mouse. n=3 at each time point. Animals were administered intravenously at a concentration of 1 nmol / ml at a dose of 5 nmol / kg of the test compound (in a buffer of 8 mM phosphate, 240 mM propylene glycol, and 0.007% polysorbate 20, pH=8.2). Approximately 100 µl of blood samples were collected from the sublingual venous plexus of conscious mice according to the following protocol: 5 min, 15 min, 30 min, 1 h, 2 h, 3 h, 4 h, 6 h, 18 h, 24 h, 30 h, 48 h, and 72 h post-administration. Blood was collected in EDTA-coated tubes (Sarstedt microvette 1293) and placed on ice until centrifuged at 6000 G for 5 min at 4 °C. Transfer the plasma (approximately 50 µl) to a 0.7 ml Micronic tube and store at -20 °C until analysis.

[0316] Bioassay: Because of the lack of immunoassays that use a combination of antibodies that react to both the GLP1 and FGF21 moieties to detect GLP-1 / FGF21 compounds in plasma, it was decided to use separate assays for GLP1 and FGF21. The selected GLP1 assay can detect GLP1 with an intact N-terminus, while the selected FGF21 assay can detect the intact C-terminus of FGF21.

[0317] FGF21 assay Sample AnalysisSamples were analyzed using a luminescent oxygen channel immunoassay (LOCI or AlphaLISA). Donor beads were coated with streptavidin (lot number 2298681, PerkinElmer, USA) according to a standard protocol, while recipient beads were conjugated with an internally generated monoclonal antibody (lot number 2356893, PerkinElmer, USA) specific for the C-terminal epitope of the FGF21 peptide of the test compound, according to a standard procedure (e.g., illustrated by Lu et al., Journal of Biomedical Science (2020) 27:1). A second antibody was a commercially available human FGF21 polyclonal antibody (lot number BAF2539, R&D systems, USA) biotinylated according to a standard procedure. The three reactants were mixed with the corresponding analytes to form a two-site immune complex. Illumination of this complex released singlet oxygen atoms from the donor beads. They are directed into acceptor beads and trigger a chemiluminescent reaction, which is measured in an EnVision plate reader (PerkinElmer, USA). The amount of light is proportional to the concentration of the test compound.

[0318] The procedure was as follows: 5 µL of plasma sample and accuracy controls (high, medium, and low concentrations within the calibrator range) were added to appropriate wells of a white 384-well plate, followed by 15 µL of a receptor bead mixture coated with the aforementioned internal monoclonal antibody (0.5 µg / well) and 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 min. The plate was read using an Envision plate reader (Perkin Elmer) at room temperature using a 520–645 nm bandwidth filter and 680 nm laser excitation. The total measurement time for each well was 210 ms, including a 70 ms excitation time.

[0319] Data analysis and reporting: Using Phoenix TM WinNonlin® (Pharsight®, St. Louis, Missouri, USA) performs non-compartmental analysis on plasma concentration-time data following a single intravenous administration. The area under the plasma concentration-time curve is calculated using the log-linear trapezoidal rule. The area under the first moment curve is also calculated using the log-linear trapezoidal rule. The mean residence time is calculated as the ratio of the area under the first moment curve to the area under the plasma concentration-time curve. The terminal half-life is calculated using the “individual best fit” of a log-linear regression of concentration-time and is reported as the harmonic mean and pseudo-standard deviation.

[0320] GLP-1 determination The assay and calculations were performed as described above for the FGF21 assay, using a biotin-labeled, internally produced monoclonal antibody against the N-terminus of GLP-1 and an internally produced monoclonal antibody against the middle portion of GLP-1 conjugated with receptor beads.

[0321] Table 8. MRT obtained by GLP-1 and FGF21 assays.

[0322] The mean residence times (MRTs) measured by GLP-1 and FGF21 were comparable, ranging from 2.5 h to 21 h (2.5 h to 17 h using GLP-1, and 2.4 h to 21.1 h using FGF21). The MRT of monoalkylated compounds increased from 2.5 h (GLP-1, Formula 21) to 9.8 h (GLP-1, Formula 23) with increasing spacer length. Dialkylated compounds showed significantly longer MRTs, with Formula 19 exhibiting the longest MRT at 17.0 h using GLP-1 and 21.1 h using FGF21.

[0323] Example 4.2: Study in miniature pigs The overall objective is to validate that the clearance and terminal half-life / MRT of compounds 16, 19 and 23 are suitable for once-weekly administration to humans.

[0324] Dosage This study was conducted on eighteen (18) female Göttingen miniature pigs from Ellegaard Göttingen Minipigs A / S, Sorø Landevej 302, DK-4261 Dalmose. At the start of the acclimation period, the pigs weighed approximately 15–20 kg and were 7–9 months old. Intravenous or subcutaneous administration was performed on the animals according to the following schedule: Dosing schedule Table 9. Dosing schedule.

[0325] Intravenous administration: Intravenous (iv) injection via an ear vein catheter. After administration of the compound, flush the catheter with 10 mL of sterile saline.

[0326] Subcutaneous administration: The compound was administered using a 1 mL or 2 mL syringe and a 5 mm deep 25Gx5 / 8 (orange needle) with a stopper or a 5 mm deep 21Gx3 / 4 butterfly needle with a stopper. In miniature pigs, subcutaneous administration was performed on the side of the neck. After administration, the needle remained in the skin for approximately 10 seconds.

[0327] Blood sampling Complete plasma concentration-time curves were obtained for each animal.

[0328] Obtain blood samples via ear vein catheter according to the following schedule.

[0329] Blood (1.3 ml) was collected in an EDTA tube (a 1.3 ml tube containing K3EDTA to obtain 1.6 mg K3EDTA / ml blood (Sarstedt, Germany)). After each blood sampling, the catheter was flushed with 10 ml of sterile 0.9% NaCl and 10 EI / ml heparin.

[0330] The sample was kept on wet ice for up to 30 minutes until centrifuged (10 min, 4°C, 2000 xg) and then transferred to a Micronic tube for exposure measurement.

[0331] Table 10. Blood sampling schedule.

[0332] Bioanalysis The GLP-1 / FGF21 fusion compound in porcine plasma was determined by immune capture, protease digestion, and liquid chromatography-mass spectrometry (LC-MS). The compound was then used to measure its concentration. N End and C The terminal substitute peptide was used to quantify the GLP-1 / FGF21 fusion compound.

[0333] In short, calibrators are prepared by incorporating a relevant fusion compound in the range of 1 to 200 nM into blank plasma. 25 μL of the calibrator, blank plasma, or study sample is 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, specific for the intermediate region of FGF21 (PGQKSPHRDPAPRGP)). The mixture is incubated at 37°C for two hours. After incubation, 25 μL of Dynabeads MyOne Streptavidin T1 magnetic beads (10 mg / ml, ThermoFisher Scientific) is added, and the mixture is incubated at room temperature for one hour. After incubation, the beads are washed three times with PBS buffer, and the GLP-1 / FGF21 fusion compound is eluted from the beads with 100 μL of elution buffer in Milli-Q water containing 10% acetonitrile, 1% formic acid, and 0.005% Tween 20. After elution, add 50 μL of trypsin or LysC (0.02 μg / μL in 1M TRIS buffer (pH 9)) to the elution buffer. After 18 hours at 37°C, terminate digestion by adding 4.5 μL of formic acid. Centrifuge the mixture and transfer the supernatant to a microtiter plate (coated with BSA). Use a Thermo Accucore 150-C4 column (100 x 2.1 mm ID; 2.6 μm) run at 60°C, or a Waters Acquity UPLC Peptide BEH C18 column (300 Å, 1.7 µm, 2.1 μm) run at 80°C. The mixture was analyzed by LC-MS (100 mm). A Nexera UHPLC system (Shimadzu) was used with gradient elution using mobile phase A (composed of milli-Q water containing 0.1% formic acid and 5% acetonitrile) and mobile phase B (composed of 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 positive electrospray ionization mode. Calibration curves were used to calculate the concentration in the plasma samples. Including quality control samples, the deviation between the nominal concentration and the calculated concentration was less than 20% (25% for LLOQ).

[0334] Pharmacokinetic analysis Plasma concentration-time data were analyzed using non-compartmental pharmacokinetic methods with Phoenix 8 (Certara, Princeton, NJ, 08540 USA).

[0335] Calculations were performed using individual concentration-time values ​​from each animal at each time point.

[0336] Each time the following pharmacokinetic parameters were calculated: AUC, AUC / dose, AUC %Extrapol , C0, C max , λ z , t max , t ½ , CL, CL / f, V z , V z / f, V ss , MRT and f.

[0337] Treatment of concentration values below the lower limit of quantification (LLOQ) The plasma concentrations below LLOQ were treated as follows: The pre-dose (0 h) plasma concentrations below the limit of detection were shown as "<LLOQ" in the data file. These values were automatically considered as zero by Phoenix.

[0338] If one post-dose concentration value was quantifiable and another value at the same dose and time was <LLOQ, the latter was set to ½LLOQ for calculating the mean concentration. If the resulting mean value was <LLOQ, the value was replaced with "<LLOQ" in the data file and thus it was not included in the calculation.

[0339] If one post-dose (mean) concentration value was <LLOQ and the subsequent (mean) value was quantifiable, the former was set to ½LLOQ for calculating the pharmacokinetic parameters.

[0340] Table 11. Pharmacokinetic profiles of GLP-1 – FGF21 fusion compounds in minipigs.

[0341] The results showed that after intravenous administration, the N-terminal peptide and the C-terminal peptide obtained the same MRT (h). After subcutaneous administration, the MRT of the N-terminal part of the monoalkylated compound of Chemical Formula 23 was longer than that of the C-terminal part. For the dialkylated compound with one extender in the spacer, the situation was opposite. Generally, the dialkylated compounds showed the longest MRT. After evaluation, all compounds had a long enough MRT in pigs to support low-dose administration in humans.

[0342] Example 4.3: Study in cynomolgus monkeys Drug administration and sampling:This study used non-juvenile (but not previously given FGF21 or GLP-1 products) female Vietnamese cynomolgus macaques (Macaca fascicularis, Nafovanny / KHI Group, Vietnam), aged at least 2 years and weighing between 2.0 and 3.0 kg at the start of treatment. Each group used n=3 monkeys. One group received intravenous administration and the other subcutaneous administration of the test compound at a concentration of 1 mg / ml (in an aqueous solution containing 10 mM phosphate, 2% (w / vol) ethylene glycol, pH=8.15). Approximately 600 µl of blood samples were collected from the femoral vein according to the following protocol: before administration, 5 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 24 hours, 48 ​​hours, 96 hours, 168 hours, 240 hours, 336 hours, 408 hours, and 504 hours after administration. Collect blood into a Teklab K3EDTA tube (part number 3K200PP) containing 1.75 mg EDTA / mL of blood and place on ice for up to 30 minutes, until centrifuged at 2000 g for 10 minutes at 2–8 °C. Transfer plasma (at least 100 µl) to a Micronic tube and store at -30–-10 °C until analysis.

[0343] Bioanalytical and pharmacokinetic analyses were performed according to the miniature pig study. Pharmacokinetic parameters are presented in Table 12.

[0344] Table 12. Pharmacokinetic profile of chemical formula 23 in cynomolgus monkeys.

[0345] For the C-terminal portion of compound formula 23, a mean residence time of 56 hours was observed in monkeys after subcutaneous administration, which was assessed to be long enough to support low-dose administration in humans.

[0346] Example 5: Evaluation of the GLP-1 PD effect of the fusion compound The aim of this study was to investigate the effect of GLP-1 on acute food intake following a single intravenous dose of the GLP-1 / FGF21 fusion compound in lean mice.

[0347] FGF21 had no effect on food intake (FI) 24 hours after a single injection. In contrast, GLP-1 is known to reduce FI. Therefore, an acute FI model was used to investigate the pharmacodynamic effective dose and in vivo efficacy of the GLP-1 moiety of the GLP-1 / FGF21 bifunctional molecule.

[0348] method: Acute fibrillation (FI) was measured using 56 eight-week-old male C57BL6J mice housed individually in a BioDaq system (New Brunswick, NJ, USA) under a reverse light / dark cycle (11 a.m. to 11 p.m. darkness) with free access to food (Altromin 1324). Mice were acclimatized to the BioDaq cages for 14 days and then randomly assigned to seven groups of eight mice each based on body weight. Baseline FI measurements were collected 24 hours before administration. One hour before the start of darkness, mice were intravenously injected via the tail vein with 10 nmol / kg of compound 23 or control compounds 24, 12, 14, 16, 19, or a carrier. FI data were collected 24 hours after administration. One animal in the compound 16 group was excluded due to technical issues.

[0349] Statistical analysis was performed using Graph Pad Prism 8.0.2, with one-way ANOVA and Dunnett correction, p < 0.05. =p<0.001, =p<0.0001, ns=not significant result Before the administration of the compound, there were no significant differences in food intake among the groups. After the administration of the active compound, food intake was significantly reduced in all groups compared with the carrier, with formula 23 reducing FI by 58%, followed by formula 12 (-52%), formula 14 (-41%), formula 16 (-39%), and formula 19 (-27%). The positive control, formula 24, showed the largest reduction (-70%) within 24 hours. Figure 1 .

[0350] All tested dual GLP-1 / FGF21 compounds significantly reduced food intake, with compounds 23 and 12 exhibiting similar in vivo efficacy against GLP-1 receptors to compound 24.

[0351] Example 6: Evaluation of the effect of the fusion compound on body weight Evaluation of the weight-reduction efficacy of the GLP-1 / FGF21 fusion compound in a low-density lipoprotein receptor-deficient mouse model (LDLr- / -) The aim of this study was to evaluate the effect of a selected GLP-1 / FGF21 fusion compound on body weight (BW) in an LDLr- / - mouse model, a pro-atherosclerotic mouse model with severe dyslipidemia and weight gain.

[0352] Research Design Eighty (80) male LDLr- / - mice (JAX, USA; STOCK:2207) were fed a high-fat, high-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 assigned to eight groups based on their morning body weight. The test substance or carrier was administered subcutaneously to the animals at noon daily for 21 days. The dose of the test substance was adjusted daily based on body weight. For group 3 (Formula 24), the dose was titrated up over five days, starting at 1.0 ml / kg equivalent to 2 nmol / kg and ending at 10 nmol / kg.

[0353] Weight data Daily baseline-corrected weight Figure 2 As shown in the figure. All compounds showed significant weight loss. Analogs 12 and 23 showed the most effective weight reduction, with a reduction of more than 20% from baseline after 7 days of administration. The weight loss induced by 12 and 23 was judged to be very effective, and the dose was reduced in all groups for ethical reasons. Analog 23 continued to cause very effective weight loss, so the dose was further reduced to 2 nmol / kg. However, this dose was judged to be too low, so the dose was adjusted back to 4 nmol / kg, which was maintained throughout the study. Notably, 25 (FGF21) and 24 showed similarly significant weight loss.

[0354] Example 7: Chemical stability: IsoAsp formation.

[0355] FGF21 contains several aspartic acid residues that readily form isoAsp (D5, D24, D25, D38, and D102). Stability studies were conducted to assess the extent of isoAsp formation.

[0356] Program stability study Formulas 12, 14, 16, 19, and 23 (20 mg / ml) were prepared in 10 mM phosphate buffer and 2% glycerol (pH 8.2); filtered and aliquoted into dust-free, sterile HPLC vials and stored at static temperatures (5°C, 25°C, 37°C) for 4 weeks for chemical and physical stability analysis.

[0357] Data analysis using peptide mapping IsoAsp formation at the aforementioned locations was determined by peptide mapping and LC-MS. Samples were digested with trypsin (E:S 1:20 w / w; pH 7.5; 37°C for 2 h, Barocycler: 72 cycles, 90 sec / 10 sec) and reduced (0.25 M DTT, pH 7.5, 37°C for 0.5 h), followed by LC-UV215-MS / MSMS analysis. The percentage of isomeric ectopic trypsin peptides with IsoAsp relative to unmodified trypsin peptides was based on the area of ​​the extracted ion chromatogram (XIC). The following IsoAsp sites, 25IsoAsp, 38IsoAsp, and 102IsoAsp, were directly identified in the isomeric ectopic trypsin peptides using electron transfer dissociation (ETD) MS / MS. For isotopic peptides containing IsoAsp24 and IsoAsp25 (amino acid positions numbered according to SEQ ID NO: 2), no ETD-reported ions were observed.

[0358] Table 13. Content of 102 isoAsp after 33 days at 0 time and 37°C.

[0359] 21 days at 5℃ result For all analogs analyzed, including reference compound Formula 25, IsoAsp formation was identified at positions D5, D24, D25, D38, and D102 (amino acid positions are numbered according to SEQ ID NO: 2). Although the isoAsp content was similar at time zero, compounds containing two side chains (Formulas 14, 16, and 19) showed higher levels of all isoAsp derivatives compared to compounds with one side chain (Formulas 12 and 23). IsoAsp formation at position 102 is shown in Table 13. After 33 days at 37°C, the IsoAsp formation at position 102 of compounds 12 and 23 was comparable to that of reference compound Formula 25.

[0360]

Claims

1. A fusion protein comprising a polypeptide of formula 1: ABC, in: (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 composed of 1-257 amino acids, and (i) C is an FGF21 polypeptide, which is an analog of FGF21 (1-181) (SEQ ID NO: 2); Or its pharmaceutically acceptable salt, amide, or ester.

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

3. The fusion protein according to claim 1 or claim 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 of the preceding claims, wherein A has 3-7 amino acid changes compared to SEQ ID NO:

1.

5. The fusion protein according to any of the preceding claims, wherein A comprises 26C, 27C, 36C or 26R.

6. The fusion protein according to any of the preceding claims, 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. The fusion protein according to any of the preceding claims, wherein C comprises 180C, 180E, or 180A.

8. The fusion protein according to any of the preceding claims, 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 1-8 and one or more substituents.

10. The fusion compound according to claim 9, wherein the substituent comprises an elongated portion and an optional linker, or is composed of an elongated portion and an optional linker, wherein the elongated portion is of formula 3: HOOC-(CH2). x -CO- , where x is an integer in the range of 10-20, and the connector comprises at least one of chemical formula 4, chemical formula 5 and chemical formula 6; Chemical formula 4 is -NH-CH(COOH)-(CH2)2-CO- , Chemical formula 5 is -NH-(CH2)2-[O-(CH2)2] k -O-[CH2] 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-(CH2) n -NH-CO-CH2- Where n is an integer in the range of 1 to 5. Formulas 4, 5, and 6 are interconnected by amide bonds and, in the order shown, are... The -NH end is connected to the CO- of the extended body (Chemical Formula 3). end, and in its CH2- An amino acid whose end is attached to A, B, or C.

11. The fusion compound according to claim 9 or claim 10, wherein the substituent is of chemical formula 7: 。 12. The fusion compound according to any one of claims 9-11, wherein the compound is selected from the list consisting of chemical formula 8, chemical formula 9, chemical formula 10, chemical formula 11, chemical formula 12, chemical formula 13, chemical formula 14, chemical formula 15, chemical formula 16, chemical formula 17, chemical formula 18, chemical formula 19, chemical formula 20, chemical formula 21, chemical formula 22 and chemical formula 23, preferably wherein the compound is chemical formula 23.

13. A fusion compound having the structure of chemical formula 23: 。 14. The fusion compound according to any one of claims 9-13, used as a drug.

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

Images