A glucagon analog for treating metabolic diseases

Abstract

The present invention relates to the field of biopharmaceuticals, and in particular to a glucagon analogue for treating metabolic diseases, having the structural formula: H-X2-X3-GTFTSD-X 10 ‑SKYLD‑X 16 ‑X 17 ‑AAQ‑DFVQWLMN‑X 29 ‑X z or H‑S‑Q‑GTFTSD‑Y‑SKYLD‑X 16 ‑X 17 ‑AAQ‑DFVQWLMN‑X 29 ‑X z The glucagon analogue of the present invention has GLP-1 / GCG / GIP triple receptor agonist activity and better enzyme stability, including resistance to neutral endopeptidase (NEP) and dipeptidyl peptidase-4 (DPP-4). Compared with natural glucagon, GLP-1, and GIP, it has a longer in vivo half-life and duration of action.

Description

Technical Field

[0001] The present invention relates to the field of biopharmaceuticals, and in particular to a glucagon analog for treating metabolic diseases, a preparation method thereof, and uses thereof. Background Art

[0002] Diabetes is a serious chronic disease that occurs when the pancreas does not produce enough insulin or the body cannot effectively use the insulin it produces. Currently, the protein-based diabetes drugs on the market are mainly GLP-1 receptor (GLP-1R) agonists, such as Dulaglutide (trade name: ), Albiglutide (trade name) ), Liraglutide (Liraglutide, trade name and used to treat obesity and diabetes, respectively), Exenatide (trade name ), Lixisenatide (trade name ) and Semaglutide, which may be on the market soon. Dulaglutide, albiglutide, liraglutide and semaglutide are all analogs of natural glucagon-like peptide-1 (GLP-1). After the GLP-1 sequence mutation, they are fused or cross-linked with the FC fragment of IgG, human albumin and fatty acids to obtain highly active and stable GLP-1R agonists. Exenatide (Exendin-4) is a small peptide of 39 amino acids derived from the salivary glands of lizards (Heloderma suspectum). Although Exendin-4 is a potent agonist of GLP-1R, its activity is higher than that of natural GLP-1 and GLP-1 analogs. Although these GLP-1R agonists can effectively lower blood sugar and control appetite, their effect on weight loss is not very significant. Among them, liraglutide (trade name Although approved for the treatment of obesity, the actual weight loss is only about 5.6 kg. Current obesity medications generally reduce weight by about 5–10% (compared to placebo), meaning the overall average weight loss 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).

[0003] Bariatric surgery can significantly improve obesity and treat diabetes, but its use is limited because most patients are reluctant to undergo it due to concerns about surgical risks and long-term consequences (Obesity and Diabetes, New Surgical and Nonsurgical Approaches, Springer, 2015). Studies have found that patients who undergo bariatric surgery experience a surge in incretin secretion (Obesity and Diabetes, New Surgical and Nonsurgical Approaches, Springer, 2015). Preclinical and clinical studies have also found that simultaneous infusion of GLP-1 / glucagon (GCG) (Tricia M. Tan etc., DIABETES, VOL. 62: 1131-1138, 2013), or GLP-1, oxyntomodulin (OXM) and PYY, has significant effects on promoting energy metabolism, suppressing appetite and controlling weight (Tricia Tan etc., J Clin Endocrinol Metab, 2017, 102(7): 2364–2372). From a clinical perspective, these peptides can be simply mixed for clinical use. However, due to the differences in the in vivo stability and degradation rates of these different types of peptides, the final in vivo efficacy is uncontrollable, and it is difficult to simply mix these peptides and use them as a compound drug. Therefore, the current development of a new generation of diabetes drugs mainly focuses on trying to concentrate the activities of these agonists in one molecule, such as GLP-1R / GIPR and GLP-1R / GCGR dual-acting agonists, and even GLP-1R / GIPR / GCGR triple-acting agonists (Chakradhar, Shraddha. All in one: researchers create combination drugs for diabetes and obesity. Nature Medicine, 22(7):694-695, 2016).

[0004] Currently, the design and development of this type of drug primarily involves the following approaches: 1. Modification of the endogenous human peptide oxyntomodulin (OXM), which possesses dual GLP-1 / GCG activity. OXM is a naturally occurring peptide with both GLP-1 and GCG activity (Diabetes, 2005, 54:2390–2395). However, OXM's activity is low (approximately 10% of GCG activity and 1% of GLP-1 activity), and its in vivo stability and half-life are poor. Therefore, OXM itself cannot be used directly in clinical practice. Instead, its in vivo activity and stability are often enhanced through the introduction of unnatural amino acids and various modifications. For example, OPKO Biologics' Mod-6030 is a long-acting OXM with a degradable PEGylated N-terminus (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 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, its clinical effect is not good, and most research has been abandoned. 2. Utilizing the homology of incretin sequences, multiple mutations, modifications, and even the introduction of non-natural amino acids based on the structures of OXM, GLP-1, and GCG are used to obtain stable hybrid peptides with multiple activities (Matthias H. et al., Unimolecular Polypharmacy for Treatment of Diabetes and Obesity, 24: 51–62, 2016). Matthias H. The review articles by et al. have introduced in detail various hybrid peptide forms currently in clinical or preclinical stages, such as the OXM-based dual-action GLP-1R / GCGR agonist 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 the GCG-based dual-action GLP-1R / GCGR agonist reported by Richard D. DiMarchi et al. (US9018164 B2) and even the triple-action GLP-1R / GCG / GIPR agonist (US9150632). Most of these multi-specific hybrid peptides are based on GLP-1 or GCG, and their activity and resistance to proteolysis are improved through sequence mutations. For example, L-type amino acids are mutated into D-type amino acids (such as D-Ser), or the non-natural amino acid Aib is introduced to improve in vivo stability. At the same time, fatty acid chain or polyethylene glycol (PEG) modification is performed to extend the half-life. The expected clinical dosing cycle is once a day (fatty acid modification) or once a week (PEG modification). In addition, Aisling M. Lynch et al. reported that the second Ser of natural GCG was mutated to D-Ser, and a GCG analogue of the C-terminal peptide of Exendin-4 (D-Ser2-glucagon-exe) was introduced at the C-terminus. The efficacy experiment was conducted in DIO, and the drug was given twice a day (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), with a significant weight loss effect.

[0005] Although the research and development of molecules with multiple agonist activities of GLP-1R, GCGR, and GIPR is very promising in clinical practice, it is actually very difficult to obtain an ideal drug of this type.

[0006] The first is safety, particularly immunogenicity. Antidiabetic and weight-loss drugs require long-term use, placing extremely high safety requirements on their development. To engineer a peptide with high GLP-1, GCG, and GIP activity and stability in vivo, existing approaches often introduce numerous mutations, often with unnatural amino acids and other modifications. These mutations and the introduction of unnatural amino acids increase the risk of potential immunogenicity. Generally, the higher the homology with the human sequence, the lower the immunogenicity risk in humans. Taspoglutide, a GLP-1 receptor agonist developed in collaboration with Ipsen (incorporating only two unnatural amino acids, Aib), has achieved an antibody production rate of 49%, and all Phase III clinical trials 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 GCG sequence before obtaining a candidate peptide MEDI0382. Among them, in order to maintain high dual activity of GLP-1 and GCG and in vivo stability, compared with GCG, MEDI0382 introduced 9 mutation sites, and the mutation rate reached about 30%; similarly, Andreas Evers et al. (J Med Chem. 2017 May 25; 60(10): 4293-4303) introduced 9 mutation sites based on the structure of Exendin-4, with a mutation rate of about 23%, and modified the fatty acid chain to obtain a hybrid peptide with high dual activity of GLP-1 and GCG; Brian Finan et al. (Brian Finan et al., Nat Med. 21: 27-36, 2015) designed a GLP-1 / GCG / GIP tripeptide by adding the GPSSGAPPPS sequence to the C-terminus of GCG and introducing 7 mutant amino acids, including the second mutation to the non-natural amino acid Aib. Therefore, existing solutions often require the introduction of numerous mutations, often unnatural amino acids, and other modifications to produce peptides with high GLP-1, GCG, and GIP activity. These mutations, modifications, and the introduction of unnatural amino acids increase the risk of potential immunogenicity. Safety is paramount for drugs treating conditions like diabetes and obesity.Therefore, it is very meaningful to develop a highly active multi-functional GLP-1 / GCG multi-agonist that does not contain unnatural amino acids and contains as few mutant amino acids as possible.

[0007] On the other hand, existing research has yet to reach a consensus on how to combine the activities of these incretins and the appropriate ratio between their activities. For example, PCT applications WO2015155139A1, WO2015155140A1, and WO201515541A1 disclose GLP-1R / GCGR dual-action agonist peptides or GLP-1R / GCG / GIPR tripartite agonist peptides modified from Exendin-4. WO201515541A1 discloses a hybrid peptide with GLP-1R / GCGR / GIPR tripartite agonist activity, which is believed to have excellent glucose-lowering and weight-loss effects. However, WO2015155139A1 and WO2015155140A1, to avoid the risk of hypoglycemia caused by GIPR agonist activity, prepare GLP-1R / GCGR dual-action agonist peptides, which instead have even better glucose-lowering and weight-loss effects. A. Seth et al. also believed that the introduction of GIP activity did not enhance the blood sugar control effect of GLP-1 (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).

[0008] Furthermore, for small peptides such as GLP-1, Exendin-4, and GCG, which have highly homologous sequences and receptors belonging to the same GPCR family, and whose peptide chains are only about 30-40 amino acids long, it is extremely difficult to predict the changes in activity at different receptors after mutation of a single site or multiple sites simultaneously. Therefore, it is extremely difficult to obtain ideal hybrid peptides with multiple agonist activities. 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) reported that after performing an alanine scan on GCG, the relative residual activity retention ranged from 0.2% to 100% after independent alanine substitution at each site of GCG. They also showed that mutations at positions 1, 2, 3, 4, 6-12, 14, 15, 22, 23, 25-27, and 29 of GCG significantly reduced the agonist activity of GCGR (Table 4 in the article). Yet we can also see in other reports that in these sites, single or several sites are taken and mutated simultaneously, and when substituted with other amino acids, the change of activity is not always consistent with the result of alanine scanning. As reported by Jonathan W Day et al. (Jonathan W Day et al., A new glucagon and GLP-1 co-agonisteliminates obesity in rodents, Nature Chemical Biology, 5:749-757,2009), different mutations such as 16S→G, 16S→T, 16S→H, 16S→E at position 16 of GCG were carried out, and its GCGR agonist activity was improved on the contrary, which is completely contradictory to the alanine scanning result of Joseph Chabenne. Secondly, Joseph Chabenne studied and believed that replacing with alanine at position 23 would cause the GCGR agonist activity to be almost completely lost (only retaining 1.1%); but Jonathan W Day et al. mutated position 23 to Ile, and its GCG activity did not decline.For example, alanine scanning results suggest that the second amino acid S is crucial for maintaining GCG activity (mutation to Ala only retains one-third of the activity). However, Brian Finan et al. (FinanB et al., A rationally designed monomeric peptide triagonist corrects obesity and diabetes in rodents. Nat Med. 2015;21:27-36.) reported that substitutions at the second amino acid position of GCG, 2S→Aib, 2S→dSer, 2S→G, and 2S→dAla, combined with mutations at other positions, actually increased the relative agonist activity of GCGR by 200%–640%. Our studies have also found that when combinations of mutations are introduced that enhance the activity of GLP-1, GCG, or GIP, the effects are often completely inconsistent with those achieved by single-site mutations. Furthermore, for peptides such as GLP-1, Exendin-4, GCG, or GIP, adding or removing amino acids at the N- or C-termini can affect their biological activity. If one or two amino acids are removed from the N-terminus, the agonist activity of GLP-1, GCG, etc. will be completely lost. For example, oxyntomodulin only has eight more amino acids, KRNRNNIA, than the C-terminus of glucagon, and its GCGR agonist 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 receptor peptide agonist in rodents and non-human primates, Diabetes Obes Metab, 2016).

[0009] For example, Joseph R. Chabenne and Richard D. DiMarchi et al. reported that adding a small C-terminal peptide cex (SEQ ID NO. 67, GPSSGAPPPS) of Exendin-4 to the C-terminus of Glucagon increased its GLP-1R agonist activity from 0.7% 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 US9018164 B2), and also lost about 50% of the GCG activity. Evers A et al. also reported (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) that after adding the cex sequence to the C-terminus of the GCG analog, the GLP-1R agonist activity actually decreased by about 2 / 3, but the agonist activity of GCG was lost by more than 90% (Table 2, peptides 7 and 8 in the article). Therefore, for small peptides with a length of 30 amino acids such as GLP-1 and Glucagon, sequence changes are extremely sensitive to changes in their activity; and for dual-active peptides, since they involve the agonism of two different receptors, the changes are even more complicated, and it is impossible to predict what consequences any amino acid change will have on the agonist activity of GLP-1R and GCGR.

[0010] The complexity of downstream signal transduction of GPCR receptors such as GCGR and GLP-1R also increases the difficulty of designing an ideal multi-active hybrid peptide. GCGR and GLP-1R receptors have multiple signal transduction pathways in the cell, including G proteins (Gαs, Gαi, Gαq, etc.) and inhibitory proteins (β-arrestin-1 and β-arrestin-2) and other downstream signal factors that form multiple different signal transduction pathways. The activation of different pathways has different physiological effects, and the relationship between the activity of some pathways and physiological functions is not even clear. For example, by introducing different mutations or different amino acid sequences into the GLP-1 sequence, different biased-agonist structures can be obtained, thereby producing 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).

[0011] Therefore, while theoretically designing a peptide with high GLP-1, glucagon, and GIP activities is clinically significant, it is also very difficult in practice. Designing a peptide with balanced activities while minimizing the introduction of mutation sites and unnatural amino acids, keeping the sequence close to the native sequence, thereby reducing potential immunogenicity and improving stability, and thus achieving multiple active hybrid peptides with excellent blood sugar and weight control, would be of great clinical significance. Summary of the Invention

[0012] In view of the above-mentioned shortcomings of the prior art, the object of the present invention is to provide a glucagon analog that exhibits GLP-1R / GCGR / GIPR triple receptor agonist activity.

[0013] Natural GCG has approximately 1% of the GLP-1R agonist activity of natural GLP-1, but no GIPR agonist activity. However, the glucagon analogs of the present invention can exhibit triple GLP-1R / GCGR / GIPR agonist activity.

[0014] To achieve the above-mentioned and other related purposes, the first aspect of the present invention provides a glucagon analog (GCG analog), wherein the structure of the glucagon analog contains a structure as shown in Formula I or Formula II, wherein the structure shown in Formula I is: HSQGTFTSD-X 10 -SKYLD-X 16 -X 17 -AA-X 20 -X 21 -FX 23 -QWLMN-X29 -X z (SEQ ID NO.1), the structure shown in Formula II is:

[0015] HSQGTFTSD-X 10 -SKYLD-X 16 -X 17 -AA-X 20 -X 21 -FX 23 -QWLMN-X 29 -X z -NH2 (SEQ ID NO. 2), wherein X 10 Any one selected from Y, K or L, X 16 Any one selected from S, E or A, X 17 Any one selected from Q, E, A or R, X 20 Any one selected from Q, R or K; X 21 Selected from any one of D, L or E; X 23 Selected from either V or I; X 29 T or missing, X z 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 NO.72) any one.

[0016] Furthermore, when the structural formula of the glucagon analog is:

[0017] HSQGTFTSD-X 10 -SKYLD-X 16 -X 17 -AA-X 20 -X 21 -FX 23 -QWLMN-X 29 -X z -NH2 (SEQ ID NO. 2), it means that the C-terminus of the glucagon analog is amidated.

[0018] As exemplified in some embodiments of the present invention, the amino acid sequence of the glucagon analog of the present invention is shown in any one of SEQ ID NOs. 6-28 and SEQ ID NOs. 47-53.

[0019] Furthermore, in a preferred embodiment, the structural formula of the glucagon analog is:

[0020] HSQGTFTSDYSKYLD-X 16 -X 17 -AAQ-DFVQWLMN-X 29 -X z (SEQ ID NO.3)

[0021] or HSQGTFTSDYSKYLD-X 16 -X 17 -AAQ-DFVQWLMN-X 29 -X z -NH2 (SEQ ID NO. 4)

[0022] Among them, X 16 Selected from either S or E, X 17 Select any one of Q or E, X 29 T or missing, X z 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 NO.72) any one.

[0023] Furthermore, when the structural formula of the glucagon analog is:

[0024] HSQGTFTSD-Y-SKYLD-X 16 -X 17 -AAQDFVQWLMN-X 29 -X z -NH2 (SEQ ID NO. 4), it means that the C-terminus of the glucagon analog is amidated.

[0025] The glucagon analogs have GCGR agonist activity similar to or better than that of natural glucagon, and GLP-1R agonist activity similar to or better than that of natural GLP-1, and additionally have increased GIPR agonist activity.

[0026] In one embodiment of the present invention, a preferred glucagon analogue incorporates GPSSGAPPPS into the C-terminus of native glucagon, with minimal mutations of 2-3 amino acids and no introduction of unnatural amino acids or modifications. This analogue retains or enhances GLP-1R and GCGR agonist activity, while also exhibiting enhanced GIPR agonist activity. The product itself exhibits excellent stability. With fewer mutation sites and no subsequent modification, the native structure is maintained to the greatest extent possible, minimizing potential immunogenicity risks.

[0027] It has been reported in the literature that high levels of GIP can cause frequent hypoglycemia symptoms in the treatment of diabetes (TMcLaughlin et al., J Clin Endocrinol Metab, 95, 1851-1855, 2010; A Hadji-Georgopoulos, J Clin Endocrinol Metab, 56, 648-652, 1983). However, in mouse animal model experiments, the glucagon analogs with GIPR agonist activity provided by the present invention can stably control blood sugar without hypoglycemia symptoms. Other literature reports that antagonizing GIPR is also a desirable method to reduce daily food intake, lose weight, and improve 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 a mouse animal model experiment, the glucagon analogues with higher GIP activity provided by the present invention have more significant effects on daily food intake control, weight loss and improved insulin sensitivity in obese mice compared with comparison analogues with lower or even no GIP activity.

[0028] The glucagon analogue of the present invention has better enzyme stability, including resistance to neutral endopeptidase (NEP) and dipeptidyl peptidase-4 (DPP-4); and has a longer in vivo half-life and duration of action compared with natural glucagon, GLP-1, and GIP.

[0029] The second aspect of the present invention provides an isolated polynucleotide encoding the aforementioned glucagon analog.

[0030] The third aspect of the present invention provides a recombinant expression vector comprising the aforementioned isolated polynucleotide.

[0031] The fourth aspect of the present invention provides a host cell, wherein the cell contains the aforementioned recombinant expression vector or the aforementioned isolated polynucleotide is integrated into its genome.

[0032] A fifth aspect of the present invention provides a method for preparing the aforementioned glucagon analog, selected from any one of the following:

[0033] (1) synthesizing the glucagon analog by a chemical synthesis method;

[0034] (2) Cultivating the aforementioned host cell under appropriate conditions to express the glucagon analog, and then isolating and purifying the glucagon analog to obtain the glucagon analog.

[0035] Specifically, the glucagon analogs of the present invention can be prepared by standard peptide synthesis methods, for example, by standard solid phase or liquid phase methods, stepwise or by fragment assembly, and isolation and purification of the final peptide compound product, or by any combination of recombinant and synthetic methods. Preferably, the glucagon analogs of the present invention can be synthesized by solid phase or liquid phase peptide synthesis methods.

[0036] The sixth aspect of the present invention provides use of the aforementioned glucagon analog in the preparation of a medicament for treating metabolic-related diseases.

[0037] The glucagon analogs provided by the present invention can be used to treat diabetes-related metabolic syndrome, such as dyslipidemia, including high triglycerides, low HDL cholesterol and high LDL cholesterol; insulin resistance or glucose intolerance, etc.

[0038] Metabolic syndrome is associated with an increased risk of other conditions related to coronary heart disease and vascular plaque accumulation, such as stroke and peripheral vascular disease, becoming atherosclerotic cardiovascular disease (ASCVD). Patients with metabolic syndrome can develop from an early stage of insulin resistance to full-blown type 2 diabetes, and the risk of ASCVD is further increased. Without being limited to any particular theory, the relationship between insulin resistance, metabolic syndrome and vascular disease may involve one or more common pathogenic mechanisms, including impaired vasodilation of insulin stimulation, reduced insulin resistance-related availability caused by enhanced oxidative stress, and abnormalities in adipocyte-derived hormones (such as adiponectin) (Lteif, Mather, Can. J. Cardiol. 20 (Suppl B): 66B-76B, 2004).

[0039] The glucagon analogs of the present invention can also be used to treat obesity. In some aspects, the glucagon analogs of the present invention treat obesity by reducing appetite, reducing food intake, reducing body fat levels, and increasing energy expenditure.

[0040] In some potential embodiments, the glucagon analogs of the present invention can be used to treat non-alcoholic fatty liver disease (NAFLD). NAFLD refers to a broad spectrum of liver diseases, ranging from simple fatty liver (steatosis) to non-alcoholic steatotic hepatitis (NASH) to cirrhosis (irreversible late scarring of the liver). All stages of NAFLD are characterized by fat accumulation in liver cells. Simple fatty liver is an abnormal accumulation of certain types of fat and triglycerides in liver cells, but without inflammation or scarring. In NASH, fat accumulation is associated with varying degrees of liver inflammation (hepatitis) and scarring (fibrosis). Inflammatory cells can destroy liver cells (hepatocellular necrosis). In the terms "steatotic hepatitis" and "steatotic necrosis", steatosis refers to fatty infiltration, hepatitis refers to inflammation in the liver, and "necrosis" refers to destroyed liver cells. NASH can ultimately lead to scarring of the liver (fibrosis) and then to irreversible advanced scarring (cirrhosis), with cirrhosis caused by NASH being the final and most severe stage within the NAFLD spectrum.

[0041] In a seventh aspect, the present invention provides a method for treating metabolic-related diseases, comprising the step of administering the aforementioned glucagon analog to a subject.

[0042] In one embodiment, the present invention uses the glucagon analogs to treat obesity, metabolic syndrome, non-alcoholic hepatitis, etc.

[0043] Researchers have discovered that the glucagon analogs of the present invention have sufficient water solubility and improved chemical stability at neutral or slightly acidic pH. In one example, an IPGTT experiment was conducted. Mice administered with the glucagon analogs of the present invention exhibited significantly smoother blood sugar fluctuations after glucose injection. Furthermore, administration of the glucagon analogs of the present invention to DIO mice induced a significant decrease in body weight. Simultaneously, various lipid-related indicators decreased significantly.

[0044] The present invention further provides a method for promoting weight loss or preventing weight gain, comprising administering the glucagon analog to a subject.

[0045] In an eighth aspect, the present invention provides a composition comprising the aforementioned glucagon analog or the aforementioned host cell culture, and a pharmaceutically acceptable carrier.

[0046] The ninth aspect of the present invention provides use of the aforementioned glucagon analog in preparing a fusion protein.

[0047] The tenth aspect of the present invention provides a fusion protein, wherein the structure of the fusion protein contains the aforementioned glucagon analog.

[0048] Furthermore, the structure of the fusion protein also contains a long-acting unit.

[0049] Furthermore, the long-acting unit is selected from covalently linked fatty acids, polyethylene glycol or its derivatives, albumin, transferrin and immunoglobulins and fragments.

[0050] The eleventh aspect of the present invention provides a modified polypeptide comprising the aforementioned glucagon analog in its structure.

[0051] Furthermore, the glucagon analog is modified with fatty acid, polyethylene glycol or its derivatives; the modified polypeptide is covalently or non-covalently bound to albumin, transferrin and immunoglobulin and its fragments.

[0052] Those skilled in the art will appreciate that the glucagon analogs of the present invention may be modified to increase their half-life or stability. For example, polyethylene glycol or a derivative thereof, a hydroxyethyl starch derivative, or a fatty acid may be covalently linked to the glucagon analogs of the present invention. In a specific embodiment, to improve stability, the glucagon analogs of the present invention may be modified by introducing a lysine residue at a position where it is not expected to affect receptor binding / activation, covalently linking to a γ-glutamic acid spacer, and adding palmitic acid to the ε-amino group.

[0053] Compared with the prior art, the present invention has the following beneficial effects:

[0054] The glucagon analogues of the present invention have GLP-1 / GCG / GIP triple receptor agonist activity and better enzyme stability, including resistance to neutral endopeptidase (NEP) and dipeptidyl peptidase-4 (DPP-4); therefore, compared with natural glucagon, GLP-1, and GIP, they have a longer in vivo half-life and duration of action. In summary, the currently reported GCG analogues generally adopt (1) single-molecule hybrid peptide cross-linked fatty acids, PEG or FC, etc., and are administered once a day or more frequently (Matthias H. et al., Unimolecular Polypharmacy for Treatment of Diabetes and Obesity, 24: 51–62, 2016); or (2) mutating 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), and administering twice a day. There are currently no reports of multi-active peptides that maintain their natural amino acid composition and are administered twice a day. The present invention provides a sufficiently stable and highly active multi-functional GCG analog that does not require cross-linking with fatty acids, PEG albumin or immunoglobulin Fc fragments, and does not require mutation of the second Ser position to a non-natural amino acid. Therefore, it can minimize the potential immunogenicity risk, omit the tedious chemical modification / cross-linking steps, simplify the preparation process, and improve product consistency. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 : HPLC spectrum of the polypeptide numbered C381 in pH 7.4 aqueous solution.

[0056] Figure 2 : HPLC spectrum of the polypeptide numbered C493 in pH 4.5 aqueous solution.

[0057] Figure 3 : HPLC spectrum of the polypeptide numbered C816 in pH 7.4 aqueous solution.

[0058] Figure 4 : HPLC spectrum of the polypeptide numbered C002 in pH 7.4 aqueous solution.

[0059] Figure 5 : HPLC spectrum of the polypeptide numbered C611 in pH 4.5 aqueous solution.

[0060] Figure 6 : HPLC spectrum of the polypeptide numbered C611 in pH 7.4 aqueous solution.

[0061] Figure 7: HPLC spectrum of C239 in pH 7.4 aqueous solution.

[0062] Figure 8A : Curve of residual activity changing with time.

[0063] Figure 8B : Curve of residual activity changing with time.

[0064] Figure 9A : Schematic diagram of the detection of GLP-1R agonist activity of several exemplary glucagon analogs.

[0065] Figure 9B : Schematic diagram of the detection of GLP-1R agonist activity of several exemplary glucagon analogs.

[0066] Figure 9C : Schematic diagram of the detection of GCGR agonist activity of several exemplary glucagon analogs.

[0067] Figure 9D : Schematic diagram of the detection of GCGR agonist activity of several exemplary glucagon analogs.

[0068] Figure 9E : The cAMP content produced by GIPR stimulated by glucagon analogs and controls at different concentration gradients.

[0069] Figure 9F : The cAMP content produced by GIPR stimulated by glucagon analogs and controls at different concentration gradients.

[0070] Figure 9G : The cAMP content produced by GIPR stimulated by glucagon analogs and controls at different concentration gradients.

[0071] Figure 9H : The cAMP content produced by GIPR stimulated by glucagon analogs and controls at different concentration gradients.

[0072] Figure 10 : The results of in vitro cell insulin secretion determination.

[0073] Figure 11A : The blood glucose change curve of IPGTT experiment in normal ICR mice.

[0074] Figure 11B : The blood glucose change curve of IPGTT experiment in normal ICR mice.

[0075] Figure 11C : Comparison results of the area under the blood glucose curve (AUC).

[0076] Figure 12A: A graph showing the relationship between body weight change (%) and time (days) in diet-induced obese (DIO) mice.

[0077] Figure 12B : A graph showing the relationship between body weight change (%) and time (days) in diet-induced obese (DIO) mice.

[0078] Figure 12C : A graph showing the relationship between body weight change (%) and time (days) in diet-induced obese (DIO) mice.

[0079] Figure 12D : Comparison of body weight loss in DIO mice.

[0080] Figure 13 : Comparison of body weight loss in DIO mice.

[0081] Figure 14 : C495 mass spectrum analysis diagram.

[0082] Figure 15 : C382 mass spectrum analysis diagram. DETAILED DESCRIPTION

[0083] Unless otherwise defined below, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0084] Glucagon analogs:

[0085] The glucagon analog provided by the present invention mutates the arginine (R) at position 18 to alanine (A). The mutation of alanine at position 18 reduces the GCGR agonist 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 a large number of combinatorial screenings, the inventors found that by making specific amino acid mutations at some specific sites, such as in combination with specific amino acid mutations at positions 16 and 17, and adding CEX or a similar sequence at the C-terminus, even if position 18 is mutated to A, its GCGR activity is not significantly reduced. More importantly, the A mutation at position 18 significantly enhances the GLP-1 and GIPR agonist activity of the glucagon analog, thereby making the glucagon analog of the present invention an effective trispecific active peptide.

[0086] The trispecific active peptides of the present invention have the ability to stimulate GLP-1R, GCGR, and GIPR, and the activity retention rate of each activity is extremely high compared to GLP-1, GCG, and GIP. Currently, most trispecific active peptides introduce multiple amino acid mutations on the basis of natural polypeptides, and even non-natural amino acids are used to become stable trispecific agonists. During the screening process of the present invention, it was found that the introduction of mutations at multiple sites does make it easier to obtain hybrid polypeptides with higher GLP-1R, GCGR, and GIPR activities. In addition, the introduction of non-natural amino acids also makes it easier to obtain polypeptides with high stability. However, the level of in vitro activity and stability is only a prerequisite for becoming a clinical drug, and safety must also be considered. The introduction of too many mutation sites or non-natural amino acids is likely to bring a higher risk of immunogenicity.

[0087] Serum stability test:

[0088] Natural GLP-1, glucagon or oxyntomodulin cannot be used as clinical drugs due to their poor serum stability and short half-life in vivo. ) can be successfully marketed due to its improved stability. In one embodiment of the present invention, the preferred glucagon analogue exhibits very high stability.

[0089] Immunogenicity experiments:

[0090] In Example 6 of the present invention, the immunogenicity of natural human Glucagon (C001) in rats is extremely low. When the glucagon analogs mutate no more than 3 amino acids relative to natural Glucagon, the antibody titer is less than <1:200, and as the number of mutated amino acids increases, the antibody titer also increases, indicating that the potential immunogenicity risk increases. For drugs to treat metabolic-related diseases, such as drugs in the fields of diabetes and obesity, safety requirements are extremely high. The glucagon analogs obtained by the present invention have lower immunogenicity and meet ideal activity and stability standards when no more than 3 mutation sites are introduced, which has never been reported.

[0091] In vivo animal pharmacodynamic studies:

[0092] In one embodiment of the present invention, preferred glucagon analogs have excellent effects on lowering blood sugar, inhibiting adipose tissue formation, and reducing weight. Although GIP, GLP-1, and Glucagon belong to the same family of incretins, it has not been widely developed as a drug. The reasons for this are, firstly, that some patients with type 2 diabetes lose sensitivity to GIP, and secondly, that GIPR activation can potentially cause obesity in rodents (Miyawaki, K. et al., Inhibition of gastric inhibitory polypeptide signaling prevents obesity. Nat. Med. 8, 738–742, 2002). However, in the embodiments of the present invention, preferred glucagon analogs with higher GIPR agonist activity significantly exhibit more significant weight loss effects.

[0093] In another in vivo animal experiment of the present invention, Example 9, the preferred GCG analogs exhibited similar weight loss effects as the corresponding GCG analogs with the same amino acid sequence and modified with fatty acids.

[0094] the term:

[0095] The term "diabetes" includes type 1 diabetes, type 2 diabetes, gestational diabetes, and other conditions that cause hyperglycemia. The term is used to describe conditions in which glucose accumulates in the blood due to metabolic disorders, such as the pancreas not producing enough insulin or the body's cells not responding appropriately to insulin. As a result, glucose is not absorbed efficiently by tissue cells, leading to a buildup of glucose in the blood.

[0096] Type 1 diabetes, also known as insulin-dependent diabetes and juvenile-onset diabetes, is caused by beta-cell destruction, typically leading to absolute insulin deficiency.

[0097] Type 2 diabetes, also known as non-insulin-dependent diabetes mellitus and adult-onset diabetes, is commonly associated with insulin resistance.

[0098] The term "obesity" refers to an excess of adipose tissue. When energy intake exceeds energy expenditure, excess calories are stored in fat, leading to obesity. Individuals with a body mass index (BMI = weight in kilograms divided by height in meters squared) exceeding 25 are considered obese herein.

[0099] The term "receptor agonist" can be defined as a polypeptide, protein, or other small molecule that binds to a receptor and triggers the usual response of a natural ligand. Incretins are gastrointestinal hormones that regulate blood glucose by enhancing glucose-stimulated insulin secretion (Drucker. DJ, 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-like peptide-1 (GLP-1), glucagon-like peptide-2 (GLP-2), and oxyntomodulin (OXM). GIP is a 42-amino acid mature peptide derived from a 133-amino acid precursor (pre-pro-GIP) by proteolytic processing. These molecules are involved in a variety of biological functions, including glucose homeostasis, insulin secretion, gastric emptying and intestinal growth, and food intake regulation.

[0100] Glucagon-like peptide-1 (GLP-1) is a 30 or 31 amino acid incretin hormone secreted from intestinal L-cells. It has two active forms: GLP-1 (7-36) and GLP-1 (7-37). GLP-1 is released into the circulation after a meal and exerts its biological activity by activating the GLP-1 receptor. GLP-1 has many biological effects, including glucose-dependent insulin secretion, inhibition of glucagon production, delayed 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.). Natural GLP-1 is rapidly degraded by dipeptidyl peptidase-4 (DPP-4), neutral endopeptidase (NEP), plasma kallikrein, or plasmin, thus limiting its therapeutic potential. Since natural GLP-1 has an ultrashort half-life of only about 2 minutes in vivo, methods have emerged to improve efficacy by utilizing chemical modifications and / or formulation forms 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).

[0101] Oxyntomodulin is a small peptide of 37 amino acids that contains the complete 29 amino acid sequence of glucagon. Oxyntomodulin is a dual agonist of GLP-1R and GCGR and 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 with equimolar doses of selective GLP-1R agonists in obese mice. It has been found that compared with selective GLP-1R agonists, oxyntomodulin has an anti-hyperglycemic effect, can significantly reduce body weight and has 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. Oxyntomodulin has also been shown to reduce food intake and increase energy expenditure in humans (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: a 14 randomized controlled trial; Wynne K et al., Int J Obes (Lond), 30: 1729-36, 2006). However, due to its small molecular weight and degradation by DPP-IV, oxyntomodulin has a short half-life. Currently, dual-acting agonists of the GLP-1 receptor (GLP-1R) and glucagon receptor (GCGR) are generally based on oxyntomodulin, and mutations have been made to improve the short-term effect and enzymatic degradation defects of oxyntomodulin (oxyntomodulin analogs). Most of them adopt the method of mutating the second serine Ser to α-aminoisobutyric acid (Aib) or D-Ser, and resist the enzymatic degradation of DPP-IV by introducing non-natural amino acids.Although oxyntomodulin analogs have shown preliminary blood sugar and fat-reducing effects, their mechanism of action is still unclear. The oxyntomodulin receptor has not been discovered. Currently, only GCGR or GLP-1R knockout mouse or cell experiments have been used to verify that oxyntomodulin can bind to these two receptors to exert its effect.

[0102] Glucagon is a 29-amino acid peptide corresponding to amino acids 53-81 of preproglucagon, with a sequence as shown in SEQ ID NO.5 (C.G. Fanelli 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 increasing blood glucose levels under hypoglycemic conditions, regulating hepatic ketone production, regulating bile acid metabolism, and a satiety effect through the vagus nerve by stimulating glycogenolysis and gluconeogenesis. Glucagon has been used in acute hypoglycemia, and glucagon receptor activation reduces food intake and promotes lipolysis and weight loss in animals and humans.

[0103] Glucose-dependent insulinotropic peptide (GIP) is a 42-amino acid polypeptide that is released from intestinal K cells after food ingestion. Its main function is to inhibit gastric acid secretion and enhance glucose-stimulated insulin secretion, hence the name gastric inhibitory peptide / glucose-dependent insulinotropic peptide.

[0104] A "GLP-1 receptor (GLP-1R) agonist" can be defined as a peptide, protein, or other small molecule that binds to the GLP-1R and can trigger the same or similar characteristic reactions as native GLP-1. GLP-1R agonists fully or partially activate the GLP-1R, triggering a series of downstream signaling pathways within the cell, resulting in corresponding cellular activity, such as insulin secretion from beta cells. Typical GLP-1R agonists include native GLP-1 and its mutants and analogs, such as exenatide and liraglutide.

[0105] A "glucagon receptor (GCGR) agonist" can be defined as a peptide, protein, or other small molecule that binds to GCGR and triggers the same or similar characteristic responses as native glucagon. GCGR agonists fully or partially activate GCGR, triggering a series of downstream signaling pathways within the cell, resulting in corresponding cellular activities, such as hepatocyte glycogenolysis, gluconeogenesis, fatty acid oxidation, and ketogenesis.

[0106] GLP-1R / GCGR dual-action agonists: The GLP-1R / GCGR dual-action agonists of the present invention include proteins or polypeptides that can simultaneously excite GLP-1R and GCGR, such as the oxyntomodulin-based dual-action agonists 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 the glucagon-based dual-action agonists reported by Richard D. DiMarchi et al. (US9018164 B2).

[0107] GLP-1R / GCGR / GIPR triple agonist: The GLP-1R / GCGR / GIPR triple agonist of the present invention includes proteins or polypeptides that can simultaneously stimulate GLP-1R, GCGR and GIPR, or are called "tri-specific agonists".

[0108] Trispecific active peptide: The preferred trispecific active peptide in the present invention refers to a polypeptide having GLP-1R / GCGR / GIPR agonist activity, or is called a "triplet active peptide".

[0109] EC 50 Concentration for 50% of maximal effect refers to the concentration of a drug or substance required to stimulate 50% of its corresponding biological response. Lower EC50 values ​​indicate a stronger stimulatory or excitatory ability of the drug or substance. For example, this can be more intuitively demonstrated by a stronger intracellular signal, thereby enhancing the ability to induce the production of a hormone.

[0110] Low-density lipoprotein (LDL): A type of plasma lipoprotein, it is the primary carrier of cholesterol in the blood. It tends to deposit cholesterol on the walls of arteries. White blood cells attempt to digest the LDL, but in the process, they turn it into a toxin. More and more white blood cells are attracted to the affected area, causing inflammation of the arterial wall. Over time, these plaque deposits can accumulate on the arterial wall, making the passage very narrow and inflexible. If too much plaque accumulates, the artery can become completely blocked. When LDL complexed with cholesterol (LDL-C) creates too much plaque on the artery wall, blood cannot flow freely through the artery. The plaque can suddenly collapse in the artery at any time, causing blockage and ultimately leading to heart disease.

[0111] High-density lipoprotein (HDL): helps clear LDL from the arteries and acts as a scavenger, clearing LDL from the arteries and returning it to the liver.

[0112] Triglycerides (TG): Another type of fat, triglycerides are used to store excess energy from the diet. High levels of triglycerides in the blood are associated with atherosclerosis. High triglycerides can be caused by being overweight and obese, physical inactivity, smoking, excessive alcohol consumption, and a high carbohydrate intake (more than 60% of total calories). Sometimes underlying medical conditions or genetic disorders are the cause of high triglycerides. People with high triglycerides often have high total cholesterol levels, including high LDL cholesterol and low HDL cholesterol. Many people with heart disease or diabetes also have high triglyceride levels.

[0113] GPCRs (G Protein-Coupled Receptors) are important proteins in cell signaling, with a topological conformation of seven transmembrane spans. When an extracellular ligand acts on the receptor, the intracellular portion of the receptor binds to the G protein, activating it. G proteins can transmit extracellular information in two ways: by opening transmembrane ion channels to allow the entry of external ions; and by activating second messengers such as cAMP and IP3 / DAG. Calcium ions are generally considered a third messenger downstream of cAMP and IP3 / DAG.

[0114] abbreviation

[0115] COMU: 1-[(1-(cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylaminomorpholinomethylene)]methylammonium hexafluorophosphate

[0116] DCM: dichloromethane

[0117] DMF: N,N-dimethylformamide

[0118] DIPEA: diisopropylethylamine

[0119] EtOH: ethanol

[0120] Et2O: ethyl ether

[0121] HATU: 2-(7-benzotriazole oxide)-N,N,N',N'-tetramethyluronium hexafluorophosphate

[0122] MeCN; acetonitrile

[0123] NMP: N-methylpyrrolidone

[0124] TFA: trifluoroacetic acid

[0125] TIS: Triisopropylsilane

[0126] The following describes the embodiments of the present invention through specific examples. Those skilled in the art will readily understand the other advantages and benefits of the present invention from the disclosure herein. The present invention may also be implemented or applied through various other specific embodiments, and the details in this specification may be modified or altered based on different viewpoints and applications without departing from the spirit of the present invention.

[0127] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing specific embodiments rather than for limiting the scope of protection of the present invention.

[0128] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art. In addition to the specific methods, devices, and materials used in the examples, any prior art methods, devices, and materials similar or equivalent to those in the examples may be used to implement the present invention, based on the knowledge of the prior art by those skilled in the art and the disclosure of this invention.

[0129] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields in the art. These techniques are well described in the literature, for example, by 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; these series METHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATINSTRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS IN ENZYMOLOGY, Vol. 304, Chromatin (PM Wassarman and AP Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol. 119, Chromatin Protocols (PB Becker, ed.) Humana Press, Totowa, 1999, etc.

[0130] Example 1: General Preparation and Purification Methods of Glucagon Analogs

[0131] Using existing technology, 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. Sci., 22: 4-27 (2015)) are used to prepare the polypeptides involved in this patent.

[0132] Specifically, solid phase peptide synthesis can be performed using the standard Fmoc method on a CEM Liberty peptide synthesizer.

[0133] Before use, Rink Amide TentaGel S Ram resin (0.25 mmol / g, 1 g) was swollen in NMP (10 mL) and added to a solid-phase synthesis apparatus. Piperidine / DMF (20%, 10 mL) was added to the resin and allowed to react for 30 minutes to remove the Fmoc protection. The resin was then drained and washed with DMF (5 × 10 mL). The resin was then drained and washed with DMF (5 × 10 mL). The first Fmoc-amino acid solution (0.2 M, NMP / DMF / DCM, 1:1:1, 5 mL) was added, along with COMU / NMP (0.5 M, 2 mL) and DIPEA / DMF (2.0 M; 1 mL). The reaction was allowed to proceed at room temperature for 1 hour. The resin was colorless and transparent after a ninhydrin test. The resin was drained and washed with DMF (5 × 10 mL). Piperidine / DMF (20%, 10 mL) was then added and allowed to react for 30 minutes to remove the Fmoc protection. The resin was drained and washed with DMF (5 × 10 mL). The resin was then drained and washed with DMF (5 × 10 mL). The above steps of adding Fmoc-amino acid for reaction and removing Fmoc protection by piperidine / DMF were repeated until the last histidine was coupled.

[0134] The resin was washed with EtOH (3 × 10 mL) and Et2O (3 × 10 mL) and dried to constant weight at room temperature. The resin was added with TFA / TIS / phenol / EDT / water (82.5 / 5 / 5 / 2.5 / 5, v / v, 40 mL) and reacted in an ice bath for 2 h. The crude peptide was cut from the resin and filtered. This step was repeated three times. The filtrates were combined, most of the TFA was removed under reduced pressure, precipitated with ether, centrifuged, and the precipitate was washed three times with ether and dried to constant weight at room temperature to obtain the crude peptide. Using a Varian SD-1 preparative liquid chromatograph equipped with a C-18 column and a fraction collector, the crude peptide was purified by preparative reverse phase HPLC to a purity greater than 97% using a gradient elution of mobile phase A (0.1% TFA, aqueous solution) and mobile phase B (0.1% TFA, 90% MeCN, aqueous solution). The resulting peptides are listed in Table 1. Among them, the peptide with C-terminal amide termination was synthesized using the above method; the remaining peptides were synthesized using Wangle Resin (0.4mmol / g, 1g) for solid phase synthesis. After the resin was swollen, Fmoc-amino acid was directly added for coupling reaction, Fmoc protection was removed, peptide was cleaved, and purified. The operation steps were the same as the synthesis steps of C-terminal amide termination peptide. The synthesis and purification of peptides with fatty acid modification are conventional techniques and can be found in the articles of 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).

[0135] The purified peptides were analyzed by LC / MS, and the analysis results are shown in Table 2. Figure 14 、 15 The mass spectrometry analysis diagrams of glucagon analogs numbered C495 and C382 are exemplary, respectively. The mass spectrometry conditions are as follows:

[0136] Instrument: Waters ZQ 2000

[0137] Mass spectrometry (Probe): ESI

[0138] Nebulizer Gas Flow: 1.5L / min

[0139] CDL: -20.0v

[0140] CDL temperature: 250℃

[0141] Heating block temperature (Block Temp): 200°C

[0142] Mass spectrometer voltage (Probe Bias): +4.5kV

[0143] Detector: 1.5kv

[0144] Mobile phase flow rate (T.Flow): 0.2ml / min

[0145] Buffer concentration (B.Conc.): 50% H2O / 50% ACN

[0146] Table 1

[0147]

[0148]

[0149]

[0150] Note: X in the table represents aminoisobutyric acid, and K' indicates that this position is a lysine residue and is covalently linked to a fatty acid. The fatty acid structure is shown in Formula I below:

[0151]

[0152] Table 2

[0153]

[0154]

[0155] Example 2: Stability Study

[0156] The purpose of this example is to study the chemical stability of various glucagon analogs prepared in Example 1 in aqueous solution.

[0157] The peptide to be tested (glucagon analog) and a control were prepared in 20 mM phosphate buffer PB or acetate buffer at the corresponding pH to a final peptide concentration of 0.2 mg / ml and sterilized by filtration using a sterile filter (0.22 μm, Millipore SLGP033RB). The prepared peptide solution was incubated at 40°C for 7 days. The supernatant was then centrifuged at 4500 rpm for 20 minutes and analyzed using RP-HPLC-UV (t7). The amount of residual intact peptide was determined, and a sample that had not been incubated (t0) was analyzed in parallel. The peak areas of the target compound at t0 and t7 were compared to obtain the "% residual peptide" according to the following equation:

[0158] Residual peptide content %=[(peptide peak area t7)×100] / peptide peak area t0.

[0159] Stability was expressed as "residual peptide content".

[0160] Detection method

[0161] Detection wavelength: 214nm;

[0162] Chromatographic column: column temperature 40 °C, Phenomenex Luna C8(2) 5 μm (150 × 4.6 mm);

[0163] Mobile phase: H2O + 0.1% TFA: ACN + 0.1% TFA (flow rate 1.0 ml / min);

[0164] Gradient: 95:5 (0 min) to 0:100 (30 min);

[0165] Analysis of experimental results: From the experimental data in 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.

[0166] Table 3

[0167]

[0168]

[0169] Figure 1-7 Liquid HPLC analysis spectra of several glucagon analogs such as C381 are shown as examples.

[0170] Figure 1 Corresponding integral data:

[0171]

[0172] Figure 2 Corresponding integral data:

[0173]

[0174] Figure 3 The corresponding original data:

[0175]

[0176] Figure 4 The corresponding original data:

[0177]

[0178] Figure 5 The corresponding original data:

[0179]

[0180] Figure 6 The corresponding original data:

[0181]

[0182] Figure 7 The corresponding original data:

[0183]

[0184] Example 3: Serum stability

[0185] (1) The corresponding polypeptides in Table 1 were prepared into a 1.0 mg / ml solution using 5 mM Tris-HCl, pH 8.5, 0.02% TWEEN80 solution, sterilized and filtered (0.22 μm, Millipore SLGP033RB), diluted 10-fold with rat serum, mixed, and dispensed into sterile centrifuge tubes;

[0186] (2) Take three tubes of each of the above samples and freeze them at -20°C as a control. The remaining samples should be placed in a 37°C incubator and sampled at different time points for activity testing.

[0187] (3) The method shown in Example 4 was used to detect the GCGR agonist activity of the polypeptide.

[0188] Relative activity: The activity value at 0 hour is 100%, and the values ​​measured at subsequent time points are compared with it. Figure 8A and 8B Serum stability can be concluded.

[0189] Table 4

[0190]

[0191]

[0192] Note: ND means below the detection limit

[0193] Example 4: Cell activity assay

[0194] (1) GLP-1R agonist activity assay:

[0195] GLP-1R agonist activity was detected using a luciferase reporter gene 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 to construct the recombinant expression plasmid pCDNA3.1-GLP-1R. Simultaneously, the full-length luciferase gene was cloned into the pCRE plasmid to generate the pCRE-Luc recombinant plasmid. The pcDNA3.1-GLP-1R and pCRE-Luc plasmids were transfected into CHO cells at a molar ratio of 1:10, and stable expression strains were screened.

[0196] Cells were cultured in a 9-cm cell culture dish using DMEM / F12 medium containing 10% FBS and 300 μg / ml G418. When the confluence reached about 90%, the supernatant was discarded and 2 ml of trypsin was added for 3 min. Then, 2 ml of DMEM / F12 medium containing 10% FBS and 300 μg / ml G418 was added to neutralize the cells. The cells were transferred to a 15 ml centrifuge tube and centrifuged at 1000 rpm for 5 min. The supernatant was discarded and 2 ml of DMEM / F12 medium containing 10% FBS and 300 μg / ml G418 was added to resuspend the cells and count them. The cells were diluted to 1×10 with DMEM / F12 medium containing 10% FBS. 5 / ml, 100 μl was plated in each well of a 96-well plate, i.e. 1×10 4 After cells adhered, the culture medium was changed to DMEM / F12 medium supplemented with 0.2% FBS. After discarding the supernatant from cells plated in 96-well plates, the purified recombinant protein was diluted to a series of specified concentrations in DMEM / F12 medium supplemented with 0.1% FBS and added to the cell culture wells at 100 μl / well. Detection was performed according to the instructions of the Luciferase reporter kit (Ray Biotech, Cat: 68-LuciR-S200). Figure 9A 、 9B This is the result of GLP-1R agonist activity detection.

[0197] (II) GCGR agonist activity detection method:

[0198] GCGR agonist activity was also detected using a luciferase reporter gene assay. The human GCGR gene was cloned into the mammalian cell expression plasmid pcDNA3.1 to construct the recombinant expression plasmid pCDNA3.1-GCGR. This plasmid was transfected into HEK 293T cells and the stable cell lines were screened and constructed as described above. Figure 9C 、 9D This is the result of GCGR agonist activity detection.

[0199] Table 5

[0200]

[0201]

[0202] (III) GIPR agonist activity detection method:

[0203] CHO cells were transfected with the pcDNA3.1-GIPR plasmid and positive stable transfected cell lines were screened. Approximately 200,000 cells / well were seeded in a 96-well cell culture plate and cultured overnight. After washing with Hanks' balanced salt solution, the test protein was diluted to a series of specified concentrations and added to the cells together with 200μM 3-isobutyl-1-methylxanthine (IBMX). After incubation at 37°C for 20 minutes, the culture supernatant was discarded, and the cells were lysed with lysis buffer. The cAMP content was measured using the cAMP Parameter assay kit (R&D Company, USA, Catalog No.: SKGE002B) according to the instructions. The results are shown in Figure 2. Figure 9E -H as shown.

[0204] Example 5: Glucose-stimulated insulin secretion experiment

[0205] This example refers to 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), using rat BRIN-BD11 cells to measure insulin release induced by active protein stimulation, but with slight modifications, namely, 1.0×10 6 cells were added to each well of a 24-well plate (Orange Scientific, Brainel'Alleud, Belgium), cultured overnight at 37° C., centrifuged, and the supernatant was removed. Then, 1.0 ml of KRB (115 mM NaCl, 4.7 mM KCl, 1.28 mM CaCl 2 , 1.2 mM MgSO 4 , 1.2 mM KH 2 PO 4 , 25 mM HEPES, 10 mM NaHCO3, NaOH adjusted to pH 7.4), 0.1% (wt / vol.) BSA, and 1.1 mM glucose. After incubating the cells at 37°C for 40 minutes, the supernatant was removed by centrifugation and replaced with 1.0 ml of fresh KRB solution and a gradient of active protein concentrations. After incubation at 37°C for 20 minutes, the buffer was removed by centrifugation and the cells were stored at -20°C overnight. Immunoradiometric analysis of insulin content was performed. The results are shown in Figure 2. Figure 10 shown.

[0206] Example 6: Mouse immunogenicity experiment

[0207] 7-week-old Balb / c mice, 6 mice in each group, were collected from the tail vein of each mouse before administration to obtain 50ul of serum as a blank control. The corresponding glucagon analog (30nmol / kg, in PBS buffer) was injected daily for 28 consecutive days. On the 45th day, blood was collected from the orbital cavity, and the serum was coagulated and separated. The antibody titer was determined by direct ELISA. The corresponding polypeptide was coated on the enzyme-labeled plate, and the mouse serum was diluted in a gradient of 1:50; 1:200, 1:1000, and 1:5000 and added to the enzyme-labeled plate. The goat anti-mouse secondary antibody was used as the detection antibody. The serum of each mouse before administration was a negative control. Under the same dilution, the OD value of the test sample was 0.05. 450 The average value was greater than the negative control serum OD 450The result that is 2.1 times the average value of the values ​​is judged as positive (+), otherwise it is judged as negative (-). The highest dilution with a positive result is the antibody titer.

[0208] Table 6

[0209]

[0210] Example 7: Glucose tolerance test (IPGTT) in normal ICR mice

[0211] Normal ICR mice were divided into 27 groups, 6 mice per group. They were fasted overnight, blood was collected from the tail (recorded as the blood glucose sample at t = 0 minutes), and the vehicle control (acetate buffer, 20 mM acetic acid, 250 mM mannitol, pH 5.0) and the glucagon analogs listed in Table 1 of the present invention (30 nmol / kg, in PBS buffer) were injected subcutaneously. Product Name 40 nmol / kg, diluted in PBS buffer). Glucose (2 g / kg body weight) was injected intraperitoneally 15 minutes later, and blood glucose levels were measured at t = 30 minutes, t = 45 minutes, t = 60 minutes, and t = 120 minutes. The animals were fasted during the experiment to prevent interference with food intake. For detailed results, see Figure 11A -C.

[0212] Example 8: Weight loss experiment in diet-induced obese (DIO) mice

[0213] Preparation of the DIO mouse model: Male C57BL / 6J mice, approximately 7 weeks old, were fed a high-fat diet (60% kcal from fat) for approximately 16 weeks (a total of 23 weeks). Experiments were performed when the mice weighed approximately 45 g. The DIO mice were randomly divided into groups of 6 mice each, with no difference in baseline body weight. Each group was subcutaneously injected daily with either the respective glucagon analog (30 nmol / kg in PBS) or an equal volume of PBS. The control group received liraglutide (trade name 30 nmol / kg), administered twice a day, and the mice were weighed every day for 30 days.

[0214] Figure 12A -C is the daily changes in body weight of DIO mice after administration of each glucagon analog, and the final percentage of body weight loss is shown in Figure 12D .

[0215] Example 9: Weight loss experiment in diet-induced obese (DIO) mice

[0216] DIO mice were randomly divided into groups of 6 mice each. There was no difference in basal body weight. Each group of mice was subcutaneously injected with each GCG analog (30 nmol / kg, in PBS) or PBS every day. The control group was given liraglutide (trade name The mice were administered with a GCG analog (30 nmol / kg, diluted in PBS) twice a day and a fatty acid-treated GCG analog (30 nmol / kg, diluted in PBS) once a day, and the mice were weighed daily for 30 days.

[0217] Figure 13 The final percentage of body weight loss in DIO mice after administration of each glucagon analog is shown in the figure. GCG analogs C381, C464, and C493, shown in the figure, exhibit similar weight-reducing effects as their fatty acid-modified counterparts with identical amino acid sequences, whereas C225 and C163, which are not fatty acid-modified, exhibit no significant weight-reducing effect.

[0218] In summary, the present invention effectively overcomes various shortcomings of the prior art and obtains a group of triple-action agonists with potential clinical value.

[0219] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the present invention. Anyone skilled in the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by one of ordinary skill in the art without departing from the spirit and technical principles disclosed herein are intended to be covered by the claims of the present invention.

Claims

1. A glucagon analog, wherein the glucagon analog has a sequence as shown in any one of the following: HSQGTFTSDYSKYLDEEAAQDFVQWLMNGPSSGAPPS-NH2; HSQGTFTSDYSKYLDEEAAQDFVQWLMNGPSSGAPPS-OH; HSQGTFTSDYSKYLDEQAAQDFVQWLMNGPSSGAPPPS-NH2; HSQGTFTSDYSKYLDEQAAQDFVQWLMNGPSSGAPPPS-OH.

2. An isolated polynucleotide encoding the glucagon analog of claim 1.

3. A recombinant expression vector comprising the isolated polynucleotide according to claim 2.

4. A host cell, comprising the recombinant expression vector according to claim 3 or an exogenous polynucleotide isolated according to claim 2 integrated into its genome.

5. The method for preparing a glucagon analogue according to claim 1, wherein: Choose from any of the following: (1) synthesizing the glucagon analog by a chemical synthesis method; (2) Cultivating the host cell according to claim 4 under appropriate conditions to express the glucagon analog, and then isolating and purifying the glucagon analog to obtain the glucagon analog.

6. A composition comprising the glucagon analog according to claim 1 or the host cell culture according to claim 4, and a pharmaceutically acceptable carrier.

7. A fusion protein, which consists of the glucagon analogue according to claim 1 and a long-acting unit, wherein the long-acting unit is selected from albumin, transferrin and immunoglobulin.

8. A modified polypeptide, comprising the glucagon analogue according to claim 1 in its structure, wherein the glucagon analogue is modified with fatty acid or polyethylene glycol.

9. Use of the glucagon analogue according to claim 1, the fusion protein according to claim 7, or the modified polypeptide according to claim 8 in the preparation of a medicament for treating a metabolic-related disease, wherein the metabolic-related disease is selected from at least one of type 2 diabetes and obesity.

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