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CN109836488B - Glucagon analogues for treating metabolic diseases - Google Patents

Glucagon analogues for treating metabolic diseases Download PDF

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CN109836488B
CN109836488B CN201711194175.0A CN201711194175A CN109836488B CN 109836488 B CN109836488 B CN 109836488B CN 201711194175 A CN201711194175 A CN 201711194175A CN 109836488 B CN109836488 B CN 109836488B
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glucagon
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CN109836488A (en
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黄岩山
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Zhejiang Doer Biologics Co Ltd
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Zhejiang Doer Biologics Co Ltd
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Abstract

The invention relates to the field of biological medicines, in particular to a glucagon analogue for treating metabolic diseases, which has a structural formula as follows: H-X 2 ‑X 3 ‑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 ‑NH 2 . The glucagon analogues of the invention have GLP-1/GCG/GIP triple receptor agonistic activity and better enzyme-resistant stability, comprise Neutral Endopeptidase (NEP) and dipeptidyl peptidase-4 (DPP-4), and have longer in vivo half-life and sustained action time compared with the natural glucagon, GLP-1 and GIP.

Description

Glucagon analogues for treating metabolic diseases
Technical Field
The invention relates to the field of biological medicines, in particular to a glucagon analogue for treating metabolic diseases and a preparation method and application thereof.
Background
Diabetes is a serious chronic disease that occurs when the pancreas does not produce enough insulin or the body cannot use the produced insulin effectively. Currently marketed proteinic diabetic drugs are mainly GLP-1 receptor (GLP-1R) agonists, such as dolaglutide (trade name:
Figure BDA0001481631680000014
) Albaglutide (trade name)
Figure BDA0001481631680000011
) Liraglutide (trade name)
Figure BDA0001481631680000012
And
Figure BDA0001481631680000013
respectively for treating obesity and diabetes), Exenatide (trade name)
Figure BDA0001481631680000015
) Lixisenatide (trade name)
Figure BDA0001481631680000016
) And Semaglutide (Semaglutide) which may be about to be marketed. Dolaglutide, albiglutide, liraglutide and somaglutide are all analogs of natural glucagon-like peptide-1(GLP-1), and are fused or crosslinked with FC fragment of IgG, human albumin and fatty acid respectively after GLP-1 sequence mutation to obtain the GLP-1R agonist with high and stable activity. Exenatide (Exendin-4) is derived from lizard (Helode)rma supectum) salivary gland 39 amino acid small peptide. Exendin-4 is a potent agonist of GLP-1R, but the activity is higher than that of native GLP-1 and GLP-1 analogues. These GLP-1R agonists, while effective in lowering blood glucose and controlling appetite, are not as significant as weight loss effects. Wherein liraglutide (trade name)
Figure BDA0001481631680000017
) Although approved for the treatment of obesity, in practice, it has only a weight loss of approximately 5.6 kg. The Weight Loss of current drugs for obesity is typically around 5-10% (compared to placebo), i.e. the average Weight Loss as a whole does not exceed 10% of the patient's body Weight (Rudolph L.Leibel et al, biological Responses to Weight Loss and Weight gain: Report From an American Diabetes Association Research Symposium, Diabetes, 64(7):2299 + 2309, 2015).
Bariatric surgery (Bariatric surgery) can significantly improve Obesity and treat Diabetes, however, its use is not widespread, as most patients are not willing to undergo surgery due to the risk of surgery and long-term sequelae (obesitiy and Diabetes, New Surgical and non-Surgical applications, Springer press 2015). It has been found that the secretion of incretins (Incretin) increases in patients undergoing Surgical bariatric surgery (obesitiy and Diabetes, New Surgical and Nonsurgical applications, Springer Press 2015). Preclinical and clinical studies also found that simultaneous infusion of GLP-1/Glucagon (GCG) (Tricia M. Tan etc., DIABETES, VOL 62: 1131. cndot. 1138,2013) or GLP-1, Oxyntomodulin (OXYNTOModulin, OXM) and PYY in patients had significant effects on promoting energy metabolism, suppressing appetite, and controlling weight (Tricia Tan etc., J Clin endothelial Metab,2017,102(7): 2364. cndot. 2372). From the clinical point of view, these polypeptides can be simply mixed directly for clinical use. However, due to the differences in vivo stability and degradation rate of these different kinds of polypeptides, the final in vivo efficacy is not controllable, and it is difficult to simply mix these polypeptides to use them as a compound drug. Therefore, the current development of new generation diabetes drugs is mainly to try to focus these agonist activities on one molecule, such as GLP-1R/GIPR and GLP-1R/GCGR dual-effect agonists, even GLP-1R/GIPR/GCGR triple-effect agonists (Chakrahard, Shradda. all in one: research yield drugs for diabetes and obesity. Nature Medicine,22(7):694-695, 2016).
At present, the design and development of the medicine mainly have the following modes: 1. based on the modification development of human endogenous polypeptide Oxyntomodulin (OXYNTMODULIN, OXM) with GLP-1/GCG dual activity. OXM is a polypeptide naturally having dual activities of GLP-1 and GCG in humans (Diabetes, 2005, 54: 2390-2395). However, OXM is not highly active (having about 10% GCG activity and about 1% GLP-1 activity) and has poor in vivo stability and half-life, so OXM cannot be directly used clinically, and it is often necessary to improve its in vivo activity and stability by introducing unnatural amino acids, various modifications, and the like. For example, Mod-6030 from OPKO Biologics is long-acting OXM (Oren Hershkovitz: Presentation Number: SAT-787. The Endocrine Society's 95th annular Meeting and Expo, June 15-18,2013-San Francisco) with degradable PEG modification at The N-terminal. TT401(LY2944876) is another PEG-modified OXM analog (Chakradhar, Shraddha. "All in one: research creation drugs for diabetes and ease." 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, OXM is limited in its low activity and stability, and its clinical effect is not good, and most studies have been abandoned. 2. Utilizes the homology of incretin (incrustin) sequence to obtain stable hybrid peptide (Matthias H) with multiple activities by multiple mutation, modification and even introduction of unnatural amino acids on the basis of structures such as OXM, GLP-1, GCG and the like.
Figure BDA0001481631680000021
Etc., molecular weights for Treatment of Diabetes and obesitiy, 24: 51-62,2016). Matthias H.
Figure BDA0001481631680000022
Etc. the various hybrid peptide forms currently in clinical or preclinical use are described in detail. As reported by Alessandro Pocai et al, an OXM-based Dual-effect GLP-1R/GCGR agonist (glucicon-Like Peptide 1/glucicon Receptor Dual Agonism reverts in Rice, Diabetes; 58(10):2258-2266, 2009), or a GCG-based Dual-effect GLP-1R/GCGR agonist (US9018164B2) reported by Richard D, DiMarchi et al, or even a triple-effect GLP-1R/GCG/GIPR agonist (US 35355091632). These multispecific hybrid peptides are mostly based on GLP-1 or GCG, and have improved activity and resistance to proteolysis by sequence mutation, such as mutation of an L-type amino acid to a D-type amino acid (e.g., D-Ser), or introduction of an unnatural amino acid Aib to improve in vivo stability, while fatty acid chain or polyethylene glycol (PEG) modification is performed to increase half-life, with clinically expected dosing cycles of once a day (fatty acid modification) or once a week (PEG modification). Furthermore, Aisling M.Lynch et al reported that the second Ser position of natural GCG was mutated to D-Ser, and a GCG analog of the C-terminal peptide of Exendin-4 was introduced at the C-terminus (D-Ser2-glucagon-exe), and pharmacodynamic experiments were performed in DIO, with twice daily administration of Novel DPP IV-resistant C-terminal extended glucose and analog peptides in high-fat-fed micro-processed glucose receptors and GLP-1 activator, Diabetologia: 57: 1927-.
Although research and development of molecules with multiple GLP-1R, GCGR and GIPR agonist activities are very promising in clinic, the actual acquisition of an ideal drug of the type is very difficult.
The first is the problem of safety, in particular immunogenicity. The hypoglycemic slimming medicine needs to be used for a long time and has extremely high requirement on safety. In order to design and obtain a polypeptide with GLP-1, GCG and GIP high activities and stability in vivo, the prior technical schemes often introduce more mutation sites, and often introduce unnatural amino acids and other modifications. Both these mutations, as well as the introduction of unnatural amino acids, increase the risk of potential immunogenicity. Generally, the higher the homology to the human sequence, the lower the relative risk of immunogenicity in humans. The GLP-1 Receptor Agonist hypoglycemic agent Taspoglutide (only 2 unnatural amino acids Aib are introduced), which is developed by Roche and Yipu in a combined way, has The antibody production rate reaching 49 percent, and has suspended all clinical stage III researches (Juuli ROSENSTOCK, etc., The face of Taspoglutide, a Weekly GLP-1 Receptor Agonist, Versus with-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 on THE basis of GCG sequence to obtain a candidate peptide MEDI 0382. Wherein, in order to maintain higher dual activity and in vivo stability of GLP-1 and GCG, compared with GCG, MEDI0382 introduces 9 mutation sites, and the mutation rate reaches about 30%; similarly, Andrea Evers et al (J Med chem.2017May 25; 60(10): 4293-; the GLP-1/GCG/GIP three active peptide designed by Brian Finan et al (Brian Finan et al, Nat Med.21:27-36, 2015) adds GPSSGAPPPS sequence at the C terminal of GCG and introduces 7 mutant amino acids, including the second position mutated into the unnatural amino acid Aib. Therefore, the existing technical scheme often introduces more mutation sites, and often introduces unnatural amino acids and other modifications to obtain the polypeptide with the high activities of GLP-1, GCG and GIP. These mutations, modifications, and introduction of unnatural amino acids all increase the risk of potential immunogenicity. The safety of the medicine for treating diabetes, obesity and other diseases is extremely important. Therefore, it would be of great interest to develop a highly active pleiotropic GLP-1/GCG multi-agonist that contains no unnatural amino acids and contains as few mutated amino acids as possible.
On the other hand, there is no general discussion of how to combine the activities of these incretins and the appropriate ratio between the activities. For example, PCT applications WO2015155139A1, WO2015155140A1 and WO201515541A1 disclose GLP-1R/GCGR double-effect agonistic peptides or GLP-1R/GCG/GIPR triple-effect agonistic peptides modified on the basis of Exendin-4. WO201515541A1 discloses a hybrid peptide with GLP-1R/GCGR/GIPR triple-effect agonistic activity, and researches prove that the peptide with triple-effect agonistic activity has good effects of reducing blood sugar and losing weight; however, WO2015155139A1 and WO2015155140A1 are to avoid the risk of hypoglycemia caused by GIPR (glucagon receptor agonistic activity), and the GLP-1R/GCGR double-effect agonistic peptide is prepared, and has better effects of reducing blood sugar and losing weight. Seth et al also believe that the effect of GLP-1 on glycemic control is not enhanced by the introduction of GIP activity (A. Seth et al, Co-administration of a qualified GIPR agonist with a GLP-1 expression no administration of HbA1 c% over GLP 1analog in db/db micro, EASD virtual meeting 2015).
Thirdly, for small peptides with the peptide chain length of only about 30-40 amino acids, such as GLP-1, Exendin-4 and GCG with highly homologous sequences, receptors belong to the GPCR family, the activity change of different receptors after single-site mutation or simultaneous mutation of a plurality of sites is very difficult to predict, so that the ideal hybrid peptide with multiple agonist activities is extremely difficult to obtain. For example, Joseph channel et al report (Joseph channel et al, A glucagon analog chemistry stabilized for organizing the Molecular species, Molecular Metabolism, 3: 293 and 300, 2014) that the relative residual activity retention span ranges from 0.2% to 100% after alanine scanning (Ala scan) of GCG, and that the 1 st, 2 nd, 3 rd, 4 th, 6 th to 12 th, 14 th, 15 th, 22 th, 23 th, 25 th to 27 th, and 29 th mutations of GCG can greatly reduce the GCGR agonistic activity (Table 4 in the article). However, in other reports, it can be seen that a single or several of the above-mentioned sites are mutated at the same time, and when other amino acids are substituted, the activity change does not always coincide 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-aginst eliminates obesities in rodents, Nature Chemical Biology, 5: 749-K757, 2009), GCG was subjected to different mutations at position 16, such as 16S → G, 16S → T, 16S → H, 16S → E, and GCGR agonistic activity was rather enhanced, which is in complete contradiction to the alanine scanning results of Joseph Chanbene. Second, the Joseph channenne study suggested that substitution with alanine at position 23 would result in almost complete loss of GCGR agonist activity (retention of only 1.1%); however, Jonathan W Day et al mutated 23 to Ile did not show a decrease in GCG activity. For example, the second position S is considered to be very important for maintaining GCG activity (the activity is only 1/3 when the mutation is Ala) as shown by alanine scanning results, but Brian Finan et al (Finan B et al, A ratio assigned monomeric peptide mutation mutants obtained from B and D in rodents. Nat. Med. 2015; 21:27-36.) report that 2S → Aib, 2S → dSer, 2S → G, 2S → dAla substitution mutations are respectively carried out on the second position amino acid of GCG, and the relative agonistic activity of GCGR is improved to 200% -640% after the mutation is combined with the mutation at other positions. It has also been found in our studies that the incorporation of a combination of mutations that is beneficial in increasing GLP-1, GCG or GIP activity is often completely inconsistent with single site mutations. In addition, polypeptides such as GLP-1, Exendin-4, GCG or GIP, in which amino acids are added or reduced at the N-and C-termini, affect their biological activities. If one or two amino acids are removed from the N-terminus, the agonistic activity of GLP-1, GCG, etc. is completely lost. For example, Oxyntomodulin has only 8 amino acids more than the C-terminus of Gluconon, and thus has about 90% of its GCGR agonistic activity lost (Alessandro Pocai et al, Gluconon-Like Peptide 1/Gluconon Receptor Dual agonist peptides in Mice, Diabetes; 58(10):2258-2266, 2009; Henderson SJ et al, Robust-object and metallic effects of a product GLP-1/Gluconon Receptor Peptide in mutants and non-human matrices, Diabetes antigens Metab, 2016).
As also reported by Joseph R.Channne and Richard D.DiMarchi et al, the addition of a small C-terminal peptide cex (SEQ ID NO.67, GPSSGAPPPS) of Exendin-4 to the C-terminus of Glucagon increased GLP-1R agonist activity from 0.7% to 1.6%, by about 2-fold (Optimization of the Native Glucagon Sequence for Medicinal peptides, J Diabetes Sci Techol.4 (6): Fr. 1331, 2010 and patent US9018164B2), and also lost about 50% of GCG activity. Evers A et al also reported (Evers A, Design of Novel Exendin-Based Dual Glucagon-like Peptide-1(GLP-1)/Glucagon Receptor peptides, J Med chem.; 60(10): 4293-4303.2017) that GLP-1R agonistic activity decreased by almost 2/3 but GCG agonistic activity was lost by more than 90% (Table 2, peptides 7and 8 in the article) after cex sequence was added to the C-terminal of GCG analogue. Thus, for small peptides of 30 amino acids in length, such as GLP-1, Glucagon, changes in sequence are extremely sensitive to changes in their activity; in the case of dual active polypeptides, however, the changes are more complex due to the involvement of agonism at two different receptors, and it is not at all predictable what consequences of GLP-1R and GCGR agonistic activity would be after any amino acid change.
The complexity of downstream signaling of GPCR receptors, such as GCGR and GLP-1R, also increases the difficulty of designing an ideal multi-active hybrid peptide. The receptors of GCGR and GLP-1R have multiple signal transduction channels in cells, downstream signal factors such as G proteins (G alpha s, G alpha i, G alpha q and the like) and arrestin (beta-arrestin-1 and beta-arrestin-2) form multiple different signal transduction channels, the physiological effects of the activation of the different channels are different, and even the relation between the activity and the physiological function of some channels is unknown. For example, different mutations or different amino acid sequences can be introduced into the GLP-1 sequence to obtain structures with different preferential agonism (biased-agonist) and thus different physiological effects (Marlies V. et al, J Am Chem Soc., 138(45): 14970-.
Thus, although it is theoretically very clinically significant to design a polypeptide having high GLP-1, Glucagon and GIP activities at the same time, it is actually very difficult. It would be of great clinical significance if a multi-active hybrid peptide could be designed with balanced activity but with as few introduction of mutated sites and unnatural amino acids as possible, as close to the natural sequence as possible, resulting in lower potential immunogenicity, improved stability, and good glycemic and weight control.
Disclosure of Invention
In view of the above-mentioned drawbacks of the prior art, it is an object of the present invention to provide a glucagon analogue exhibiting GLP-1R/GCGR/GIPR triple receptor agonistic activity.
Native GCG has approximately 1% GLP-1R agonistic activity relative to native GLP-1, but no GIPR agonistic activity. The glucagon analogues of the invention can show GLP-1R/GCGR/GIPR triple receptor agonistic activity.
In order to achieve the above objects and other related objects, a first aspect of the present invention provides a glucagon analog (GCG analog), wherein the structure of the glucagon analog comprises a structure represented by formula I or formula II, and the structure represented by formula I is:
HSQGTFTSD-X 10 -SKYLD-X 16 -X 17 -AA-X 20 -X 21 -F-X 23 -QWLMN-X 29 -X z (SEQ ID NO.1), the structure shown in formula II is:
HSQGTFTSD-X 10 -SKYLD-X 16 -X 17 -AA-X 20 -X 21 -F-X 23 -QWLMN-X 29 -X z -NH 2 (SEQ ID NO.2) wherein X 10 Selected from any one of Y, K or L, X 16 Selected from any one of S, E or A, X 17 Selected from any one of Q, E, A or R, X 20 Selected from any one of 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 Is T or deleted, X z Selected from any one of 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).
Further, when the glucagon analog has the structural formula:
HSQGTFTSD-X 10 -SKYLD-X 16 -X 17 -AA-X 20 -X 21 -F-X 23 -QWLMN-X 29 -X z -NH 2 (SEQ ID NO.2) means that the C-terminal of the glucagon analogue is amidated and modified.
As set forth in some embodiments of the invention, the amino acid sequence of the glucagon analogues of the invention is as set forth 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 Selected from any one of S or E, X 17 Selected from any one of Q or E, X 29 Is T or deleted, X z Selected from any one of 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).
Further, when the structural formula of the glucagon analog is:
HSQGTFTSD-Y-SKYLD-X 16 -X 17 -AAQDFVQWLMN-X 29 -X z -NH 2 (SEQ ID NO.4) means that the C-terminal of the glucagon analogue is amidated and modified.
The glucagon analogs described above have GCGR agonist activity similar to or superior to native glucagon, and GLP-1R agonist activity similar to or superior to native GLP-1, and additionally increased GIPR agonist activity.
In one embodiment of the invention, the preferred glucagon analog is to add GPSSGAPPPS to the C terminal of the natural glucagon, only 2-3 amino acids are mutated at least, no unnatural amino acid is introduced, no modification is needed, GLP-1R and GCGR agonistic activity can be retained or improved, and the additional GIPR agonistic activity is increased, and the product has good stability. Less mutation sites, no subsequent modification, natural structure maintenance as much as possible and potential immunogenicity risk reduction.
High levels of GIP have been reported in the literature to cause frequent hypoglycemic symptoms in the treatment of diabetes (T McLaughlin et al, J Clin endocrine Metab,95, 1851-. However, in a mouse animal model test, the glucagon analogue with the GIPR (glucokinase) agonistic activity provided by the invention can smoothly control blood sugar and has no hypoglycemic symptom. It has also been reported that antagonism of the GIPR is also a desirable approach for reducing daily food intake, weight loss, increasing insulin sensitivity and energy expenditure (Irwin et al, Diabetologia 2007,50, 1532-. In a mouse animal model test, the glucagon analogue with higher GIP activity provided by the invention has more obvious effects on daily feeding control, weight loss and insulin sensitivity improvement of obese mice compared with a comparative analogue with lower GIP activity or even without GIP activity.
The glucagon analogues of the invention have better enzyme-resistant stability, and comprise Neutral Endopeptidase (NEP) and dipeptidyl peptidase-4 (DPP-4); has longer half-life and duration of action in vivo compared with native glucagon, GLP-1, GIP.
In a second aspect of the invention, there is provided an isolated polynucleotide encoding a glucagon analogue as hereinbefore described.
In a third aspect of the invention, there is provided a recombinant expression vector comprising the aforementioned isolated polynucleotide.
In a fourth aspect of the invention, there is provided a host cell comprising the recombinant expression vector or the isolated polynucleotide having an exogenous sequence integrated into its genome.
In a fifth aspect of the present invention, there is provided a method for preparing the glucagon analogue, selected from any one of the following:
(1) synthesizing the glucagon analog by a chemical synthesis method;
(2) culturing the aforementioned host cell under suitable conditions to allow expression of the glucagon analog, and isolating and purifying to obtain the glucagon analog.
In particular, the glucagon analogs of the present invention can be prepared by standard peptide synthesis methods, e.g., by standard solid 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. The glucagon analogs of the present invention can be synthesized, preferably, by solid-phase or liquid-phase peptide synthesis methods.
In a sixth aspect of the invention there is provided the use of a glucagon analogue as hereinbefore defined in the manufacture of a medicament for the treatment of a metabolic-related disorder.
The glucagon analogs provided herein can be used to treat diabetes-related metabolic syndrome, such as dyslipidemia, including hypertriglyceridemia, low HDL cholesterol, and high LDL cholesterol; insulin resistance or glucose intolerance, etc.
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, as atherosclerotic cardiovascular disease (ASCVD). Patients with metabolic syndrome may progress from an insulin resistant state in the early stages to fully mature type ii 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 disorder, decreased availability of insulin resistance associated with increased oxidative stress, and abnormalities in adipocyte-derived hormones, such as adiponectin (Lteif, Mather, can.j. cardio.20 (suppl B): 66B-76B, 2004).
The glucagon analogs of the invention are also useful for treating obesity. In some aspects, the glucagon analogs of the present invention treat obesity by mechanisms that reduce appetite, reduce food intake, reduce fat levels in the body of a patient, increase energy expenditure, and the like.
In some potential embodiments, the glucagon analogs of the present invention are useful for treating non-alcoholic fatty liver disease (NAFLD). NAFLD refers to a broad spectrum of liver diseases ranging from simple fatty liver (steatosis) to nonalcoholic steatohepatitis (NASH) to cirrhosis (irreversible late scarring of the liver). All stages of NAFLD show fat accumulation in liver cells. Simple fatty liver is an abnormal accumulation of certain types of fat, 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 (hepatocyte necrosis). In the terms "steatosis hepatitis" and "steatosis necrosis", steatosis refers to fatty infiltration, hepatitis refers to inflammation in the liver, and "necrosis" refers to destroyed liver cells. NASH can eventually lead to scarring of the liver (fibrosis) and then irreversible late scarring (cirrhosis), the cirrhosis caused by NASH being the last and most severe stage within the NAFLD spectrum.
In a seventh aspect of the invention, there is provided a method of treating a metabolic-related disorder, comprising the steps of: administering the foregoing glucagon analog to the subject.
In one embodiment, the glucagon analogs are used in the present invention to treat obesity, metabolic syndrome, nonalcoholic hepatitis, and the like.
Researchers of the present invention have found that the glucagon analogs of the present invention have sufficient water solubility at neutral pH or slightly acidic pH and have improved chemical stability. In one of the examples, IPGTT experiments were performed. Mice administered with the glucagon analogs of the invention exhibited very smooth blood glucose excursions following glucose injection. In addition, the glucagon analogs of the invention induced a significant decrease in body weight following DIO mouse administration. Meanwhile, various indexes related to blood fat are obviously reduced.
The invention further provides a method of promoting weight loss or preventing weight gain comprising administering said glucagon analog in a subject.
In an eighth aspect of the present invention, there is provided a composition comprising a culture of the aforementioned glucagon analogues or the aforementioned host cells, and a pharmaceutically acceptable carrier.
In a ninth aspect of the present invention, there is provided a use of the aforementioned glucagon analogues in the preparation of fusion proteins.
In the tenth aspect of the present invention, a fusion protein is provided, wherein the structure of the fusion protein contains the glucagon analogue.
Further, the structure of the fusion protein also comprises 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 the eleventh aspect of the present invention, there is provided a modified polypeptide, wherein the structure of the modified polypeptide comprises the glucagon analog.
Further, the glucagon analogues are modified by fatty acids, polyethylene glycol or derivatives thereof; the modified polypeptide is combined with albumin, transferrin and immunoglobulin and fragments in a covalent or non-covalent mode.
One skilled in the art will appreciate that modifications may be made to increase the half-life or stability of the glucagon analogs of the present invention, for example polyethylene glycol or derivatives thereof, hydroxyethyl starch derivatives or fatty acids may be covalently linked to the glucagon analogs of the present invention. In a particular embodiment, for improved stability, a lysine residue may be introduced at a position of the glucagon analog of the invention that is not expected to affect receptor binding/activation, covalently linked to a gamma-glutamic acid spacer and modified by the addition of palmitic acid to the epsilon-amino group.
Compared with the prior art, the invention has the following beneficial effects:
the glucagon analogues of the invention have GLP-1/GCG/GIP triple receptor agonistic activity and better enzyme-resistant stability, including resistance to Neutral Endopeptidase (NEP) and dipeptidyl peptidase-4 (DPP-4); thus having a longer half-life and duration of action in vivo compared to native glucagon, GLP-1, GIP. In summary, the GCG analogues reported so far generally use (1) single-molecule hybrid peptide-crosslinked fatty acids, PEG, FC, or the like, and are administered at a frequency of once a day or more (mathias H).
Figure BDA0001481631680000091
Etc., molecular weights for Treatment of Diabetes and obesitiy, 24: 51-62,2016); or (2) the second Ser position of the natural GCG is mutated into an unnatural amino acid such as D-Ser to resist DPP-IV degradation (Novel DPP IV-resistant C-terminated glucose and amino acids with high weight and solubility-protective effects in high-fat-fed micro-treated glucose and GLP-1 receptor activation, diabetes: 57: 1927-. Multiple active polypeptides that retain the natural amino acid composition and are administered twice daily are not currently reported. The invention provides a multi-effect GCG analogue with sufficient stability and high activity, which does not need to crosslink fatty acid, PEG albumin or immunoglobulin Fc fragment and does not need to mutate the second-position Ser into non-natural amino acid, thereby reducing potential immunogenicity risk to the maximum extent, omitting fussy chemical modification/crosslinking steps, simplifying preparation process and improving product consistency.
Drawings
FIG. 1: HPLC profile of polypeptide No. C381 in aqueous pH 7.4.
FIG. 2: HPLC of polypeptide No. C493 in aqueous solution at pH 4.5.
FIG. 3: HPLC profile of polypeptide No. C816 in aqueous pH 7.4.
FIG. 4: HPLC profile of polypeptide No. C002 in aqueous pH 7.4 solution.
FIG. 5: HPLC profile of polypeptide No. C611 in aqueous pH 4.5.
FIG. 6: HPLC profile of polypeptide No. C611 in aqueous pH 7.4.
FIG. 7 is a schematic view of: is the HPLC spectrum of C239 in a pH 7.4 aqueous solution.
FIG. 8A: the residual activity is plotted as a function of time.
FIG. 8B: the residual activity is plotted as a function of time.
FIG. 9A: exemplary are several glucagon analogs with detectable GLP-1R agonistic activity.
FIG. 9B: exemplary are several glucagon analogs with detectable GLP-1R agonistic activity.
FIG. 9C: exemplary glucagon analogs are assays for GCGR agonistic activity.
FIG. 9D: exemplary glucagon analogs are assays for GCGR agonistic activity.
FIG. 9E: the cAMP content stimulated by GIPR production for the glucagon analogs and controls at different concentration gradients.
FIG. 9F: the cAMP content stimulated by GIPR production for the glucagon analogs and controls at different concentration gradients.
FIG. 9G: the glucagon analogues and the control stimulated the cAMP content produced by the GIPR at different concentration gradients.
FIG. 9H: the cAMP content stimulated by GIPR production for the glucagon analogs and controls at different concentration gradients.
FIG. 10: is the result of in vitro cell insulin secretion assay.
FIG. 11A: is a graph of IPGTT experimental blood glucose changes of normal ICR mice.
FIG. 11B: is a graph of IPGTT experimental blood glucose changes of normal ICR mice.
FIG. 11C: results are area under the blood glucose curve (AUC) comparisons.
FIG. 12A: graph of weight change (%) versus time (days) in diet-induced obese (DIO) mice.
FIG. 12B: graph of weight change (%) versus time (days) in diet-induced obese (DIO) mice.
FIG. 12C: graph of weight change (%) versus time (days) in diet-induced obese (DIO) mice.
FIG. 12D: the graph compares the weight loss in DIO mice.
FIG. 13 is a schematic view of: compare the weight loss in DIO mice.
FIG. 14: is a C495 mass spectrometry plot.
FIG. 15: is a C382 mass spectrum analysis chart.
Detailed Description
Unless defined otherwise 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.
Glucagon analogs:
the glucagon analogues provided by the invention mutate arginine (R) at position 18 to alanine (A). A mutation of the alanine at position 18 reduced the GCGR agonistic activity by about 30% (Joseph channel et al, A Gluguan analog chemical stabilized for immunization therapy of life-treating hypoglycemia, Molecular Metabolism, 3: 293-300, 2014). However, after extensive combinatorial screening by the present inventors, it was found that the GCGR activity was not significantly reduced even if the 18-position was mutated to A by the addition of CEX or the like at the C-terminus after specific amino acid mutations at some specific sites, such as in combination with specific amino acid mutations at positions 16 and 17. More importantly, the mutation at the 18-position A can obviously improve the GLP-1 and GIPR agonistic activities of the glucagon analogues, so that the glucagon analogues become effective tri-specific active peptides.
The tri-specific active peptide has the efficacy of stimulating GLP-1R, GCGR and GIPR, and the activity retention rate of each activity is extremely high relative to GLP-1, GCG and GIP. At present, most of the tri-active peptides introduce a plurality of amino acid mutations on the basis of natural polypeptides, and even unnatural amino acids can become stable tri-potent agonists. In the screening process of the invention, the hybrid polypeptide with higher GLP-1R, GCGR and GIPR activities can be more easily obtained by introducing mutation at multiple sites, and the polypeptide with high stability can be more easily obtained by introducing unnatural amino acids. However, the in vitro activity and stability are only a prerequisite for clinical drugs, and safety needs to be concerned. Introducing too many mutation sites or unnatural amino acids easily brings higher immunogenicity risk.
Serum stability experiments:
natural GLP-1, Glucagon or Oxyntomodulin (Oxyntomodulin) cannot be truly clinical drugs due to poor serum stability and too short in vivo half-life. While the same small peptide Exenatide (Exenatide, a commercial product) only has 39 amino acidsName (name)
Figure BDA0001481631680000111
) But successfully marketed due to the improved stability. In one of the embodiments of the present invention, the preferred glucagon analogs exhibit very high stability.
Immunogenicity experiments:
in example 6 of the present invention, the immunogenicity of native human Glucagon (C001) in rats was very low. When the Glucagon analogue is mutated no more than 3 amino acids relative to the native Glucagon, the antibody titers are all less than < 1: 200, and as the number of mutated amino acids increases, so does the antibody titer, indicating an increased risk of potential immunogenicity. The safety requirements for drugs for treating metabolic-related diseases, such as drugs in the fields of diabetes, obesity, and the like, are extremely high. The glucagon analogues obtained by the invention have lower immunogenicity under the condition of introducing no more than 3 mutation sites, and reach the ideal activity and stability standards, which has never been reported.
Pharmacodynamic study in animals:
in one embodiment of the present invention, the preferred glucagon analogs have good blood glucose lowering, adipose tissue formation inhibiting, and weight loss effects. Although GIP, GLP-1 and Glucagon belong to the family of incretins (Incretins), there is no trend toward the widespread development of drugs. One reason for this is the loss of GIP sensitivity in some type of diabetic patients, and the other is the potential obesity of GIPR activation in rodents (Miyawaki, k. et al, Inhibition of pathological Inhibition signalling: med.8, 738-742, 2002). However, in the present examples, the preferred glucagon analogues with higher GIPR agonistic activity clearly have a more pronounced weight loss effect.
In another in vivo example 9 of the animals of the present invention, the preferred GCG analogs exhibit similar weight loss effects to the corresponding GCG analogs of the same amino acid sequence and modified with fatty acids.
The terms:
the term "diabetes" includes type one diabetes, type two diabetes, gestational diabetes, and other symptoms that cause hyperglycemia. The term is used to refer to the condition in which the pancreas does not produce enough insulin, or the body's cells fail to respond properly to insulin, due to metabolic disturbances, and thus the efficiency of glucose uptake by the tissue cells decreases, resulting in the accumulation of glucose in the blood.
Type one diabetes, also known as insulin-dependent diabetes and juvenile onset diabetes, is caused by beta cell destruction, often resulting in absolute insulin deficiency.
Type ii diabetes, also known as non-insulin dependent diabetes mellitus and adult-onset diabetes, is commonly associated with insulin resistance.
The term "obesity" means an excess of adipose tissue, and when energy intake exceeds energy consumption, excess calories are stored in fat, resulting in obesity. Individuals with a body mass index (BMI ═ body weight (kilograms) divided by height (meters) squared) of more than 25 are considered herein as obese.
The term "receptor agonist" may be defined as a polypeptide, protein or other small molecule that binds to a receptor and elicits the usual response of a natural ligand. Incretins are gastrointestinal hormones that regulate blood glucose by enhancing glucose-stimulated insulin secretion (Drucker. D J, Nauck, MA, Lancet 368: 1696-. There are two known incretins to date: glucagon-like peptide-1(GLP-1) and glucose-dependent insulinotropic polypeptide (GIP). Preproglucagon (preproglucagon) is a precursor polypeptide of 158 amino acids that is differentially processed in tissues to form a variety of structurally related Glucagon-derived peptides, including Glucagon (Glucagon), Glucagon-like peptide-1(GLP-1), Glucagon-like peptide-2 (GLP-2), and Oxyntomodulin (Oxyntomodulin, OXM). GIP is a 42 amino acid mature peptide derived from a 133 amino acid precursor (pre-pro-GIP) by proteolytic processing, and these molecules are involved in a variety of biological functions, including glucose homeostasis, insulin secretion, gastric emptying and intestinal growth, and regulation of food intake.
Glucagon-like peptide-1(GLP-1) is an incretin hormone of 30 or 31 amino acids secreted from intestinal L-cells, and 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 biological activity by activating the GLP-1 receptor. GLP-1 has a number of biological effects, including glucose-dependent insulinotropic secretion, inhibition of glucagon production, retardation of gastric emptying and appetite suppression (Tharakan G, Tan T, Bloom S. emitting therapeutics in the intestinal tract of diabetes': beyond GLP-1.Trends Pharmacol Sci 2011; 32(1): 8-15), etc. Native GLP-1 has limited its therapeutic potential due to its ability to be rapidly degraded by dipeptidyl peptidase-4 (DPP-4), Neutral Endopeptidase (NEP), plasma kallikrein or plasmin, etc. Since native GLP-1 has an ultra-short half-life of only about 2 minutes in vivo, methods have emerged to improve efficacy by using chemical modifications and/or formulation formats to treat diabetes and obesity (Lorenz M, Evers A, Wagner M.Recent progress and future options in the development of GLP-1 receptor assays for the development of diabetes. biological Chem Lett 2013; 23(14) 4011-8.Tomlinson B, HuM, Zhang Y, Chan P, LiZmu.an overview of new GLP-1 receptor assays for type 2 diabetes. Expert Opin Drugs 2016; 25(2) 145-58).
Oxyntomodulin (Oxyntomodulin) is a small 37 amino acid peptide comprising the entire 29 amino acid sequence of glucagon. Oxyntomodulin is a dual agonist of GLP-1R and GCGR, secreted with GLP-1 by intestinal L-cells after a meal. Like glucagon, oxyntomodulin produces significant weight loss in humans and rodents. The slimming activity of oxyntomodulin has been compared in obese mice with equimolar doses of selective GLP-1R agonists. Oxyntomodulin has been found to have anti-hyperglycemic effects, be able to significantly reduce body weight and have lipid lowering activity compared to selective GLP-1R agonists (The glucose receptor is involved in mediating The body weight-lowering effects of oxyntomodulin, Kosinski JR et al, obesity (silver spring), 20):1566-71, 2012). Subcutaneous administration of natural oxyntomodulin reduced body weight by 1.7 kg over four weeks in overweight and obese patients. Oxyntomodulin has also been shown to reduce food intake and increase energy expenditure in humans (Subcutaneomodulatory protein recovery body weight in excess weight and object subjects: a double-blind, randomized, controlled trial, Wynne K et al, Diabetes, 54:2390-5, 2005; Oxyntomodulin involved genes ex-dependent in addition detail in addition to optimization of energy intake and object humankind: a 14 andoded controlled trial; Wne K et al, Int J Obes (Lond), 30: 9-36, 2006). But also due to the smaller molecular weight and degradation of DPP-IV, oxyntomodulin has a shorter half-life. At present, double-effect agonists of a GLP-1 receptor (GLP-1R) and a glucagon receptor (GCGR) are generally based on oxyntomodulin, mutations are made for improving the short-acting effect and the enzymolysis defect of the oxyntomodulin (oxyntomodulin analogues), a method for mutating a second-position serine Ser into alpha-amino isobutyric acid (Aib) or D-Ser is mostly adopted, and the enzymolysis of DPP-IV is resisted by introducing non-natural amino acid. Although the oxyntomodulin analogue shows primary blood sugar reducing and fat reducing effects, the action mechanism of the oxyntomodulin analogue is still uncertain, and the oxyntomodulin receptor has been only verified to be capable of binding with the 2 receptors to act through GCGR or GLP-1R knockout mice or cell experiments.
Glucagon (Glucagon) is a 29 amino acid peptide corresponding to amino acids 53-81 of preproglucagon, and has the sequence shown in SEQ ID No.5 (c.g. fanelli et al, Nutrition, Metabolism & cardiovacular Diseases (2006)16, S28-S34). Glucagon receptor activation has been shown to increase energy expenditure and decrease food intake in both rodents and humans (Habegger k.m. et al, The metabolic actions of glucose consumption, nat. rev. endocrinol.2010,6, 689-. Glucagon has many physiological effects, such as by stimulating glycogenolysis and gluconeogenesis, increasing blood glucose levels in hypoglycemic conditions, regulating hepatic ketogenesis, regulating bile acid metabolism, and satiety effects through the vagus nerve. Glucagon has been used in acute hypoglycemia, with glucagon receptor activation reducing food intake and promoting lipolysis and weight loss in animals and humans.
Glucose-dependent insulinotropic peptide (GIP) is a 42 amino acid polypeptide which is released from K cells in the small intestine after food intake, and its main functions are to inhibit gastric acid secretion and to enhance glucose-stimulated insulin secretion, and is therefore called gastric inhibitory peptide (gastric-inhibitory insulin peptide)/glucose-dependent insulinotropic polypeptide (glucose-dependent insulin 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 the same or a similar characteristic response as native GLP-1. GLP-1R agonists produce corresponding cellular activities by fully or partially activating GLP-1R, which in turn elicits a series of intracellular downstream signaling pathway responses: such as insulin secretion by beta cells; typical GLP-1R agonists include native GLP-1 and mutants, analogs thereof, such as exenatide, Liraglutide, and the like.
A "glucagon receptor (GCGR) agonist" may be defined as a polypeptide, protein, or other small molecule that binds to GCGR and is capable of eliciting a characteristic response that is the same as or similar to native glucagon (glucagon). GCGR agonists produce corresponding cellular activities by fully or partially activating GCGR, which in turn elicits a series of intracellular downstream signaling pathway responses: such as hepatic cell glycogenolysis, carbohydrate neogenesis, fatty acid oxidation, ketogenesis, etc.
GLP-1R/GCGR dual-effect agonist: the GLP-1R/GCGR dual-effect agonist comprises a protein or polypeptide which can stimulate GLP-1R and GCGR at the same time. Oxyntomodulin-based Dual agonists as reported by Alessandro Pocai et al (Alessandro Pocai et al, Glucaon-Like Peptide 1/Glucaon Receptor Dual agonist epitopes in Mice, Diabetes; 58(10):2258-2266, 2009), or Glucaon-based Dual agonists as reported by Richard D. DiMarchi et al (US9018164B 2).
GLP-1R/GCGR/GIPR triple-acting agonists: the GLP-1R/GCGR/GIPR three-way agonist comprises a protein or polypeptide which can stimulate GLP-1R, GCGR and GIPR at the same time, or is called a three-specificity agonist.
Trispecific active peptides: the preferred trispecific active peptide of the present invention refers to a polypeptide having GLP-1R/GCGR/GIPR agonistic activity at the same time, otherwise known as a "trispecific active peptide".
EC 50 (concentration for 50% of maximum effect) refers to the concentration of a drug or substance required to stimulate 50% of its corresponding biological response. Lower EC50 values indicate greater stimulation or stimulation of the drug or substance, e.g., more intuitively it may appear that a greater intracellular signal is elicited, and thus greater ability to induce the production of a certain hormone.
Low Density Lipoprotein (LDL): belongs to one of plasma lipoproteins, and is a main carrier of cholesterol in blood. It tends to deposit cholesterol on the arterial wall. Leukocytes attempt to digest the low density lipoproteins, but in the process turn them into toxins. More and more leukocytes are attracted to the changed area, causing inflammation of the arterial wall. Over time, these plaque deposits can accumulate on the arterial wall, causing the channel to become very narrow and inflexible. If too much plaque accumulates, the artery may become completely occluded. When the complex formed by LDL and cholesterol (LDL-C) creates too many plaques on the arterial wall, blood will not flow freely through the artery. The plaque can collapse suddenly in the artery from time to time, causing blockage of the blood vessel, ultimately resulting in heart disease.
High density protein (HDL): helps to clear LDL from the arteries, acts as a scavenger, and clears LDL from the arteries back to the liver.
Triglyceride (TG): is another type of fat used to store energy that is too much 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 physical exercise, smoking, excessive alcohol consumption and high carbohydrate (over 60% of total calories) intake. Sometimes the underlying disease or genetic disease is the cause of high triglycerides. People with high triglycerides often have high total cholesterol levels, including high LDL cholesterol and low HDL cholesterol, as do many people with heart disease or diabetes.
GPCR: the G Protein Coupled Receptor (G Protein-Coupled Receptor) is an important Protein in cell signaling, and the topological conformation thereof is a 7-transmembrane Receptor. When an extramembranous ligand acts on the receptor, the intramembrane portion of the receptor binds to the G protein, activating the G protein. G proteins can transmit extracellular information via two pathways: the first way is to open the transmembrane ion channel to let the external ions enter; the second way is to activate a second messenger, e.g. cAMP, IP 3 DAG, etc. Calcium ions are commonly known as cAMP, IP 3 Third messenger downstream of DAG.
Abbreviations
COMU: 1- [ (1- (cyano-2-ethoxy-2-oxoethyleneaminooxy) dimethylaminomethylolmorpholine) ] methylammonium hexafluorophosphate
DCM: methylene dichloride
DMF: n, N-dimethylformamide
DIPEA: diisopropylethylamine
EtOH: ethanol
Et 2 O: ether (A)
HATU: 2- (7-benzotriazole oxide) -N, N, N ', N' -tetramethyluronium hexafluorophosphate
MeCN; acetonitrile
NMP: n-methyl pyrrolidone
TFA: trifluoroacetic acid
And (3) TIS: tri-isopropyl silane
The following embodiments of the present invention are provided by way of specific examples, and other advantages and effects of the present invention will be readily apparent to those skilled in the art from the disclosure herein. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.
Before the present embodiments are further described, it is to be understood that the scope of the invention is not to be limited to the specific embodiments described below; it is also to be understood that the terminology used in the examples herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention.
Unless defined otherwise, 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. In addition to the specific methods, devices, and materials used in the examples, the invention may be practiced using any method, device, and material that is similar or equivalent to the methods, devices, and materials described in examples herein, in addition to those described in prior art practice and the description herein.
Unless otherwise indicated, the methods of testing, methods of preparation, and methods of preparation disclosed herein employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related arts. These techniques are well described in the literature, and may be found in particular in the study of the MOLECULAR CLONING, Sambrook et al: 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, 1987and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; (iii) METHODS IN ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol.119, chromatography Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999, etc.
Example 1: general preparation and purification methods for glucagon analogues
By using the prior art, e.g. the prior documents (
Figure BDA0001481631680000171
V. et al, Beilstein J.org.chem.,10: 1197-1212 (2014); palomo, j.m., RSC adv.,4: 32658-; behredt, R. et al, J.Pept.Sci.,22:4-27(2015)) The polypeptide solid phase synthesis and modification method in (1) to prepare each polypeptide involved in the present patent.
In particular, solid phase peptide synthesis can be performed using standard Fmoc methods on a CEM Liberty peptide synthesizer.
Rink Amide TentaGel S Ram resin (0.25mmol/g, 1g) was swollen in NMP (10mL) before use, added to a solid phase synthesis apparatus, piperidine/DMF (20%, 10mL) was added to the resin for 30min for Fmoc deprotection, pumped dry, washed with DMF (5X 10mL), pumped dry. The first Fmoc-amino acid solution (0.2M, NMP/DMF/DCM, 1:1:1, 5mL) was added along with COMU/NMP (0.5M, 2mL) and DIPEA/DMF (2.0M; 1mL) and reacted for 1h at room temperature, ninhydrin assay resin was colorless and clear, aspirated, and washed with DMF (5X 10 mL). piperidine/DMF (20%, 10mL) was then added for 30min for Fmoc deprotection, suction dried, washed with DMF (5X 10mL), suction dried. The above steps of Fmoc-amino acid addition for reaction and piperidine/DMF deprotection for Fmoc protection were repeated until the final histidine coupling was completed.
Resin was washed with EtOH (3X 10mL) and Et 2 O (3X 10mL) and dried to constant weight at room temperature. The resin was reacted in an ice bath for 2h with the addition of TFA/TIS/phenol/EDT/water (82.5/5/5/2.5/5, v/v, 40mL), the crude peptide was cleaved from the resin, filtered, and the procedure was repeated three times. The filtrates were combined, most of the TFA was removed under reduced pressure, precipitated with diethyl ether, centrifuged, the precipitate was washed three times with diethyl ether and dried at room temperature to constant weight to give the crude peptide. Crude peptide was purified by preparative reverse phase HPLC using a preparative liquid chromatograph equipped with a C-18 column and a partial trap, model warian SD-1, eluting with a gradient of mobile phase a (0.1% TFA, aqueous solution) and mobile phase B (0.1% TFA, 90% MeCN, aqueous solution) to a purity of greater than 97%, and the resulting peptides are listed in table 1. Wherein, the polypeptide with C-terminal amide end is synthesized by the method; and (3) performing solid-phase synthesis on the rest polypeptides by using Wangle Resin (0.4mmol/g, 1g), directly adding Fmoc-amino acid after Resin swelling for coupling reaction, removing Fmoc protection, performing polypeptide cutting, and purifying, wherein the operation steps are the same as the steps for synthesizing the C-terminal amide-terminated polypeptide. Synthesis and purification of polypeptides with fatty acid modifications is a routine technique and can be found in Finan B et al (Finan B et al, A ratio designed monomeric peptides)triparenist solids and diabetes in rodents.nat. med.2015; 21:27-36.) or Chhabr et al (Chhabr et al, applied of New Variants of Dde Amine Protecting Group for Solid Phase Peptide Synthesis, Tetrahedron Lett.1998,39(12), 1603-1606).
The purified peptides were analyzed by LC/MS and the results are shown in Table 2. Wherein figures 14, 15 are exemplary mass spectrometric profiles of glucagon analogs numbered C495 and C382, respectively. Mass spectrometry conditions were as follows:
the instrument comprises the following steps: waters ZQ 2000
Mass spectrum (Probe): ESI
Nebulizer Flow rate (Nebulizer Gas Flow): 1.5L/min
CDL：-20.0v
CDL temperature: 250 ℃ C
Heating Block temperature (Block Temp): 200 deg.C
Mass spectrometry voltage (Probe Bias): +4.5kv
Detector (Detector): 1.5kv
Mobile phase flow rate (t.flow): 0.2ml/min
Buffer concentration (b.conc.): 50% H2O/50% ACN
TABLE 1
Figure BDA0001481631680000181
Figure BDA0001481631680000191
Figure BDA0001481631680000201
Note: x in the table is aminoisobutyric acid, K' indicates that this position is a lysine residue and is covalently linked to a fatty acid of the structure shown in formula I below:
Figure BDA0001481631680000202
TABLE 2
Figure BDA0001481631680000203
Figure BDA0001481631680000211
Example 2: stability study
The purpose of this example was to investigate the chemical stability of the various glucagon analogues prepared in example 1 in aqueous solution.
The test polypeptide (glucagon analogue) and the control were prepared in 20mM phosphate buffer PB or acetate buffer at the respective pH to a final concentration of 0.2mg/ml and sterile filtered through a sterile filter (0.22 μm, Millipore SLGP033 RB). The prepared polypeptide solution was left at 40 ℃ for 7 days. The supernatant was then centrifuged at 4500rpm for 20 minutes and analyzed using RP-HPLC-UV (t 7). The amount of residual intact peptide was determined and the uninsulated samples were analyzed in parallel (t 0). Comparing the peak areas of the target compound 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 t 0.
Stability is expressed as "residual peptide content".
Detection method
Detection wavelength: 214 nm;
and (3) chromatographic column: column temperature 40 deg.C, Phenomenex Luna C8(2)5 μm (150X 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 min) to 0:100(30 min);
and (3) analyzing an experimental result: from the experimental data in table 3, it can be seen that the preferred glucagon analogues (polypeptides) of the present invention have high stability in both neutral and weakly acidic aqueous solutions.
TABLE 3
Figure BDA0001481631680000221
Figure BDA0001481631680000231
Figures 1-7 schematically show liquid phase HPLC analysis profiles of several glucagon analogs such as C381. The corresponding integrated data of fig. 1:
Figure BDA0001481631680000232
the corresponding integrated data of fig. 2:
Figure BDA0001481631680000233
raw data corresponding to fig. 3:
Figure BDA0001481631680000241
raw data corresponding to fig. 4:
Figure BDA0001481631680000242
raw data corresponding to fig. 5:
Figure BDA0001481631680000243
raw data corresponding to fig. 6:
Figure BDA0001481631680000251
raw data for fig. 7:
Figure BDA0001481631680000252
example 3: serum stability
(1) The corresponding polypeptide in Table 1 was prepared into 1.0mg/ml solution with 5mM Tris-HCl, pH8.5, 0.02% TWEEN80 solution, sterilized and filtered (0.22 μm, Millipore SLGP033RB), diluted 10 times with rat serum, mixed well and dispensed into sterile centrifuge tubes;
(2) taking 3 tubes of the samples from each sample, freezing at-20 ℃ for serving as a reference, placing the rest samples in a 37 ℃ thermostat, and sampling at different time points for detecting activity;
(3) the GCGR agonistic activity of the polypeptide was measured using the method described in example 4.
Relative activity: activity value at 0 hour is 100%, and the values measured at subsequent time points are compared. And (3) analyzing an experimental result: serum stability can be derived from table 4and figures 8A and 8B.
TABLE 4
Figure BDA0001481631680000261
Figure BDA0001481631680000271
Note: n.d. indicates below the lower limit of detection
Example 4: cell activity assay
(I) GLP-1R agonistic activity assay:
the GLP-1R agonistic activity was measured by luciferase reporter assay (Jonathan W Day et al: Nat Chem biol.2009 Oct; 5(10): 749-57). Cloning the human GLP-1R gene into a mammalian cell expression plasmid pCDNA3.1 to construct a recombinant expression plasmid pCDNA3.1-GLP-1R, and simultaneously cloning the luciferase (luciferase) full-length gene into a pCRE plasmid to obtain the pCRE-Luc recombinant plasmid. The pcDNA3.1-GLP-1R and the pCRE-Luc plasmids transfect CHO cells according to the molar ratio of 1:10, and stably-transformed expression strains are screened.
Culturing cells in a 9-cm cell culture dish by using DMEM/F12 culture medium containing 10% FBS and 300 mu G/ml G418, when the confluency is about 90%, discarding culture supernatant, adding 2ml pancreatin for digestion for 3min, adding 2ml DMEM/F12 culture medium containing 10% FBS and 300 mu G/ml G418 for neutralization, transferring to a 15ml centrifuge tube, centrifuging at 1000rpm for 5min, discarding supernatant, adding 2ml DMEM/F12 culture medium containing 10% FBS and 300 mu G/ml G418 for resuspension, and counting. Cells were diluted to 1X 10 with DMEM/F12 medium containing 10% FBS 5/ ml, 100. mu.l per well in 96-well plates, i.e.1X 10 4 Per well, after adherence, DMEM/F12 medium containing 0.2% FBS was used. After the supernatant was discarded from the cells plated in the 96-well plate, the purified recombinant protein was diluted to a series of prescribed concentrations with DMEM/F12 medium containing 0.1% FBS, added to the cell culture wells at 100. mu.l/well, and assayed after 6h of stimulation. Detection was performed according to the lucifersae reporter kit (Ray Biotech, Cat:68-Lucir-S200) instructions. FIGS. 9A and 9B show the results of GLP-1R agonistic activity assays.
(II) a GCGR agonistic activity detection method:
the GCGR agonistic activity assay also employs the luciferase reporter assay. The human GCGR gene is cloned into a mammalian cell expression plasmid pcDNA3.1 to construct a recombinant expression plasmid pCDNA3.1-GCGR, and the screening construction of the transfected HEK 293T and the stable transfer cell strain is the same as the above. FIGS. 9C and 9D show the results of GCGR agonistic activity assays.
TABLE 5
Figure BDA0001481631680000281
Figure BDA0001481631680000291
(III) GIPR agonist activity detection method:
the pcDNA3.1-GIPR plasmid was transfected into CHO cells and positive stable transgenic cell lines were selected. About 200,000 cells/well were seeded in a 96-well cell culture plate, cultured overnight, washed with Hanks' balanced salt solution, and the test proteins were diluted to a series of prescribed concentrations and added to the cells together with 200. mu.M 3-isobutyl-1-methylxanthine (IBMX), cultured at 37 ℃ for 20min, the culture supernatant was discarded, and the cells were lysed by adding a lysate, and the cAMP content was determined using cAMP Parameter assay kit reference (R & D, USA: SKGE 002B). The results are shown in FIGS. 9E-H.
Example 5: glucose stimulated insulin secretion assay
This example refers to the method of Aisling M.Lynch et al (A novel DPP IV-resistant C-terminated dextran on analog insulin weights in high-fat-fed micro-peptide-mediated insulin Glucaging and GLP-1 receptor activation, Aisling M.Lynch et al, Diabetologia, 57: 1927- 2 、1.2mM MgSO 4 、1.2mM KH 2 PO 4 、25mM HEPES、10mM NaHCO 3 NaOH adjusted pH to 7.4), 0.1% (wt/vol.) BSA, and 1.1mM glucose. After incubation of the cells at 37 ℃ for 40 minutes, the supernatant was centrifuged off and replaced with 1.0ml of fresh KRB solution and a gradient of active protein. After incubation at 37 ℃ for 20 minutes, the buffer was removed by centrifugation and stored overnight at-20 ℃ before immunoradiometric detection of insulin content. The results are shown in FIG. 10.
Example 6: mouse immunogenicity experiments
7 weeks old Balb/c mice, each group of 6, before the administration of each rat tail vein blood sampling 50ul serum as blank control. The corresponding glucagon analogue (30nmol/kg in PBS buffer) was injected daily for 28 consecutive days. Blood was collected from the orbit by day 45, and serum was isolated by coagulation. The antibody titer was determined by direct ELISA. Corresponding polypeptide coated enzyme label plate, and mouse serum is added according to the proportion of 1: 50; 1: 200,1: 1000,1: 5000 gradient dilution enzyme label plateAnd the goat anti-mouse secondary antibody is a detection antibody. Serum was used as negative control before each mouse was administered, and OD was measured for each mouse at the same dilution 450 The value average value is larger than the negative control serum OD 450 The result of the average value of the values being 2.1 times is judged to be positive (+), otherwise, the result is judged to be negative (-), and the highest dilution with the positive result is the antibody titer.
TABLE 6
Figure BDA0001481631680000301
Example 7: glucose Tolerance Test (IPGTT) in Normal ICR mice
Normal ICR mice were divided into 27 groups of 6 mice each. Fasting overnight, tail bleed (denoted as t ═ 0min glycemia), subcutaneous injection of vehicle control (acetate buffer, 20mM acetic acid, 250mM mannitol, ph5.0) and glucagon analogues of table 1 of the invention (30nmol/kg in PBS buffer) and vehicle control, liraglutide (r), (g), (d) b), (d) b) and (d), b) and (d) b, d (d, d (d, b) and (d) b (d) b)Trade name
Figure BDA0001481631680000302
Figure BDA0001481631680000303
40nmol/kg, diluted into 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, t-120 minutes. Animals were still fasted during the experiment to prevent interference with food intake. See FIGS. 11A-C for specific results.
Example 8: weight loss experiments in diet-induced obese (DIO) mice
Preparation of DIO mouse model: male C57BL/6J male mice of about 7 weeks of age were given a high fat diet (60% kcal from fat) and kept on for about 16 weeks (23 weeks total) until they had a body weight of about 45g for the test. DIO mice were randomly divided into groups of 6 mice each with no difference in basal body weight, each group of mice was injected subcutaneously with glucagon analogues (30nmol/kg in PBS) or PBS of equal volume per day, and a control group of liraglutide (trade name)
Figure BDA0001481631680000311
30nmol/kg), 2 times daily, weighing to 30 days daily.
Fig. 12A-C are graphs showing the daily change in body weight of DIO mice after administration of each glucagon analog, and the final percent weight loss is shown in fig. 12D.
Example 9: weight loss experiments in diet-induced obese (DIO) mice
DIO mice were randomly divided into groups of 6 mice each with no difference in basal body weight, and each group of mice was injected subcutaneously with each GCG analogue (30nmol/kg in PBS) or PBS, respectively, and a control group of liraglutide (trade name: Liraglutide)
Figure BDA0001481631680000312
30nmol/kg, diluted in PBS) 2 times daily and the fatty-acidified GCG analogue (30nmol/kg, in PBS) 1 time daily, weighing to 30 days daily.
Figure 13 is a graph of the final percent weight loss in DIO mice following administration of each glucagon analog. The GCG analogues C381, C464 and C493 shown in the figure show similar weight loss effects to the corresponding GCG analogues with the same amino acid sequence and modified by fatty acids, whereas C225, C163 show no significant weight loss effect without fatty acidification.
In view of the foregoing, the present invention effectively overcomes the disadvantages of the prior art to provide a group of three-way agonists with potential clinical utility.
The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.
Sequence listing
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His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser
1 5 10 15
Gln Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 9
<211> 39
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 9
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser
1 5 10 15
Gln Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 10
<211> 39
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 10
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser
1 5 10 15
Glu Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 11
<211> 39
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 11
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser
1 5 10 15
Glu Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 12
<211> 39
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 12
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser
1 5 10 15
Gln Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 13
<211> 39
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 13
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser
1 5 10 15
Gln Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 14
<211> 38
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 14
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Glu Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Ser
35
<210> 15
<211> 38
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 15
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Glu Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Ser
35
<210> 16
<211> 39
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 16
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Glu Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 17
<211> 39
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 17
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Glu Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 18
<211> 38
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 18
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Glu Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Ser
35
<210> 19
<211> 38
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 19
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Glu Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Ser
35
<210> 20
<211> 39
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 20
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Glu Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 21
<211> 39
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 21
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Glu Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 22
<211> 36
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 22
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Gln Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Ser Ser Gly
20 25 30
Ala Pro Pro Ser
35
<210> 23
<211> 39
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 23
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Gln Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 24
<211> 38
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 24
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Gln Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Ser
35
<210> 25
<211> 39
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 25
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Gln Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 26
<211> 39
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 26
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Gln Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 27
<211> 37
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 27
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Gln Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Ser Ser Gly
20 25 30
Ala Pro Pro Pro Ser
35
<210> 28
<211> 37
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 28
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Gln Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Ser Ser Gly
20 25 30
Ala Pro Pro Pro Ser
35
<210> 29
<211> 40
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 29
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser
1 5 10 15
Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Gly Gly Pro
20 25 30
Ser Ser Gly Ala Pro Pro Pro Ser
35 40
<210> 30
<211> 39
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 30
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Arg Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 31
<211> 39
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 31
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Arg Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 32
<211> 39
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 32
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser
1 5 10 15
Arg Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 33
<211> 39
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 33
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser
1 5 10 15
Arg Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 34
<211> 39
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 34
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ala
1 5 10 15
Arg Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 35
<211> 39
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 35
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ala
1 5 10 15
Arg Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 36
<211> 39
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 36
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser
1 5 10 15
Glu Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 37
<211> 39
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 37
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser
1 5 10 15
Glu Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 38
<211> 39
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 38
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Glu Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 39
<211> 39
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 39
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ala
1 5 10 15
Glu Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 40
<211> 39
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 40
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser
1 5 10 15
Gln Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 41
<211> 39
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 41
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Gln Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 42
<211> 39
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 42
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ala
1 5 10 15
Gln Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 43
<211> 39
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 43
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ala
1 5 10 15
Ala Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 44
<211> 39
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 44
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Arg Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 45
<211> 39
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 45
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser
1 5 10 15
Arg Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 46
<211> 39
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 46
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ala
1 5 10 15
Arg Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 47
<211> 39
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 47
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Gln Ala Ala Arg Glu Phe Val Gln Trp Leu Met Asn Thr Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 48
<211> 39
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 48
His Ser Gln Gly Thr Phe Thr Ser Asp Leu Ser Lys Tyr Leu Asp Glu
1 5 10 15
Glu Ala Ala Gln Asp Phe Ile Gln Trp Leu Met Asn Thr Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 49
<211> 39
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 49
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Gln Ala Ala Arg Asp Phe Ile Gln Trp Leu Met Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 50
<211> 39
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 50
His Ser Gln Gly Thr Phe Thr Ser Asp Leu Ser Lys Tyr Leu Asp Glu
1 5 10 15
Glu Ala Ala Gln Glu Phe Ile Gln Trp Leu Met Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 51
<211> 39
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 51
His Ser Gln Gly Thr Phe Thr Ser Asp Leu Ser Lys Tyr Leu Asp Glu
1 5 10 15
Glu Ala Ala Arg Glu Phe Val Gln Trp Leu Met Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 52
<211> 39
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 52
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
1 5 10 15
Gln Ala Ala Arg Glu Phe Val Gln Trp Leu Met Asn Thr Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 53
<211> 39
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 53
His Ser Gln Gly Thr Phe Thr Ser Asp Leu Ser Lys Tyr Leu Asp Glu
1 5 10 15
Glu Ala Ala Arg Glu Phe Val Gln Trp Leu Met Asn Thr Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 54
<211> 29
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 54
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Ser Gln
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Cys Trp Leu Met Asn Thr
20 25
<210> 55
<211> 39
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 55
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Ser Gln
1 5 10 15
Ala Ala Val Arg Leu Phe Ile Gln Trp Leu Met Asn Thr Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 56
<211> 30
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 56
His Ser Gln Gly Thr Phe Thr Ser Asp Lys Ser Glu Tyr Leu Asp Glu
1 5 10 15
Glu Ala Ala Arg Asp Phe Val Ala Trp Leu Glu Ala Gly Gly
20 25 30
<210> 57
<211> 39
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 57
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Met Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 58
<211> 38
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 58
His Xaa Gln Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Asp Glu
1 5 10 15
Gln Ala Ala Gln Asp Phe Val Gln Trp Leu Leu Asp Gly Pro Ser Ser
20 25 30
Gly Ala Pro Pro Pro Ser
35
<210> 59
<211> 39
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 59
His Ser Gln Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Asp Ser
1 5 10 15
Gln Ala Ala Gln Asp Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 60
<211> 39
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 60
His Ser Gln Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Asp Ser
1 5 10 15
Gln Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 61
<211> 39
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 61
His Ser Gln Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Asp Glu
1 5 10 15
Glu Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 62
<211> 39
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 62
His Ser Gln Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Asp Glu
1 5 10 15
Gln Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 63
<211> 39
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 63
His Ser Gln Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Asp Glu
1 5 10 15
Arg Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 64
<211> 39
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 64
His Ser Gln Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Asp Ser
1 5 10 15
Arg Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 65
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 65
Gly Gly Pro Ser Ser Gly Ala Pro Pro Pro Ser
1 5 10
<210> 66
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 66
Gly Gly Pro Ser Ser Gly Ala Pro Pro Ser
1 5 10
<210> 67
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 67
Gly Pro Ser Ser Gly Ala Pro Pro Pro Ser
1 5 10
<210> 68
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 68
Gly Pro Ser Ser Gly Ala Pro Pro Ser
1 5
<210> 69
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 69
Pro Ser Ser Gly Ala Pro Pro Pro Ser
1 5
<210> 70
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 70
Pro Ser Ser Gly Ala Pro Pro Ser
1 5
<210> 71
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 71
Ser Ser Gly Ala Pro Pro Pro Ser
1 5
<210> 72
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 72
Ser Ser Gly Ala Pro Pro Ser
1 5

Claims (11)

1. A glucagon analogue is characterized in that the sequence of the glucagon analogue is shown in SEQ ID NO 12-13.
2. The glucagon analog of claim 1, wherein the glucagon analog has GLP-1/GCG/GIP triple receptor agonistic activity.
3. An isolated polynucleotide encoding a glucagon analog of any of claims 1-2.
4. A recombinant expression vector comprising the isolated polynucleotide of claim 3.
5. A host cell comprising the recombinant expression vector of claim 4 or having integrated into its genome the exogenous isolated polynucleotide of claim 3.
6. The method for the preparation of a glucagon analog of any of claims 1 to 2, selected from any of the following:
(1) synthesizing the glucagon analog by a chemical synthesis method;
(2) culturing the host cell of claim 5 under suitable conditions to allow expression of said glucagon analog, followed by isolation and purification to obtain said glucagon analog.
7. Use of a glucagon analogue of any one of claims 1-2 in the manufacture of a medicament for the treatment of type ii diabetes and obesity.
8. A composition comprising a culture of a glucagon analog of any one of claims 1 to 2 or a host cell of claim 5, and a pharmaceutically acceptable carrier.
9. Use of a glucagon analogue according to any of claims 1-2 for the preparation of a fusion protein.
10. A fusion protein consisting of a glucagon analogue according to any of claims 1-2 and a long acting unit selected from the group consisting of albumin, transferrin and an immunoglobulin.
11. A modified polypeptide comprising a glucagon analog of any one of claims 1-2 in its structure, said glucagon analog being modified with a fatty acid, polyethylene glycol, albumin, transferrin, or an immunoglobulin.
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Families Citing this family (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111349155B (en) * 2018-12-24 2022-04-05 浙江和泽医药科技股份有限公司 Glucagon analogue and preparation method and application thereof
CN112898404B (en) * 2019-12-03 2024-11-12 天津药物研究院有限公司 A long-acting modified glucagon peptide analog or its salt and its use
CN111040022B (en) * 2019-12-23 2021-12-14 万新医药科技(苏州)有限公司 Triple agonist targeting glucagon-like peptide-1 receptor, glucagon receptor, and gastric inhibitory peptide receptor
EP3842060A1 (en) * 2019-12-23 2021-06-30 Merck Sharp & Dohme Corp. Stapled lactam co-agonists of the glucagon and glp-1 receptors
EP3842061A1 (en) * 2019-12-23 2021-06-30 Merck Sharp & Dohme Corp. Stapled triazole co-agonists of the glucagon and glp-1 receptors
CN113493503B (en) * 2020-04-08 2022-08-05 浙江道尔生物科技有限公司 Incretin analogue and preparation method and application thereof
CN113278060B (en) * 2020-05-29 2022-03-25 东莞云璟生物技术有限公司 GLP-1/glucagon dual agonist fusion proteins
EP4249505A4 (en) * 2020-12-23 2024-08-21 Zhejiang Doer Biologics Co., Ltd. Long-acting glucagon derivative
CN114685642B (en) * 2020-12-29 2024-03-29 浙江和泽医药科技股份有限公司 Pharmaceutically acceptable salt of incretin analogue, and preparation method and application thereof
US20240116999A1 (en) * 2021-02-09 2024-04-11 The Trustees Of Indiana University Conformationally constrained glucagon analogues and their use in glucagon-single chain insulin fusion proteins
EP4357358A4 (en) * 2021-06-18 2024-11-06 Beijing Tuo Jie Biopharmaceutical Co. Ltd. Glucagon analog and medical use thereof
WO2024252366A1 (en) * 2023-06-09 2024-12-12 Sun Pharmaceutical Industries Limited Glp-1/gip dual, glp-1/gcg dual and glp-1/gip/gcg triple receptor agonists
CN117586374B (en) * 2023-10-07 2024-11-29 深圳湾实验室 GLP-1R/GIPR/GCGR triple agonist analogs and their uses
CN119930789A (en) * 2023-11-06 2025-05-06 成都奥达生物科技有限公司 A GCG agonist compound

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101534846A (en) * 2005-11-07 2009-09-16 印第安纳大学研究及科技有限公司 Glucagon analogs exhibiting physiological solubility and stability
CN102459325A (en) * 2009-06-16 2012-05-16 印第安纳大学科技研究有限公司 Gastrostatin receptor activated glucagon compounds
CN103857408A (en) * 2011-06-22 2014-06-11 印第安那大学科技研究公司 Glucagon/glp-1 receptor co-agonists
CN104902919A (en) * 2012-12-21 2015-09-09 赛诺菲 Dual GLP1/GIP or trigonal GLP1/GIP/Glucagon agonists
CN106414488A (en) * 2014-04-07 2017-02-15 赛诺菲 Peptide dual GLP-1/glucagon receptor agonists derived from exendin-4
WO2017189342A1 (en) * 2016-04-26 2017-11-02 Merck Sharp & Dohme Corp. Insulin dimer-incretin conjugates

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101663317A (en) * 2007-01-05 2010-03-03 CovX科技爱尔兰有限公司 glucagon-like protein-1 receptor (glp-1r) agonist compounds
BRPI0807728A2 (en) * 2007-02-15 2012-04-17 Univ Indiana Res & Tech Corp glucagon / glp-1 receptor co-agonists
MX2011006314A (en) * 2008-12-15 2011-09-22 Zealand Pharma As Glucagon analogues.
WO2010096052A1 (en) * 2009-02-19 2010-08-26 Merck Sharp & Dohme Corp. Oxyntomodulin analogs
US20130157953A1 (en) * 2010-01-20 2013-06-20 Zealand Pharma A/S Treatment of cardiac conditions
AR091478A1 (en) * 2012-06-21 2015-02-04 Univ Indiana Res & Tech Corp GLUCAGON ANALOGS EXHIBITING GIP RECEIVER ACTIVITY (GLUCOSE DEPENDENT INSULINOTROPIC PEPTIDE)
AR092873A1 (en) * 2012-09-26 2015-05-06 Cadila Healthcare Ltd PEPTIDES AS TRIPLE AGONISTS OF GIP, GLP-1 AND GLUGAGON RECEPTORS

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101534846A (en) * 2005-11-07 2009-09-16 印第安纳大学研究及科技有限公司 Glucagon analogs exhibiting physiological solubility and stability
CN102459325A (en) * 2009-06-16 2012-05-16 印第安纳大学科技研究有限公司 Gastrostatin receptor activated glucagon compounds
CN103857408A (en) * 2011-06-22 2014-06-11 印第安那大学科技研究公司 Glucagon/glp-1 receptor co-agonists
CN104902919A (en) * 2012-12-21 2015-09-09 赛诺菲 Dual GLP1/GIP or trigonal GLP1/GIP/Glucagon agonists
CN106414488A (en) * 2014-04-07 2017-02-15 赛诺菲 Peptide dual GLP-1/glucagon receptor agonists derived from exendin-4
WO2017189342A1 (en) * 2016-04-26 2017-11-02 Merck Sharp & Dohme Corp. Insulin dimer-incretin conjugates

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
A novel GIP-oxyntomodulin hybrid peptide acting through GIP，glucagon and GLP-1 receptors exhibits weight reducing and anti-diabetic properties;Vikas K. Bhat等;《Biochemical Pharmacology》;20130318;第85卷;第1655-1662页 *
A Novel Glucagon-like Peptide-1 (GLP-1)/Glucagon Hybrid Peptide with Triple-acting Agonist Activity at Glucose-dependent Insulinotropic Polypeptide, GLP-1,and Glucagon Receptors and Therapeutic Potential in and Glucagon Receptors and Therapeutic Potential;Victor A. Gault等;《THE JOURNAL OF BIOLOGICAL CHEMISTRY》;20131206;第288卷(第49期);第35581-35591页 *
AN OPTIMIZED NOVEL GLP-1-GIP RECEPTOR DUAL AGONIST WITH POTENT EFFECTS ON BODY WEIGHT AND GLUCOSE CONTROL IN MICE HAS THE POTENTIAL FOR ONCE-WEEKLY ADMINISTRATION IN HUMANS;Carsten Boye Knudsen等;《ZEALAND PHARMA A/S》;20150609;第1页 *
Characterization of insulin and atypically processed proglucagon-derived peptides from the Surinam toad Pipa pipa (Anura:Pipidae);Beverly Matutte等;《Peptides 》;20001231;第21卷;第1355-1360页 *
Design of Novel Exendin-Based Dual Glucagon-like Peptide 1 (GLP-1)/Glucagon Receptor Agonists;Andreas Evers等;《J. Med. Chem.》;20170427;第60卷;第4293-4303页 *
GLP-1/GIP/Gcg三受体激动剂改善阿尔茨海默病三转基因小鼠的认知行为;焦娟娟等;《生理学报》;20170425;第69卷(第2期);第135-145页 *

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