WO2019101035A1 - Glucagon analogue for treating metabolic diseases - Google Patents

Abstract

A glucagon analogue for treating metabolic diseases, the structure formula is as follows: H-X2-X3-GTFTSD-X10-SKYLD-X16-X17-AAQ-DFVQWLMN-X29-Xz or H-S-Q-GTFTSD-Y-SKYLD-X16-X17-AAQ-DFVQWLMN-X29-Xz-NH2. The described glucagon analogue has a GLP-1/GCG/GIP triple receptor agonist activity and better enzyme resistant stability for neutral endopeptidase (NEP) and dipeptidyl peptidase-4(DPP-4), and has a longer half-life in vivo and duration of action compared with natural glucagon, GLP-1 and GIP.

Description

Glucagon analog for treating metabolic diseases Technical field

The invention relates to the field of biopharmaceuticals, in particular to a glucagon analog for treating metabolic diseases, a preparation method thereof and use thereof.

Background technique

Diabetes is a serious chronic disease that occurs when the pancreas does not produce enough insulin or the body cannot effectively use the insulin produced. The currently marketed protein-based diabetes drugs are mainly GLP-1 receptor (GLP-1R) agonists, such as Duraglutide (trade name:

), Albiglutide (trade name)

), liraglutide (Liraglutide, trade name

and

For the treatment of obesity and diabetes, respectively, Exenatide (trade name)

), lixisenatide (Lixisenatide, trade name

) and may be listed soon, such as Semaglutide. Duraglutide, albudide, liraglutide and somaglutide are all analogs of native glucagon-like peptide-1 (GLP-1), which are separately IgG after GLP-1 sequence mutation. The FC fragment, human albumin and fatty acid are fused or crosslinked to obtain a highly active and stable GLP-1R agonist. Exenatide (Exendin-4) is a 39-amino acid small peptide derived from the salivary gland of the lizard (Heloderma suspectum). Although Exendin-4 is a potent agonist of GLP-1R, it is more active than native GLP-1 and GLP-1 analogs. Although these agonists of GLP-1R can effectively lower blood sugar and control appetite, the effect on weight loss is not too significant. Liraglutide (trade name)

Although it has been approved for the treatment of obesity, its weight loss is actually only 5.6 kg. The current weight loss for obesity drugs is generally around 5–10% (compared to placebo), ie the overall average weight loss ratio does not exceed 10% of the patient's body weight (Rudolph L. Leibel et al, Biologic Responses to Weight Loss) And Weight Regain: Report From an American Diabetes Association Research Symposium, Diabetes, 64(7): 2299-2309, 2015).

Bariatric surgery can significantly improve obesity and treat diabetes, but its use is not widespread, as most patients are reluctant to undergo such surgery for surgical risks and long-term sequelae (Obesity and Diabetes, New Surgical and Nonsurgical Approaches, Springer Press, 2015). The study found that incretin secretion increased dramatically in patients undergoing surgical bariatric surgery (Obesity and Diabetes, New Surgical and Nonsurgical Approaches, Springer, 2015). Preclinical and clinical studies have also found that patients are simultaneously infused with GLP-1/Glucagon (GCG) (Tricia M.Tan etc., DIABETES, VOL. 62: 1131-1138, 2013), or GLP- 1, Oxyntomodulin (OXM) and PYY, are important for promoting energy metabolism, suppressing appetite, and controlling body weight (Tricia Tan etc., J Clin Endocrinol Metab, 2017, 102(7): 2364–2372) effect. These polypeptides can be simply mixed for clinical use in terms of clinical needs. However, due to the difference in the in vivo stability and degradation rate of these different kinds of polypeptides, the final in vivo efficacy is uncontrollable, and it is actually difficult to simply mix these polypeptides and use them as a combination drug. Therefore, the current development of a new generation of diabetes drugs is mainly to try to concentrate these agonist activities in one molecule, such as GLP-1R/GIPR and GLP-1R/GCGR double-effect agonists, and even GLP-1R/GIPR/GCGR three-effects. Agonist (Chakradhar, Shraddha. All in one: researchers to create a combination drugs for diabetes and obesity. Nature Medicine, 22(7): 694-695, 2016).

At present, the design and development of such drugs are mainly in the following ways: 1. The development of modification of human endogenous polypeptide Oxyntomodulin (OXM) based on dual activity of GLP-1/GCG. OXM is a polypeptide naturally occurring in humans with dual activities of GLP-1 and GCG (Diabetes, 2005, 54: 2390-2395). However, the activity of OXM is not high (having about 10% of GCG activity and about 1% of GLP-1 activity), and its stability and half-life are very poor in vivo. Therefore, OXM itself cannot be directly used in clinical practice, and it is often necessary to introduce non-natural. Amino acids, various modifications, etc. to enhance their activity and stability in vivo. For example, OPKO Biologics' Mod-6030 is a long-acting OXM with a degradable PEG modification at the N-term (Oren Hershkovitz: Presentation Number: SAT-787. The Endocrine Society's 95th Annual Meeting and Expo, June 15–18, 2013-San Francisco) . TT401 (LY2944876) is another PEG-modified OXM analog (Chakradhar, Shraddha. "All in one: researchers to create combination drugs for diabetes and obesity." Nature Medicine, vol. 22, no. 7, 2016: 694- 5). PSA-OXM is an OXM analog modified with polysialic acid (Vorobiev I et al, Biochimie, 2013, 95(2): 264-70). However, due to the low activity and poor stability of OXM, the clinical effect is not good, and most of the research has been abandoned. 2. Using the homology of incretin sequence to obtain multiple active stable hybrid peptides by multiple mutations, modifications, and even introduction of non-natural amino acids on the basis of structures such as OXM, GLP-1 and GCG ( Matthias H.

Etc. Unimolecular Polypharmacy for Treatment of Diabetes and Obesity, 24: 51–62, 2016). Matthias H.

The review articles in the article provide a detailed description of the various forms of hybrid peptides that are currently in clinical or clinical settings. OXM-based double-effect GLP-1R/GCGR agonists as reported by Alessandro Pocai et al. (Glucagon-Like Peptide 1/Glucagon Receptor Dual Agonism Reverses Obesity in Mice, Diabetes; 58(10): 2258-2266, 2009), or Richard D. DiMarchi et al. reported a GCG-based double-effect GLP-1R/GCGR agonist (US9018164B2) or even a three-way GLP-1R/GCG/GIPR agonist (US9150632). These multispecific hybrid peptides are mostly based on GLP-1 or GCG, which enhance activity and resist protease hydrolysis by sequence mutations, such as mutating L-form amino acids to D-amino acids (such as D-Ser), or introducing unnatural amino acids Aib. To improve the stability of the body, while fatty acid chain or polyethylene glycol (PEG) modification, etc. to extend the half-life, the clinical expected delivery cycle is once a day (fatty acid modification) or once a week (PEG modification). In addition, Aisling M. Lynch et al. reported a GCG analog (D-Ser2-glucagon-exe) that mutates the second Ser of native GCG to D-Ser and introduces the C-terminal peptide of Exendin-4 at the C-terminus, and Pharmacodynamic experiments in DIO, twice daily dosing (Novel DPP IV-resistant C-terminally extended glucagon analogue exhibits weight-lowering and diabetes-protective effects in high-fat-fed mice mediated through glucagon and GLP-1 receptor Activation, Diabetologia: 57: 1927–1936, 2014), the weight loss effect is obvious.

Although the development of molecules with multiple agonistic activities of GLP-1R, GCGR, and GIPR is very promising, it is actually very difficult to obtain an ideal drug of this kind.

The first is the issue of safety, especially the issue of immunogenicity. The hypoglycemic and weight-loss drugs need to be used for a long time, and the safety requirements are extremely high. In order to design a polypeptide with high activity of GLP-1, GCG and GIP and stable in vivo, the prior art schemes often introduce more mutation sites, and often introduce non-natural amino acids and other modifications. These mutations and the introduction of unnatural amino acids increase the risk of potential immunogenicity. In general, the higher homology with human sequences, the lower the risk of immunogenicity in humans. The GLP-1 receptor agonist Taspoglutide, which was developed by Roche and Epson, introduced only two unnatural amino acids, Aib, with an antibody production rate of 49%. All clinical phase III studies have been suspended ( JULIO ROSENSTOCK et al, The Fate of Taspoglutide, a Weekly GLP-1 Receptor Agonist, Versus wice-Daily Exenatide for Type 2, DIABETES CARE, 36: 498-504, 2013). PHIL AMBERY et al. (THE ENDOCRINOLOGIST, SPRING, 2017: 12-13) screened more than 500 structures based on the sequence of GCG to obtain a candidate peptide MEDI0382. Among them, in order to maintain high GLP-1 and GCG dual activity and in vivo stability, compared with GCG, MEDI0382 introduced 9 mutation sites, the mutation rate reached about 30%; likewise, Andreas Evers et al (J Med Chem .2017May 25;60(10):4293-4303) Introduced 9 mutation sites based on the structure of Exendin-4, the mutation rate reached about 23%, and the fatty acid chain modification was carried out. Hybrid peptide with high GLP-1 and GCG dual activity; GLP-1/GCG/GIP triactive peptide designed by Brian Finan et al. (Brian Finan et al., Nat Med. 21:27-36, 2015) is at the C-terminus of GCG The GPSSGAPPPS sequence was added and seven mutant amino acids were introduced, including a second mutation to the unnatural amino acid Aib. Therefore, the existing technical solutions often introduce more mutation sites, and often introduce non-natural amino acids and other modifications to obtain polypeptides with high activity of GLP-1, GCG and GIP. These mutations, modifications, and introduction of unnatural amino acids increase the risk of potential immunogenicity. For drugs that treat diabetes and obesity, safety is extremely important. Therefore, it is of great interest to develop a highly active multi-effect GLP-1/GCG multiple agonist that contains no unnatural amino acids and contains as few mutant amino acids as possible.

On the other hand, the existing research on how to combine the activity of these incretins and the appropriate ratio between the activities has not been widely discussed. GLP-1R/GCGR double-acting agonist or GLP-1R/GCG/GIPR triple-acting agonist modified on Exendin-4 is disclosed, for example, in PCT applications WO2015155139A1, WO2015155140A1 and WO201515541A1. Among them, WO201515541A1 discloses a hybrid peptide having GLP-1R/GCGR/GIPR triple-acting agonistic activity, and it is considered that the three-way agonistic active peptide has a good hypoglycemic weight-loss effect; however, WO2015155139A1 and WO2015155140A1 are for avoiding GIPR. The risk of hypoglycemia caused by agonistic activity is prepared as a GLP-1R/GCGR double-acting agonist, which in turn has a better hypoglycemic weight loss effect. A.Seth et al. also suggested that the introduction of GIP activity does not enhance the effect of GLP-1 on blood glucose control (A.Seth et al, Co-administration of a lipidated GIPR agonist with a GLP-1 analogue provides no additional benefit on HbA1c% over GLP1 analogue In db/db mice, EASD virtual meeting, 2015).

Again, for sequences such as GLP-1, Exendin-4, and GCG, which are highly homologous, the receptor belongs to the GPCR family, and the peptide has a peptide length of only 30-40 amino acids, a single site mutation or several bits. The change in activity of different receptors after simultaneous mutation is very difficult to predict, so it is extremely difficult to obtain the desired multiple agonistic activity hybrid peptide. For example, Joseph Chabenne et al. (Joseph Chabenne et al., A glucagon analog chemically stabilized for immediate treatment of life-threatening hypoglycemia, Molecular Metabolism, 3: 293-300, 2014) performed Ala scan on GCG, GCG After each site is independently substituted with alanine, the relative residual activity retention span is from 0.2% to 100%, and represents the 1, 2, 3, 4, 6-12, 14, 15, 22, 23, 25 of the GCG. Both the -27 and 29 mutations significantly attenuated the GCGR agonistic activity (Table 4 in the article). However, we can also see in other reports that single or several sites in these sites are simultaneously mutated. When substituted with other amino acids, the change in activity is not always consistent with the results of alanine scanning. . As reported by Jonathan W Day et al. (Jonathan W Day et al., A new glucagon and GLP-1 co-agonist eliminates obesity in rodents, Nature Chemical Biology, 5: 749-757, 2009), 16 bits of GCG are 16S→G, Different mutations such as 16S→T, 16S→H, 16S→E, etc., the GCGR agonistic activity is increased, which is completely contradictory to the alanine scan result of Joseph Chabenne. Second, the Joseph Chabenne study concluded that substitution with alanine at position 23 would result in almost complete loss of GCGR agonistic activity (only 1.1% retained); however, Jonathan W Day et al. mutated 23 to Ile with no decrease in GCG activity. Another example is the alanine scan, which suggests that the second S is very important for retaining GCG activity (only 1/3 of the activity is retained when the mutation is Ala), but Brian Finan et al. (Finan B et al., A rationally designed monomeric peptide triagonist Corrects obesity and diabetes in rodents. Nat Med. 2015;21:27-36.), 2 S→Aib, 2 S→dSer, 2 S→G, 2 S→dAla substitution mutations for the second amino acid of GCG, After combining the mutations at other sites, the relative agonistic activity of GCGR increased to 200%-640%. It has also been found in our study that when a combination of mutations that promote GLP-1, GCG or GIP activity is introduced, the effect is often completely inconsistent with a single site mutation. Moreover, polypeptides such as GLP-1, Exendin-4, GCG or GIP, which increase or decrease amino acids at the N and C terminals, affect their biological activities. If one or two amino acids are removed at the N-terminus, the agonistic activity of GLP-1, GCG, etc. is completely lost. For example, Oxyntomodulin only has 8 amino acids of KRNRNNIA at the C-terminus of Glucagon, and its GCGR agonistic activity is lost by about 90% (Alessandro Pocai et al., Glucagon-Like Peptide 1/Glucagon Receptor Dual Agonism Reverses Obesity in Mice, Diabetes; 58 ( 10): 2258-2266, 2009; Henderson SJ et al, Robust anti-obesity and metabolic effects of a dual GLP-1/Glucagon antibody peptide agonist in rodents and non-human primates, Diabetes Obes Metab, 2016).

As reported by Joseph R. Chabenne and Richard D. DiMarchi, the C-terminal small peptide cex (SEQ ID NO. 67, GPSSGAPPPS) of Exendin-4 was added to the C-terminus of Glucagon to make its GLP-1R agonistic activity. 0.7% increased to 1.6%, an increase of about 2 times (Optimization of the Native Glucagon Sequence for Medicinal Purposes, J Diabetes Sci Technol. 4 (6): 1322–1331, 2010 and patent US9018164B2), and also lost about 50 % GCG activity. Evers A et al. (Evers A, Design of Novel Exendin-Based Dual Glucagon-like Peptide-1 (GLP-1)/Glucagon Receptor Agonists, J Med Chem.; 60(10): 4293-4303.2017) in GCG analogues After the C-terminus plus the cex sequence, the GLP-1R agonistic activity decreased by about 2/3, but the agonistic activity of GCG was more than 90% lost (Table 2, peptides 7 and 8 in the article). Therefore, for GLP-1, a small peptide of 30 amino acids in length such as Glucagon, the sequence change is extremely sensitive to changes in its activity; for dual active polypeptides, the change is due to the excitation of two different receptors. More complex, it is impossible to predict the consequences of GLP-1R and GCGR agonistic activity after any amino acid change.

The complexity of signaling downstream of GPCR receptors such as GCGR and GLP-1R also increases the difficulty of designing an ideal multiplex active hybrid peptide. The receptors of GCGR and GLP-1R have multiple signaling pathways in the cell, including downstream proteins such as G protein (Gαs, Gαi, Gαq, etc.) and inhibitory proteins (β-arrestin-1 and β-arrestin-2). A number of different signal transduction channels, the physiological effects of activation of different channels are not the same, and even the relationship between channel activity and physiological function is not clear. For example, by introducing different mutations or different amino acid sequences into the GLP-1 sequence, different biased-agonist structures can be obtained, resulting in different physiological effects (Marlies V. et al., J Am Chem Soc., 138(45): 14970-14979, 2016; Hongkai Zhang et al., Nat Commun., 6:8918, 2015).

Therefore, although it is theoretically very useful to design a polypeptide that has both high GLP-1, Glucagon, and GIP activities, it is actually very difficult. If you can design an activity balance, but introduce minimal mutation sites and unnatural amino acids, try to be close to the natural sequence to make it less potential immunogenic, and improve stability, with good glycemic control and weight control multiple Active hybrid peptides are very clinically relevant.

Summary of the invention

In view of the above-discussed deficiencies of the prior art, it is an object of the present invention to provide a glucagon analog which exhibits GLP-1R/GCGR/GIPR triple receptor agonistic activity.

Native GCG has approximately 1% GLP-1R agonistic activity relative to native GLP-1, but does not have GIPR agonistic activity. The glucagon analog of the present invention can exhibit GLP-1R/GCGR/GIPR triple receptor agonistic activity.

To achieve the above and other related objects, a first aspect of the present invention provides a glucagon analog (GCG analog) having a structure as shown in Formula I or Formula II The structure of the formula I is: HSQGTFTSD-X ₁₀ -SKYLD-X ₁₆ -X ₁₇ -AA-X ₂₀ -X ₂₁ -FX ₂₃ -QWLMN-X ₂₉ -X ^z (SEQ ID NO.1), The structure shown in II is:

HSQGTFTSD-X ₁₀ -SKYLD-X ₁₆ -X ₁₇ -AA-X ₂₀ -X ₂₁ -FX ₂₃ -QWLMN-X ₂₉ -X ^z -NH ₂ (SEQ ID NO. 2), wherein X _{10 is} selected from Y, Any one of K or L, X _{16 is} selected from any one of S, E or A, X _{17 is} selected from any one of Q, E, A or R, and X _{20 is} selected from any one of Q, R or K; X ₂₁ Any one selected from D, L or E; X _{23 is} selected from any one of V or I; X ₂₉ is T or a deletion, and X ^{z is} selected from GGPSSGAPPPS (SEQ ID NO. 65), GGPSSGAPPS (SEQ ID NO. 66) , GPSSGAPPPS (SEQ ID NO. 67), GPSSGAPPS (SEQ ID NO. 68), PSSGAPPPS (SEQ ID NO. 69), PSSGAPPS (SEQ ID NO. 70), SSGAPPPS (SEQ ID NO. 71) or SSGAPPS (SEQ ID Any of NO.72).

Further, when the glucagon analog has the structural formula:

HSQGTFTSD-X ₁₀ -SKYLD-X ₁₆ -X ₁₇ -AA-X ₂₀ -X ₂₁ -FX ₂₃ -QWLMN-X ₂₉ -X ^z -NH ₂ (SEQ ID NO.2) means the hyperglycemia of the pancreas The C-terminus of the analog was subjected to amidation modification.

As exemplified by some embodiments of the present invention, the amino acid sequence of the glucagon analog of the present invention is as shown in any one of SEQ ID NO. 6-28 and SEQ ID NO. 47-53.

Further, in a preferred embodiment, the glucagon analog has the structural formula:

HSQGTFTSDYSKYLD-X ₁₆ -X ₁₇ -AAQ-DFVQWLMN-X ₂₉ -X ^z (SEQ ID NO.3) or HSQGTFTSDYSKYLD-X ₁₆ -X ₁₇ -AAQ-DFVQWLMN-X ₂₉ -X ^z -NH ₂ (SEQ ID NO. 4)

Wherein X _{16 is} selected from any one of S or E, X _{17 is} selected from any one of Q or E, X ₂₉ is T or a deletion, and X ^{z is} selected from GGPSSGAPPPS (SEQ ID NO. 65), GGPSSGAPPS (SEQ ID NO. 66), GPSSGAPPPS (SEQ ID NO. 67), GPSSGAPPS (SEQ ID NO. 68), PSSGAPPPS (SEQ ID NO. 69), PSSGAPPS (SEQ ID NO. 70), SSGAPPPS (SEQ ID NO. 71) or SSGAPPS ( Any of SEQ ID NO. 72).

Further, when the glucagon analog has the structural formula:

HSQGTFTSD-Y-SKYLD-X ₁₆ -X ₁₇ -AAQDFVQWLMN-X ₂₉ -X ^z -NH ₂ (SEQ ID NO. 4) means that the C-terminus of the glucagon analog is amidated.

The above glucagon analog has GCGR agonistic activity similar to or superior to native glucagon, and has GLP-1R agonistic activity similar to or superior to native GLP-1, and an additional increased GIPR agonistic activity.

In one embodiment of the invention, the preferred glucagon analog is added to GPSSGAPPPS at the C-terminus based on native glucagon, at least 2-3 amino acids are mutated and no unnatural amino acid is introduced, nor is any modification required. It can retain or enhance the agonistic activity of GLP-1R and GCGR, and also has an additional GIPR agonistic activity, and the product itself has good stability. Fewer mutation sites, without subsequent modifications, try to maintain the natural structure and reduce the potential for immunogenicity.

It has been reported in the literature that high levels of GIP cause frequent hypoglycemia in the treatment of diabetes (T McLaughlin et al, J Clin Endocrinol Metab, 95, 1851-1855, 2010; A Hadji-Georgopoulos, Jclin Endocrinol Metab, 56, 648- 652, 1983). However, in the mouse animal model test, the glucagon analog having the GIPR agonistic activity provided by the present invention can smoothly control blood sugar without hypoglycemia symptoms. In addition, it has been reported in the literature that it is also advisable to antagonize GIPR in order to reduce daily food intake, weight loss, insulin sensitivity and energy expenditure (Irwin et al, Diabetologia 2007, 50, 1532-1540; Althage et al, J Biol Chem, 2008, 283(26): 18365–18376). In the mouse animal model test, the glucagon analog having higher GIP activity provided by the present invention has a daily feeding control and body weight for obese mice relative to a comparative analog having a lower GIP activity or no GIP activity. Both mitigation and increased insulin sensitivity have a more pronounced effect.

The glucagon analog of the invention has better enzyme stability, including neutral endopeptidase (NEP) and dipeptidyl peptidase-4 (DPP-4); and natural glucagon, GLP- 1, GIP has a longer in vivo half-life and duration of action.

In a second aspect of the invention, an isolated polynucleotide is provided, the isolated polynucleotide encoding the aforementioned glucagon analog.

In a third aspect of the invention, a recombinant expression vector comprising the isolated polynucleotide described above is provided.

In a fourth aspect of the invention, a host cell comprising the aforementioned recombinant expression vector or the above-described isolated polynucleotide integrated with exogenous in the genome is provided.

According to a fifth aspect of the present invention, there is provided a method for producing the aforementioned glucagon analog, which is selected from any one of the following:

(1) synthesizing the glucagon analog by a chemical synthesis method;

(2) cultivating the aforementioned host cell under appropriate conditions to express the glucagon analog, followed by isolation and purification to obtain the glucagon analog.

In particular, the glucagon analogs of the invention can be prepared by standard peptide synthesis methods, for example, by standard solid or liquid phase methods, stepwise or by fragment assembly, and isolation and purification of the final peptide compound product, or by Recombination and synthesis methods are arbitrarily combined. The glucagon analog of the present invention can be preferably synthesized by a solid phase or liquid phase peptide synthesis method.

In a sixth aspect of the invention, there is provided the use of the aforementioned glucagon analog in the manufacture of a medicament for the treatment of a metabolic related disorder.

The glucagon analogs provided by the present invention can be used for the treatment of diabetes-related metabolic syndromes, such as dyslipidemia, including high triglycerides, low HDL cholesterol and high LDL cholesterol; insulin resistance or glucose intolerance.

Metabolic syndrome is associated with an increased risk of coronary heart disease and other conditions associated with vascular plaque accumulation, such as stroke and peripheral vascular disease, becoming atherosclerotic cardiovascular disease (ASCVD). Patients with metabolic syndrome can progress from being in an early stage of insulin resistance to fully mature type 2 diabetes, and the risk of ASCVD is further increased. Without being bound by any particular theory, the relationship between insulin resistance, metabolic syndrome, and vascular disease may involve one or more common pathogenesis, including insulin-stimulated vasodilation, insulin resistance due to increased oxidative stress. Reduced sexual relevance, as well as abnormalities in fat cell-derived hormones such as adiponectin (Lteif, Mather, Can. J. Cardiol. 20 (Supp. B): 66B-76B, 2004).

The glucagon analogs of the invention are also useful in the treatment of obesity. In some aspects, the glucagon analogs of the invention treat obesity by reducing appetite, reducing food intake, reducing fat levels in a patient, and increasing energy expenditure.

In some potential embodiments, the glucagon analogs of the invention are useful for the treatment of nonalcoholic fatty liver disease (NAFLD). NAFLD refers to a broad spectrum of liver disease ranging from simple fatty liver (steatosis) to nonalcoholic steatosis hepatitis (NASH) to cirrhosis (reversible late scar formation of the liver). All stages of NAFLD show accumulation of fat in liver cells. Simple fatty liver is an abnormal accumulation of certain types of fats and triglycerides in liver cells, but no inflammation or scar formation. In NASH, fat accumulation is associated with varying degrees of liver inflammation (hepatitis) and scar formation (fibrosis). Inflammatory cells destroy liver cells (hepatocyte necrosis). In the terms "steatogenic hepatitis" and "fatty degeneration necrosis", steatosis refers to fat infiltration, hepatitis refers to inflammation in the liver, and "necrosis" refers to damaged liver cells. NASH can eventually lead to liver scar formation (fibrosis) and then to irreversible late scar formation (cirrhosis), and cirrhosis caused by NASH is the last and most serious stage within the NAFLD spectrum.

According to a seventh aspect of the invention, a method of treating a metabolic-related disease, comprising the step of administering the aforementioned glucagon analog to a subject.

In one embodiment, the invention uses the glucagon analog for the treatment of obesity, metabolic syndrome, non-alcoholic hepatitis, and the like.

The researchers of the present invention have found that the glucagon analogs of the present invention are sufficiently water soluble and have improved chemical stability at neutral or slightly acidic pH. In one of the examples, an IPGTT experiment was performed. Mice administered the glucagon analog of the present invention exhibited extremely stable blood glucose fluctuations after glucose injection. Furthermore, the glucagon analog of the present invention induced a significant decrease in body weight after administration in DIO mice. At the same time, various indicators related to blood lipids decreased significantly.

The invention further provides a method of promoting weight loss or preventing weight gain comprising administering the glucagon analog in a subject.

In an eighth aspect of the invention, there is provided a composition comprising the aforementioned glucagon analog or a culture of the aforementioned host cell, and a pharmaceutically acceptable carrier.

In a ninth aspect of the invention, the use of the aforementioned glucagon analog in the preparation of a fusion protein is provided.

In a tenth aspect of the invention, a fusion protein comprising the aforementioned glucagon analog is provided in the structure of the fusion protein.

Further, the structure of the fusion protein further contains a long-acting unit.

Further, the long acting unit is selected from the group consisting of covalently linked fatty acids, polyethylene glycol or derivatives thereof, albumin, transferrin and immunoglobulins and fragments.

In an eleventh aspect of the present invention, a modified polypeptide comprising the aforementioned glucagon analog is provided in the structure.

Further, the glucagon analog is modified by a fatty acid, polyethylene glycol or a derivative thereof; the modified polypeptide is covalently or non-covalently associated with albumin, transferrin and immunization The combination of globulin and fragment.

It is known to those skilled in the art that in order to increase the half-life or stability of the glucagon analog of the present invention, it may be modified, for example, polyethylene glycol or a derivative thereof, a hydroxyethyl starch derivative or a fatty acid may be covalently linked. To the glucagon analog of the present invention. In a specific embodiment, to increase stability, a lysine residue can be introduced at a position where the glucagon analog of the present invention is expected to not affect receptor binding/activation, covalently linked to the gamma-valley The amino spacer was modified by adding palmitic acid to the ε-amino group.

Compared with the prior art, the present invention has the following beneficial effects:

The glucagon analog of the present invention has GLP-1/GCG/GIP triple receptor agonistic activity and better enzyme stability, including neutral endopeptidase (NEP) and dipeptidyl peptidase Resistance of 4 (DPP-4); therefore, it has a longer in vivo half-life and duration of action compared to native glucagon, GLP-1, GIP. In summary, currently reported GCG analogs are commonly used (1) single-molecule hybrid peptide cross-linked fatty acid, PEG or FC, etc., administered once a day or more (Matthias H.

Etc., Unimolecular Polypharmacy for Treatment of Diabetes and Obesity, 24:51–62, 2016); or (2) Mutation of the second Ser of natural GCG to non-natural amino acids such as D-Ser to resist DPP-IV degradation (Novel DPP IV -resistant C-terminally extended glucagon analogue exhibits weight-lowering and diabetes-protective effects in high-fat-fed mice mediated through glucagon and GLP-1 receptor activation, Diabetologia: 57:1927–1936, 2014), administered twice daily . Multiple active polypeptides that retain the natural amino acid composition and are administered twice daily are not currently reported. The present invention provides a sufficiently stable and highly active multi-effect GCG analog without cross-linking fatty acid, PEG albumin or immunoglobulin Fc fragment, and without mutating the second Ser to an unnatural amino acid, thereby Minimize potential immunogenicity risks and omit cumbersome chemical modification/crosslinking steps, simplifying the preparation process and increasing product consistency.

DRAWINGS

Figure 1: HPLC chromatogram of the polypeptide numbered C381 in an aqueous solution of pH 7.4.

Figure 2: HPLC chromatogram of the polypeptide numbered C493 in an aqueous solution of pH 4.5.

Figure 3: HPLC chromatogram of the polypeptide numbered C816 in aqueous pH 7.4.

Figure 4: HPLC chromatogram of the polypeptide numbered C002 in an aqueous solution of pH 7.4.

Figure 5: HPLC chromatogram of the polypeptide numbered C611 in aqueous pH 4.5 solution.

Figure 6: HPLC chromatogram of the polypeptide numbered C611 in an aqueous solution of pH 7.4.

Figure 7: HPLC chromatogram of C239 in aqueous pH 7.4.

Figure 8A is a graph of residual activity versus time.

Figure 8B is a graph showing residual activity as a function of time.

Figure 9A is a graph showing the detection of GLP-1R agonistic activity by several exemplary glucagon analogs.

Figure 9B is a graph showing the GLP-1R agonistic activity of several exemplary glucagon analogs.

Figure 9C is a graph showing the detection of GCGR agonistic activity by several exemplary glucagon analogs.

Figure 9D is a graph showing the detection of GCGR agonistic activity by several exemplary glucagon analogs.

Figure 9E: cAMP levels produced by glucagon analogs and control stimulated GIPR for different concentration gradients.

Figure 9F: cAMP levels produced by glucagon analogs and control stimulated GIPR for different concentration gradients.

Figure 9G: cAMP content produced by stimulation of GIPR for glucagon analogs and controls at different concentration gradients.

Figure 9H: cAMP levels produced by glucagon analogs and control stimulated GIPR for different concentration gradients.

Figure 10: Results of in vitro cellular insulin secretion assay.

Figure 11A is a graph showing blood glucose changes in IPGTT experiments in normal ICR mice.

Figure 11B is a graph showing blood glucose changes in IPGTT experiments for normal ICR mice.

Figure 11C: Comparison of area under the blood glucose curve (AUC).

Figure 12A is a graph showing the relationship between body weight change (%) and time (days) in diet-induced obesity (DIO) mice.

Figure 12B is a graph showing the relationship between body weight change (%) and time (days) in diet-induced obesity (DIO) mice.

Figure 12C is a graph showing the relationship between body weight change (%) and time (days) in diet-induced obesity (DIO) mice.

Figure 12D: Comparison of body weight loss in DIO mice.

Figure 13: Comparison of body weight loss in DIO mice.

Figure 14: Analysis of the mass spectrum of C495.

Figure 15: Analysis of the C382 mass spectrometry.

Detailed ways

All technical and scientific terms mentioned herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs, unless otherwise defined.

Glucagon analogues:

The glucagon analog provided by the present invention mutates the arginine (R) at position 18 to alanine (A). Mutation of the 18-alanine attenuates GCGR agonistic activity by about 30% (Joseph Chabenne et al, A Glucagon analog chemically stabilized for immediate treatment of life-threatening hypoglycemia, Molecular Metabolism, 3: 293-300, 2014). However, after extensive screening by the present inventors, it was found that specific amino acid mutations were made at specific sites, such as combination with specific amino acid mutations at positions 16 and 17, and CEX or similar sequences were added at the C-terminus, even at 18 positions. When mutated to A, there was no significant decrease in GCGR activity. More importantly, the 18-position A mutation significantly increased the GLP-1 and GIPR agonistic activity of the glucagon analog, thereby making the glucagon analog of the present invention an effective trispecific active peptide.

The trispecific active peptide of the present invention has potency of agonizing GLP-1R, GCGR and GIPR, and each activity has an extremely high activity retention rate relative to GLP-1, GCG and GIP. At present, most of the tri-active peptides introduce multiple amino acid mutations on the basis of natural polypeptides, and even unnatural amino acids can become stable three-way agonists. In the screening process of the present invention, it was found that multi-site introduction mutations are more likely to obtain hybrid polypeptides having higher GLP-1R, GCGR and GIPR activities, and the introduction of non-natural amino acids is more likely to obtain polypeptides with high stability. However, the level of activity and stability in vitro is only a prerequisite for clinical drugs, and attention needs to be paid to safety. Introducing too many mutation sites or unnatural amino acids tends to pose a higher risk of immunogenicity.

Serum stability test:

Natural GLP-1, glucagon Glucagon, or Oxyntomodulin are not truly clinical drugs due to poor serum stability and too short in vivo half-life. And the only peptide amino acid Essinide (Exenatide, only 39 amino acids)

) It can be successfully listed because of the stability improvement. In one of the embodiments of the invention, the preferred glucagon analog exhibits a very high stability.

Immunogenicity experiment:

In Example 6 of the present invention, the natural human Glucagon (C001) was extremely low in immunogenicity in rats. When the glucagon analog is less than 3 amino acids relative to the native Glucagon mutation, the antibody titer is less than <1:200, and as the number of mutated amino acids increases, the antibody titer increases, indicating potential immunity. The original risk is increased. For drugs that treat metabolic-related diseases, such as diabetes, obesity, and other fields, the safety requirements are extremely high. However, the glucagon analog obtained by the present invention has low immunogenicity when introduced into no more than 3 mutation sites, and achieves an ideal activity and stability standard, which has never been reported. of.

Pharmacodynamic studies in animals:

In one embodiment of the invention, the preferred glucagon analog has a good effect of lowering blood glucose, inhibiting adipose tissue formation, and reducing body weight. Although GIP and GLP-1 and Glucagon belong to the same family of incretins, they have not been widely developed into drugs. One reason is that some patients with type 2 diabetes lose sensitivity to GIP, and another reason is that in rodents, GIPR activation has potential obesity (Miyawaki, K. et al., Inhibition of gastric inhibitory polypeptide signaling prevents obesity.Nat.Med. 8, 738–742, 2002). However, in the examples of the present invention, preferred glucagon analogs having a higher GIPR agonistic activity clearly have a more significant weight loss effect.

In Example 9 of another animal of the invention, preferred GCG analogs exhibit similar weight-reducing effects as the corresponding GCG analogs of the same amino acid sequence and modified with fatty acids.

the term:

The term "diabetes" includes type 1 diabetes, type 2 diabetes, gestational diabetes, and other symptoms that cause hyperglycemia. The term is used to mean that the pancreas does not produce enough insulin due to metabolic disorders, or that the cells of the body fail to respond appropriately to insulin, and thus the decrease in the efficiency of absorption of glucose by tissue cells results in the accumulation of glucose in the blood.

Type 1 diabetes, also known as insulin-dependent diabetes and juvenile onset diabetes, is caused by beta cell destruction and usually leads to absolute insulin deficiency.

Type 2 diabetes, also known as non-insulin-dependent diabetes and adult-onset diabetes, is generally associated with insulin resistance.

The term "obesity" means an excess of adipose tissue, and when energy intake exceeds energy expenditure, excess calories are stored in fat, resulting in obesity. Individuals with a body mass index (BMI = body weight (kg) divided by height (m) square) of more than 25 are considered obese herein.

The term "receptor agonist" can be defined as a polypeptide, protein or other small molecule that binds to a receptor and elicits a usual response to a natural ligand. Incretin is a gut hormone that regulates blood sugar by enhancing glucose-stimulated insulin secretion (Drucker. D J, Nauck, MA, Lancet 368: 1696-705, 2006). To date, there are two known incretins: glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP). Preproglucagon is a 158 amino acid precursor polypeptide that is differentially processed in tissues to form a variety of structurally related proglucagon-derived peptides, including glucagon (Glucagon). ), glucagon-like peptide-1 (GLP-1), glucagon-like peptide-2 (GLP-2), and Oxyntomodulin (OXM). GIP is a 42 amino acid mature peptide obtained by proteolytic processing of a 133 amino acid precursor (pre-pro-GIP) involved in various biological functions including glucose homeostasis, insulin secretion, gastric emptying and intestine Growth and food intake regulation.

Glucagon-like peptide-1 (GLP-1) is an incretin hormone secreted by intestinal L-cells of 30 or 31 amino acids, with GLP-1 (7-36) and GLP-1 (7- 37) Two active forms. GLP-1 is released into the circulation after a meal and exerts biological activity by activating the GLP-1 receptor. GLP-1 has many biological effects, including glucose-dependent insulin secretion, inhibition of glucagon production, delay of gastric emptying and appetite suppression (Tharakan G, Tan T, Bloom S. Emerging therapies in the treatment of 'diabesity ':beyond GLP-1.Trends Pharmacol Sci 2011; 32(1): 8-15.) et al. Native GLP-1 limits its therapeutic potential due to its rapid degradation by dipeptidyl peptidase-4 (DPP-4), neutral endopeptidase (NEP), plasma kallikrein or plasmin. Since native GLP-1 has an ultra-short half-life of only about 2 minutes in the body, there has been a method of improving the efficacy by using chemical modification and/or formulation to treat diabetes and obesity (Lorenz M, Evers A, Wagner M) .Recent progress and future options in the development of GLP-1 receptor agonists for the treatment of diabesity.Bioorg Med Chem Lett 2013;23(14):4011-8.Tomlinson B,Hu M,Zhang Y,Chan P,Liu ZM .An overview of new GLP-1 receptor agonists for type 2 diabetes. Expert Opin Investig Drugs 2016;25(2):145-58).

Oxyntomodulin is a 37 amino acid small peptide comprising the complete 29 amino acid sequence of glucagon. Glutathione is a dual agonist of GLP-1R and GCGR that is secreted together with GLP-1 by intestinal L-cells after a meal. Similar to glucagon, oxyntomodulin produces significant weight loss in humans and rodents. The weight loss activity of oxyntomodulin has been compared to equimolar doses of selective GLP-1R agonists in obese mice. It has been found that oxyntomodulin has an antihyperglycemic effect compared to a selective GLP-1R agonist, which is capable of significantly reducing body weight and having lipid lowering activity (The glucagon receptor is involved in mediating the body weight-lowering effects Of oxyntomodulin, Kosinski JR et al, Obesity (Silver Spring), 20): 1566-71, 2012). In overweight and obese patients, subcutaneous administration of natural oxyntomodulin reduced body weight by 1.7 kg in four weeks. Oxytocin is also shown to reduce human food intake and increase energy expenditure (Subcutaneous oxyntomodulin reduces body weight in overweight and obese subjects: a double-blind, randomized, controlled trial, Wynne K et al, Diabetes, 54: 2390- 5,2005; Oxyntomodulin increases energy expenditure in addition to decreasing energy intake in overweight and obese humans: a14 andomized controlled trial; Wynne K et al, Int J Obes (Lond), 30: 1729-36, 2006). However, due to the small molecular weight and degradation of DPP-IV, oxyntomodulin has a shorter half-life. Currently, double-acting agonists of GLP-1 receptor (GLP-1R) and glucagon receptor (GCGR) are generally based on oxyntomodulin, and in order to improve the short-acting and enzymes of oxyntomodulin The mutation is mutated (the oxyntomodulin analog), and most of them use the second serine Ser mutation to α-aminoisobutyric acid (Aib) or D-Ser, which is resistant by introducing non-natural amino acids. Enzymatic hydrolysis of DPP-IV. Although the oxyntomodulin analogue showed initial hypoglycemic and lipid-lowering effects, its mechanism of action was still inaccurate. The oxyntomodulin receptor has not been found and is currently only knocked out by GCGR or GLP-1R. Mouse or cell assays have demonstrated that oxyntomodulin binds to these two receptors.

Glucagon is a 29 amino acid peptide corresponding to amino acids 53-81 of proglucagon, and the sequence is shown in SEQ ID NO. 5 (CGFanelli et al., Nutrition, Metabolism & Cardiovascular Diseases). (2006) 16, S28-S34). Glucagon receptor activation has been shown to increase energy expenditure and reduce food intake in both rodents and humans (Habegger KM et al, The metabolic actions of glucagon revisited, Nat. Rev. Endocrinol. 2010, 6, 689-697). And these effects are stable and sustained in rodents. Glucagon has many physiological effects, such as by stimulating glycogenolysis and gluconeogenesis, increasing blood glucose levels under hypoglycemia, regulating hepatic ketone production, regulating bile acid metabolism, and satiety through the vagus nerve. Glucagon has been used in acute hypoglycemia, and glucagon receptor activation reduces food intake and promotes lipolysis and weight loss in animals and humans.

Glucose-dependent insulinotropic peptide (GIP) is a 42 amino acid polypeptide that is released from small intestinal K cells after food intake. Its main function is to inhibit gastric acid secretion and enhance glucose-stimulated insulin secretion, so it is called gastric inhibitory peptide. (gastric inhibitory peptide) / glucose-dependent insulinotropic peptide.

A "GLP-1 receptor (GLP-1R) agonist" can be defined as a polypeptide, protein or other small molecule that binds to GLP-1R and is capable of eliciting a characteristic response similar or similar to native GLP-1. GLP-1R agonists activate GLP-1R in whole or in part, which in turn causes a series of downstream signaling pathways in the cell to produce corresponding cellular activities: such as beta cells secreting insulin; typical GLP-1R agonists include native GLP-1 And mutants thereof, analogs such as exenatide, liraglutide and the like.

A "glucagon receptor (GCGR) agonist" can be defined as a polypeptide, protein or other small molecule that binds to GCGR and is capable of eliciting the same or similar characteristic response as native glucagon. GCGR agonists activate GCGR in whole or in part, which in turn induces a series of downstream signaling pathways in cells that produce corresponding cellular activities such as hepatocyte glycogenolysis, saccharification, fatty acid oxidation, and ketogenic effects.

GLP-1R/GCGR double-acting agonist: The GLP-1R/GCGR double-acting agonist of the present invention includes a protein or polypeptide capable of simultaneously agonizing GLP-1R and GCGR. Oxyntomodulin-based double-effect agonists as reported by Alessandro Pocai et al. (Alessandro Pocai et al, Glucagon-Like Peptide 1/Glucagon Receptor Dual Agonism Reverses Obesity in Mice, Diabetes; 58(10): 2258-2266, 2009), or Richard D Glucagon-based double-effect agonist (US9018164B2) reported by DiMarchi et al.

GLP-1R/GCGR/GIPR Triple-acting Agonist: The GLP-1R/GCGR/GIPR triple-acting agonist of the present invention includes a protein or polypeptide capable of simultaneously agonizing GLP-1R, GCGR and GIPR, or "trispecific activation" Agent".

Trispecific Active Peptide: A preferred trispecific active peptide in the present invention refers to a polypeptide having both GLP-1R/GCGR/GIPR agonistic activity, or a "triple-acting active peptide". .

EC ₅₀ (concentration for 50% of maximal effect) refers to the concentration required for a drug or substance to stimulate 50% of its corresponding biological response. The lower the EC50 value, the stronger the stimulating or stimulating ability of the drug or substance, for example, the more intuitively, the stronger the intracellular signal that is caused, the better the ability to induce the production of a hormone.

Low-density lipoprotein (LDL): a type of plasma lipoprotein that is the main carrier of cholesterol in the blood. It tends to deposit cholesterol on the arterial wall. White blood cells try to digest low-density lipoproteins, but in the process they turn them into toxins. More and more white blood cells are attracted to the changing areas, causing inflammation of the arterial wall. Over time, these plaque deposits can accumulate on the arterial wall, making the channel very narrow and lacking toughness. If too many plaques accumulate, the artery can be completely blocked. When the complex of LDL and cholesterol (LDL-C) produces too many plaques on the arterial wall, blood will not flow freely through the artery. Plaques can suddenly collapse in the arteries at any time, causing blood vessels to clog and eventually lead to heart disease.

High-density protein (HDL): Helps clear LDL on the arteries, acts as a scavenger, clears LDL from the artery and returns to the liver.

Triglyceride (TG): Another type of fat used to store excess energy in the diet. High levels of triglycerides in the blood are associated with atherosclerosis. High triglycerides can be caused by overweight and obesity, lack of exercise, smoking, excessive alcohol consumption, and high carbohydrate intake (more than 60% of total calories). Sometimes basic or genetic diseases are the cause of high triglycerides. People with high triglycerides typically have high total cholesterol levels, including high LDL cholesterol and low HDL cholesterol, and many people with heart disease or diabetes also have high triglyceride levels.

GPCR: G Protein-Coupled Receptor, which is an important protein in cell signaling, and its topological conformation is a 7-transmembrane receptor. When an extracellular ligand acts on the receptor, the intramembrane portion of the receptor binds to the G protein to activate the G protein. G protein can transmit extracellular information in two ways: the first way is to open the transmembrane ion channel, allowing the outside ions to enter; the second way is to activate the second messenger, such as cAMP, IP ₃ /DAG. Calcium ions are generally considered to be third messengers downstream of cAMP, IP ₃ /DAG.

abbreviation

COMU: 1-[(1-(Cyano-2-ethoxy-2-oxoethylaminooxy)dimethylaminomorpholinomethylene)]methylammonium hexafluorophosphate

DCM: dichloromethane

DMF: N,N-dimethylformamide

DIPEA: diisopropylethylamine

EtOH: ethanol

Et ₂ O: ether

HATU: 2-(7-oxobenzotriazole)-N,N,N',N'-tetramethyluron hexafluorophosphate

MeCN; acetonitrile

NMP: N-methylpyrrolidone

TFA: trifluoroacetic acid

TIS: Triisopropylsilane

The embodiments of the present invention are described below by way of specific examples, and those skilled in the art can readily understand other advantages and effects of the present invention from the disclosure of the present disclosure. The present invention may be embodied or applied in various other specific embodiments, and various modifications and changes can be made without departing from the spirit and scope of the invention.

Before the present invention is further described, it is to be understood that the scope of the present invention is not limited to the specific embodiments described below; It is not intended to limit the scope of the invention.

Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning meaning In addition to the specific methods, devices, and materials used in the embodiments, the methods, devices, and materials described in the embodiments of the present invention may also be used according to the prior art and the description of the present invention by those skilled in the art. Any method, apparatus, and material of the prior art, similar or equivalent, is used to practice the invention.

Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention employ molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields conventional in the art. Conventional technology. These techniques are well described in the prior literature, see Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001; Ausubel et al, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987 and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS IN ENZYMOLOGY, Vol. Chromatin (PM Wassarman and AP Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol. 119, Chromatin Protocols (PBBecker, ed.) Humana Press, Totowa, 1999, and the like.

Example 1: General preparation and purification method of glucagon analog

Using existing technologies, such as existing literature (

V. et al., Beilstein J. Org. Chem., 10: 1197–1212 (2014); Palomo, JM, RSC Adv., 4: 32658-32672 (2014); Behrendt, R. et al., J. Pept. The polypeptide solid phase synthesis and modification methods in Sci., 22: 4-27 (2015)) are used to prepare each polypeptide involved in the present patent.

Specifically, solid phase peptide synthesis can be performed on a CEM Liberty peptide synthesizer using standard Fmoc methods.

Rink Amide TentaGel S Ram resin (0.25 mmol/g, 1 g) was swollen in NMP (10 mL) before use, and added to a solid phase synthesis apparatus, and piperidine/DMF (20%, 10 mL) was added to the resin for 30 min. Deprotected by Fmoc, drained, washed with DMF (5×10 mL) and dried. Add the first Fmoc-amino acid solution (0.2M, NMP/DMF/DCM, 1:1:1, 5mL) and COMU/NMP (0.5M, 2mL) and DIPEA/DMF (2.0M; 1mL), react at room temperature for 1h The ninhydrin detection resin was colorless and transparent, drained, and washed with DMF (5×10 mL). Then, piperidine/DMF (20%, 10 mL) was added and reacted for 30 min for Fmoc protection, drained, washed with DMF (5×10 mL), and dried. The above procedure of adding Fmoc-amino acid for the reaction and piperidine/DMF for Fmoc protection was repeated until the coupling of the last histidine was completed.

The resin was washed with EtOH (3×10 mL) and Et ₂ O (3×10 mL) and dried at room temperature to constant weight. The resin was added to TFA/TIS/phenol/EDT/water (82.5/5/5/2.5/5, v/v, 40 mL) for 2 h in an ice bath, the crude peptide was cleaved from the resin, filtered, and the procedure was repeated three times. The filtrates were combined, and most of the TFA was removed under reduced pressure, which was crystallised from diethyl ether. Prepare a liquid chromatograph using a Varian SD-1 model equipped with a C-18 column and a partial collector, using mobile phase A (0.1% TFA, aqueous solution) and mobile phase B (0.1% TFA, 90% MeCN, aqueous solution) Gradient elution of the crude peptide was purified by preparative reverse phase HPLC to a purity greater than 97%. The resulting peptides are listed in Table 1. Among them, the C-terminal amide-terminated polypeptide was synthesized by the above method; the other peptides were synthesized by Wangle Resin (0.4 mmol/g, 1 g), and the resin was swollen and directly added with Fmoc-amino acid for coupling reaction, Fmoc protection, polypeptide The steps of cleavage, purification, and operation are the same as those of the C-terminal amide-end polypeptide. The synthesis and purification of polypeptides with fatty acid modifications is conventional and can be found in Finan B et al. (Finan B et al, A rationally designed monomeric peptide triagonist corrects obesity and diabetes in rodents. Nat Med. 2015; 21: 27-36.) or Chhabr et al. (Chhabr et al., Appraisal of New Variants of Dde Amine Protecting Group for Solid Phase Peptide Synthesis. Tetrahedron Lett. 1998, 39(12), 1603–1606).

The purified peptide was analyzed by LC/MS, and the results of the analysis are shown in Table 2. 14 and 15 are mass spectrograms of exemplary glucagon analogs numbered C495 and C382, respectively. The mass spectrometry conditions are as follows:

Instrument: Waters ZQ 2000

Mass Spectrometry (Probe): ESI

Nebulizer Gas Flow: 1.5L/min

CDL: -20.0v

CDL temperature: 250 ° C

Block Temperature: 200 ° C

Spectrum Bias: +4.5kv

Detector: 1.5kv

Mobile phase flow rate (T.Flow): 0.2 ml/min

Buffer concentration (B.Conc.): 50% H2O/50% ACN

Table 1

Numbering	sequence	SEQ ID NO.
C001	HSQGTFTSDYSKYLDSRRAQDFVQWLMNT-NH2	5
C326	HSQGTFTSDYSKYLDSQAAQDFVQWLMNGGPSSGAPPS-NH2	6
C327	HSQGTFTSDYSKYLDSQAAQDFVQWLMNGGPSSGAPPS-OH	7
C231	HSQGTFTSDYSKYLDSQAAQDFVQWLMNTGPSSGAPPPS-NH2	8
C232	HSQGTFTSDYSKYLDSQAAQDFVQWLMNTGPSSGAPPPS-OH	9
C365	HSQGTFTSDYSKYLDSEAAQDFVQWLMN GGPSSGAPPPS-NH2	10
C366	HSQGTFTSDYSKYLDSEAAQDFVQWLMNTGPSSGAPPPS-NH2	11
C381	HSQGTFTSDYSKYLDSQAAQDFVQWLMNGGPSSGAPPPS-NH2	12
C382	HSQGTFTSDYSKYLDSQAAQDFVQWLMNGGPSSGAPPPS-OH	13
C392	HSQGTFTSDYSKYLDEEAAQDFVQWLMNTGPSSGAPPS-OH	14
C393	HSQGTFTSDYSKYLDEEAAQDFVQWLMNTGPSSGAPPS-NH2	15
C355	HSQGTFTSDYSKYLDEEAAQDFVQWLMNTGPSSGAPPPS-NH2	16
C356	HSQGTFTSDYSKYLDEEAAQDFVQWLMNTGPSSGAPPPS-OH	17

C463	HSQGTFTSDYSKYLDEEAAQDFVQWLMNGGPSSGAPPS-NH2	18
C462	HSQGTFTSDYSKYLDEEAAQDFVQWLMNGGPSSGAPPS-OH	19
C464	HSQGTFTSDYSKYLDEEAAQDFVQWLMNGGPSSGAPPPS-NH2	20
C465	HSQGTFTSDYSKYLDEEAAQDFVQWLMNGGPSSGAPPPS-OH	twenty one
C372	HSQGTFTSDYSKYLDEQAAQDFVQWLMNTSSGAPPS-NH2	twenty two
C369	HSQGTFTSDYSKYLDEQAAQDFVQWLMNTGPSSGAPPPS-NH2	twenty three
C494	HSQGTFTSDYSKYLDEQAAQDFVQWLMNGGPSSGAPPS-NH2	twenty four
C493	HSQGTFTSDYSKYLDEQAAQDFVQWLMNGGPSSGAPPPS-NH2	25
C495	HSQGTFTSDYSKYLDEQAAQDFVQWLMNGGPSSGAPPPS-OH	26
C358	HSQGTFTSDYSKYLDEQAAQDFVQWLMNTSSGAPPPS-NH2	27
C359	HSQGTFTSDYSKYLDEQAAQDFVQWLMNTSSGAPPPS-OH	28
C002	H-dSer-QGTFTSDYSKYLDSRRAQDFVQWLMNTGGPSSGAPPPS-OH	29
C225	HSQGTFTSDYSKYLDERAAQDFVQWLMNTGPSSGAPPPS-OH	30
C226	HSQGTFTSDYSKYLDERAAQDFVQWLMNTGPSSGAPPPS-NH2	31
C163	HSQGTFTSDYSKYLDSRAAQDFVQWLMNTGPSSGAPPPS-OH	32
C164	HSQGTFTSDYSKYLDSRAAQDFVQWLMNTGPSSGAPPPS-NH2	33
C271	HSQGTFTSDYSKYLDARAAQDFVQWLMNTGPSSGAPPPS-OH	34
C272	HSQGTFTSDYSKYLDARAAQDFVQWLMNTGPSSGAPPPS-NH2	35
C138	HSQGTFTSDYSKYLDSERAQDFVQWLMNTGPSSGAPPPS-NH2	36
C137	HSQGTFTSDYSKYLDSERAQDFVQWLMNTGPSSGAPPPS-OH	37
C239	HSQGTFTSDYSKYLDEERAQDFVQWLMNTGPSSGAPPPS-NH2	38
C277	HSQGTFTSDYSKYLDAERAQDFVQWLMNTGPSSGAPPPS-NH2	39
C139	HSQGTFTSDYSKYLDSQRAQDFVQWLMNTGPSSGAPPPS-NH2	40
C287	HSQGTFTSDYSKYLDEQRAQDFVQWLMNTGPSSGAPPPS-NH2	41
C245	HSQGTFTSDYSKYLDAQRAQDFVQWLMNTGPSSGAPPPS-NH2	42
C291	HSQGTFTSDYSKYLDAARAQDFVQWLMNTGPSSGAPPPS-NH2	43
C240	HSQGTFTSDYSKYLDERAAQDFVQWLMNGGPSSGAPPPS-NH2	44
C276	HSQGTFTSDYSKYLDSRAAQDFVQWLMNGGPSSGAPPPS-NH2	45
C236	HSQGTFTSDYSKYLDARAAQDFVQWLMNGGPSSGAPPPS-NH2	46
C539	HSQGTFTSDYSKYLDEQAAREFVQWLMNTGPSSGAPPPS-NH2	47
C456	HSQGTFTSDLSKYLDEEAAQDFIQWLMNTGPSSGAPPPS-NH2	48
C649	HSQGTFTSDYSKYLDEQAARDFIQWLMNGGPSSGAPPPS-NH2	49
C580	HSQGTFTSDLSKYLDEEAAQEFIQWLMNGGPSSGAPPPS-NH2	50

C581	HSQGTFTSDLSKYLDEEAAREFVQWLMNGGPSSGAPPPS-OH	51
C578	HSQGTFTSDYSKYLDEQAAREFVQWLMNTGPSSGAPPPS-NH2	52
C481	HSQGTFTSDLSKYLDEEAAREFVQWLMNTGPSSGAPPPS-NH2	53
G490	HSQGTFTSDYSKYLSQEAVRLFICWLMNT	54
C824	HSQGTFTSDYSKYLSQAAVRLFIQWLMNTGPSSGAPPPS	55
C721	HSQGTFTSD K' SEYLDEEAARDFVAWLEAGG	56
C816	HSQGTFTSDYSKYLDSQAAKEFIAWLMNGGPSSGAPPPS-NH2	57
C632	HXQGTFTSD K' SKYLDEQAAQDFVQWLLDGPSSGAPPPS-NH2	58
C611	HSQGTFTSDLSKQMDSQAAQDFIEWLKNGGPSSGAPPPS-NH2	59
FC381	HSQGTFTSD K' SKYLDSQAAQDFVQWLMNGGPSSGAPPPS-NH2	60
FC464	HSQGTFTSD K' SKYLDEEAAQDFVQWLMNGGPSSGAPPPS-NH2	61
FC493	HSQGTFTSD K' SKYLDEQAAQDFVQWLMNGGPSSGAPPPS-NH2	62
FC225	HSQGTFTSD K' SKYLDERAAQDFVQWLMNTGPSSGAPPPS-OH	63
FC163	HSQGTFTSD K' SKYLDSRAAQDFVQWLMNTGPSSGAPPPS-OH	64

Note: X in the table is aminoisobutyric acid, and K' indicates that this position is a lysine residue and is covalently linked to a fatty acid having the following structure:

Table 2

Numbering	Theoretical molecular weight (Da)	Detection of molecular weight (Da)
C326	4062.4	4062.5
C327	4063.3	4063.3
C231	4203.5	4203.5
C232	4204.5	4204.6
C365	4005.3	4005.3
C366	4006.3	4006.4
C381	4159.5	4159.5
C382	4160.5	4160.5
C392	4150.4	4150.4
C393	4149.4	4149.3
C355	4246.6	4246.6
C356	4247.5	4247.5
C463	4105.4	4105.4
C462	4106.4	4106.3
C464	4202.5	4202.5
C465	4203.5	4203.5
C372	3994.3	3994.3

C369	4245.6	4245.6
C494	4104.4	4104.4
C493	4201.5	4201.5
C495	4202.5	4202.5
C358	4091.4	4091.4
C359	4092.4	4092.3
C002	4374.7	4374.6
C225	4274.6	4274.6
C226	4273.6	4273.6
C163	4232.6	4232.7
C164	4231.6	4231.6
C271	4216.6	4216.6
C272	4215.6	4215.5
C138	4289.6	4289.5
C137	4290.6	4290.5
C239	4331.7	4331.6
C277	4273.6	4273.5
C139	4288.6	4288.6
C287	4330.7	4330.6
C245	4272.6	4272.5
C291	4215.6	4215.7
C240	4229.6	4229.6
C276	4187.5	4187.4
C236	4171.5	4171.5
C539	4216.5	4216.4
C456	4196.5	4196.6
C649	4172.5	4172.5
C580	4152.5	4152.5
C581	4153.5	4153.4
C578	4246.6	4246.6
C481	4196.5	4196.6

Example 2: Stability study

The purpose of this example was to investigate the chemical stability of various glucagon analogs obtained in the preparation of Example 1 in aqueous solution.

The polypeptide to be tested (glucagon analog) and the control substance were prepared in a 20 mM phosphate buffer PB or acetate buffer at a corresponding pH, and the final concentration of the polypeptide was 0.2 mg/ml and a sterile filter (0.22 μm) was used. , Millipore SLGP033RB) for filtration sterilization. The prepared polypeptide solution was allowed to stand at 40 ° C for 7 days. It was then centrifuged at 4500 rpm for 20 minutes and the supernatant (t7) was analyzed using RP-HPLC-UV. The amount of residual intact peptide was determined, and the sample (t0) which was not incubated was analyzed in parallel. Comparing the peak areas of the target compounds at t0 and t7, the "residual peptide %" was obtained according to the following equation:

Residual peptide content % = [(peptide peak area t7) × 100] / peptide peak area t0.

Stability is expressed as "residual peptide content".

Detection method

Detection wavelength: 214 nm;

Column: column temperature 40 ° C, Phenomenex Luna C8 (2) 5 μm (150 × 4.6 mm);

Mobile phase: H ₂ O + 0.1% TFA: ACN + 0.1% TFA (flow rate 1.0 ml / min);

Gradient: 95:5 (0 minutes) to 0:100 (30 minutes);

Analysis of experimental results: From the experimental data of Table 3, it can be concluded that the preferred glucagon analog (polypeptide) of the present invention has high stability in both neutral and weakly acidic aqueous solutions.

table 3

Numbering	Residual peptide content pH 4.5 (%)	Residual peptide content pH 7.4 (%)
C326	96.8	95.6
C327	97.5	98.9
C231	96.7	96.9
C232	95.2	97.8
C365	98.2	96.4
C366	97.5	96.8
C381	98.6	99.5
C382	98.9	97.5
C392	95.9	96.8
C393	96.4	95.7
C355	97.5	97.3
C356	98.1	97.7
C463	96.6	97.1
C462	97.1	96.7
C464	95.1	96.7
C465	95.6	96.8
C372	96.3	97.4
C369	96.9	97.0
C494	96.8	95.9
C493	97.9	98.3
C495	98.4	96.7
C358	97.5	96.8
C359	98.1	96.9
C002	78.4	81.3
C225	83.7	82.3
C226	82.4	84.7
C163	81.8	84.5
C164	80.6	84.2
C271	83.7	82.8
C272	84.9	84.2
C138	81.9	83.4
C137	82.4	84.8
C239	83.8	86.7
C277	82.8	82.4

C139	81.4	84.1
C287	83.8	82.8
C245	84.7	82.5
C291	82.9	83.8
C240	84.7	83.1
C276	83.1	83.2
C236	79.2	80.3
C539	93.4	94.5
C456	93.7	95.1
C649	94.8	94.1
C580	93.7	92.8
C581	92.4	91.8
C578	94.7	94.5
C481	95.7	93.7
C490	92.2	95.2
C824	91.2	95.4
C721	95.8	96.7
C816	90.1	91.8
C632	96.4	96.9
C611	87.8	89.3

Figures 1-7 exemplarily show liquid phase HPLC analytical spectra of several glucagon analogs such as C381.

Figure 1 corresponds to the integral data:

Figure 2 corresponds to the integral data:

The raw data corresponding to Figure 3:

The raw data corresponding to Figure 4:

The raw data corresponding to Figure 5:

Figure 6 corresponds to the original data:

The raw data corresponding to Figure 7:

Example 3: Serum stability

(1) The corresponding polypeptide in Table 1 was formulated into a solution of 1.0 mg/ml with 5 mM Tris-HCl, pH 8.5, 0.02% TWEEN80 solution, and sterilized by filtration (0.22 μm, Millipore SLGP033RB), and diluted with rat serum. 10 times, mix and dispense into a sterile centrifuge tube;

(2) Three samples of the above samples were frozen at -20 °C as a control, and the other was placed in a 37 ° C incubator, and the activity was sampled at different time points;

(3) Using the method shown in Example 4, the polypeptide GCGR agonistic activity was examined.

Relative activity: The activity value at 0 hours was 100%, and the value measured at the subsequent time point was obtained. Analysis of experimental results: Serum stability can be obtained from Table 4 and Figures 8A and 8B.

Table 4

Note: N.D. means lower than the lower limit of detection

Example 4: Determination of cell viability

(1) Determination of GLP-1R agonistic activity:

The luciferase reporter assay was used for GLP-1R agonistic activity assay (Jonathan W Day et al: Nat Chem Biol. 2009 Oct; 5(10): 749-57). The human GLP-1R gene was cloned into the mammalian cell expression plasmid pCDNA3.1, and the recombinant expression plasmid pCDNA3.1-GLP-1R was constructed, and the full-length gene of luciferase was cloned into the pCRE plasmid to obtain pCRE-Luc. Recombinant plasmid. The pcDNA3.1-GLP-1R and pCRE-Luc plasmids were transfected into CHO cells at a ratio of 1:10, and the stably transfected expression strains were screened.

The cells were cultured in a 9-cm cell culture dish in DMEM/F12 medium containing 10% FBS and 300 μg/ml G418. When the confluency was about 90%, the culture supernatant was discarded, and after 2 ml trypsin digestion for 3 min, Add 2 ml of DMEM/F12 medium containing 10% FBS and 300 μg/ml G418, transfer to a 15 ml centrifuge tube, centrifuge at 1000 rpm for 5 min, discard the supernatant, and add 2 ml of DMEM containing 10% FBS and 300 μg/ml G418. /F12 medium was resuspended and counted. The cells were diluted to 1 × 10 ^{5 /} ml with DMEM/F12 medium containing 10% FBS, 100 μl per well in a 96-well plate, ie 1 × 10 ⁴ /well, and then replaced with DMEM/ containing 0.2% FBS. F12 medium culture. After discarding the supernatant in the 96-well plate, the purified recombinant protein was diluted to a specified concentration in DMEM/F12 medium containing 0.1% FBS, and added to the cell culture well, 100 μl/well, and stimulated for 6 hours. Detection. Detection was carried out according to the instructions of the lucifersae reporter kit (Ray Biotech, Cat: 68-LuciR-S200). Figures 9A and 9B show the results of GLP-1R agonistic activity assay.

(B) GCGR agonistic activity detection method:

The luciferase reporter assay is also used for GCGR agonistic activity assays. The human GCGR gene was cloned into the mammalian cell expression plasmid pcDNA3.1 to construct a recombinant expression plasmid pCDNA3.1-GCGR, and the transfected HEK 293T and stably transformed cell lines were screened as above. Figures 9C and 9D show the results of GCGR agonistic activity assay.

table 5

(C) GIPR agonistic activity detection method:

The pcDNA3.1-GIPR plasmid was transfected into CHO cells and screened for positive stable cell lines. Approximately 200,000 cells/well were seeded in 96-well cell culture plates, cultured overnight, and washed with Hanks' balanced salt solution, and the protein to be tested was diluted to a specified concentration of 200 μM 3-isobutyl-1-methylxanthine. (IBMX) was added to the cells, cultured at 37 ° C for 20 min, the culture supernatant was discarded, the lysate was added to lyse the cells, and cAMP content was determined by cAMP Parameter assay kit (American R&D Company, article number: SKGE002B). The results are shown in Figures 9E-H.

Example 5: Glucose-stimulated insulin secretion assay

In this embodiment, the method of Aisling M. Lynch et al. (A novel DPP IV-resistant C-terminally extended Glucagon analogue exhibits weight-lowering and diabetes-protective effects in high-fat-fed mice mediated through Glucagon and GLP-1 receptor activation , Aisling M. Lynch et al, Diabetologia, 57: 1927–1936, 2014) Rat BRIN-BD11 cells were used to determine insulin release induced by active protein stimulation, but with minor modifications, ie in a 24-well plate (Orange Scientific, Brainel) Add 1.0×106 cells to each well in 'Alleud, Belgium, incubate at 37 ° C overnight, centrifuge to remove the supernatant, and add 1.0 ml of KRB (115 mM NaCl, 4.7 mM KCl, 1.28 mM CaCl ₂ , 1.2 mM MgSO ₄ , per well). 1.2 mM KH ₂ PO ₄ , 25 mM HEPES, 10 mM NaHCO ₃ , NaOH adjusted to pH 7.4), 0.1% (wt/vol.) BSA and 1.1 mM glucose. After the cells were incubated at 37 ° C for 40 minutes, the supernatant was centrifuged and replaced with 1.0 ml of fresh KRB solution and a gradient concentration of active protein. After incubation at 37 ° C for 20 minutes, the buffer was removed by centrifugation and stored at -20 ° C overnight, and then subjected to immunoradiometric detection of insulin content. The result is shown in Figure 10.

Example 6: Mouse immunogenicity experiment

7-week-old Balb/c mice, 6 in each group, were collected from the tail vein of each rat before administration to obtain 50 ul of serum as a blank control. A corresponding glucagon analog (30 nmol/kg in PBS buffer) was injected daily for 28 consecutive days. On the 45th day, blood was collected from the eyelids and the serum was coagulated. The antibody titer was determined by direct ELISA. The corresponding polypeptide coated with the enzyme plate, the mouse serum was diluted 1:50; 1:200, 1:1000, 1:5000 into the enzyme plate, and the goat anti-mouse secondary antibody was the detection antibody. Before the administration of each mouse, the serum was a negative control. Under the same dilution, the average value of the OD ₄₅₀ value of the test sample was greater than 2.1 times the average value of the OD ₄₅₀ value of the negative control serum, and the result was judged as positive (+), and vice versa (negative). -), the highest dilution that results in a positive is the antibody titer.

Table 6

Example 7: Glucose tolerance test (IPGTT) in normal ICR mice

Normal ICR mice were divided into 27 groups of 6 animals each. Fasted overnight, blood was collected from the tail (denoted as t=0 minute blood glucose), subcutaneously injected with media control (acetate buffer, 20 mM acetic acid, 250 mM mannitol, pH 5.0) and pancreas in Table 1 of the present invention. Glucagon analog (30nmol/kg in PBS buffer) and vehicle control, liraglutide ( trade name)

40 nmol/kg, diluted into PBS buffer). After 15 minutes, glucose (2 g/kg body weight) was intraperitoneally injected, and blood glucose levels were measured at t = 30 minutes, t = 45 minutes, t = 60 minutes, and t = 120 minutes. Animals were still fasted during the experiment to prevent interference with food intake. See Figures 11A-C for specific results.

Example 8: Weight loss experiment in diet-induced obesity (DIO) mice

Preparation of the DIO mouse model: Male C57BL/6J male mice, approximately 7 weeks old, were fed a high fat diet (60% kcal from fat) for about 16 weeks (23 weeks total) and tested at a body weight of approximately 45 g. DIO mice were randomly divided into groups, with 6 rats in each group. There was no difference in basal body weight. Each group of mice was injected subcutaneously with each glucagon analog (30nmol/kg, PBS) or PBS for the same time. Rupeptide (trade name)

30 nmol/kg), administered twice a day, weighing to 30 days per day.

12A-C are daily changes in body weight of DIO mice after administration of each glucagon analog, and the final percentage of weight loss is shown in Figure 12D.

Example 9: Weight loss experiment in diet-induced obesity (DIO) mice

DIO mice were randomly divided into groups, with 6 rats in each group. There was no difference in basal body weight. Each group of mice was injected subcutaneously with each GCG analog (30 nmol/kg in PBS) or PBS, and the control group liraglutide (trade name).

30 nmol/kg, diluted to PBS) was administered twice a day, and the fatty acidified GCG analog (30 nmol/kg in PBS) was administered once a day and weighed to 30 days per day.

Figure 13 is the percentage of final body weight loss in DIO mice after administration of each glucagon analog. The GCG analogs C381, C464 and C493 shown in the figure have the same weight loss effect as the corresponding amino acid sequence and the fatty acid modified corresponding GCG analog, while C225 and C163 have no significant weight loss effect without fatty acidification.

In summary, the present invention effectively overcomes various shortcomings in the prior art and obtains a group of three-way agonists with potential clinical value.

The above-described embodiments are merely illustrative of the principles of the invention and its effects, and are not intended to limit the invention. Modifications or variations of the above-described embodiments may be made by those skilled in the art without departing from the spirit and scope of the invention. Therefore, all equivalent modifications or changes made by those skilled in the art without departing from the spirit and scope of the invention will be covered by the appended claims.

Claims (16)

1. A glucagon analog having a structure as shown in Formula I or Formula II, wherein the structure of Formula I is: HSQGTFTSD-X ₁₀ -SKYLD-X ₁₆ -X ₁₇ -AA-X ₂₀ -X ₂₁ -FX ₂₃ -QWLMN-X ₂₉ -X ^z , the structure shown in formula II is:

   HSQGTFTSD-X ₁₀ -SKYLD-X ₁₆ -X ₁₇ -AA-X ₂₀ -X ₂₁ -FX ₂₃ -QWLMN-X ₂₉ -X ^z -NH ₂ , wherein X _{10 is} selected from any one of Y, K or L, X _{16 is} selected from any one of S, E or A, X _{17 is} selected from any one of Q, E, A or R, X _{20 is} selected from any one of Q, R or K; X _{21 is} selected from D, L or E Any one; X _{23 is} selected from any one of V or I; X ₂₉ is T or a deletion, and X ^{z is} selected from any one of GGPSSGAPPPS, GGPSSGAPPS, GPSSGAPPPS, GPSSGAPPS, PSSGAPPPS, PSSGAPPS, SSGAPPPS or SSGAPPS.
2. The glucagon analog according to claim 1, wherein the glucagon analog comprises a structure as shown in Formula III or Formula IV, and the structure shown in Formula III is:

   HSQGTFTSDYSKYLD-X ₁₆ -X ₁₇ -AAQ-DFVQWLMN-X ₂₉ -X ^z ,

   The structure shown in formula IV is:

   HSQGTFTSDYSKYLD-X ₁₆ -X ₁₇ -AAQ-DFVQWLMN-X ₂₉ -X ^z -NH ₂ ,

   Wherein X _{16 is} selected from any one of S or E, X _{17 is} selected from any one of Q or E, X ₂₉ is T or a deletion, and X ^{z is} selected from GGPSSGAPPPS, GGPSSGAPPS, GPSSGAPPPS, GPSSGAPPS, PSSGAPPPS, PSSGAPPS, SSGAPPPS or SSGAPPS Any one.
3. The glucagon analog according to any one of claims 1 to 2, wherein the glucagon analog has GLP-1/GCG/GIP tri-receptor agonistic activity.
4. An isolated polynucleotide encoding the glucagon analog of any one of claims 1-3.
5. A recombinant expression vector comprising the polynucleotide isolated according to claim 4.
6. A host cell comprising the recombinant expression vector of claim 5 or a polynucleotide isolated according to claim 4 integrated in the genome.
7. A method for producing a glucagon analog according to any one of claims 1 to 3, which is characterized by being selected from any one of the following:

   (1) synthesizing the glucagon analog by a chemical synthesis method;

   (2) cultivating the host cell of claim 6 under appropriate conditions to express the glucagon analog, and then isolating and purifying to obtain the glucagon analog.
8. Use of a glucagon analog according to any one of claims 1 to 3 for the preparation of a medicament for treating a metabolic related disease.
9. A method of promoting weight loss or preventing weight gain, comprising administering a glucagon analog according to any one of claims 1-3 to a subject.
10. A composition comprising a glucagon analog according to any one of claims 1 to 3 or a culture of a host cell according to claim 6, and a pharmaceutically acceptable carrier.
11. Use of a glucagon analog according to any one of claims 1 to 3 for the preparation of a fusion protein.
12. A fusion protein comprising a glucagon analog according to any one of claims 1-3.
13. The fusion protein according to claim 12, wherein the fusion protein further comprises a long-acting unit in its structure.
14. The fusion protein according to claim 13, wherein the long acting unit is selected from the group consisting of albumin, transferrin and immunoglobulins and fragments.
15. A modified polypeptide comprising a glucagon analog according to any one of claims 1 to 3 in the structure.
16. The modified polypeptide according to claim 15, wherein the glucagon analog is modified with a fatty acid, polyethylene glycol, albumin, transferrin or immunoglobulin and a fragment.

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