CN115304666A - Glucagon analogue for treating metabolic diseases - Google Patents

Glucagon analogue for treating metabolic diseases Download PDF

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CN115304666A
CN115304666A CN202210863408.6A CN202210863408A CN115304666A CN 115304666 A CN115304666 A CN 115304666A CN 202210863408 A CN202210863408 A CN 202210863408A CN 115304666 A CN115304666 A CN 115304666A
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glucagon
glp
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黄岩山
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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 the structural formula: 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, 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-1receptor (GLP-1R) agonists, such as dolaglutide (trade name:
Figure BDA0003757568040000011
) Albaglutide (trade name)
Figure BDA0003757568040000012
) Liraglutide (trade name)
Figure BDA0003757568040000013
And
Figure BDA0003757568040000014
respectively for treating obesity and diabetes), exenatide (trade name)
Figure BDA0003757568040000015
) Lixisenatide (trade name)
Figure BDA0003757568040000016
) And Semaglutide (Semaglutide) which may be about to be marketed. The dolaglutide, the albiglutide, the liraglutide and the somaglutide are all analogues 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 GLP with high and stable activity-1R agonist. Exenatide (Exendide-4) is a 39 amino acid small peptide from the salivary gland of lizards (Heloderma supectum). Exendin-4 is a potent agonist of GLP-1R, but the activity is higher than that of natural 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 BDA0003757568040000017
) 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 have also found that simultaneous infusion of GLP-1/Glucagon (GCG) (Tricia m. Tan etc., DIABETES, vol. 62, 1131-1138, 2013), or GLP-1, oxyntomodulin (Oxyntomodulin, OXM) and PYY, in patients has significant effects on promoting energy metabolism, suppressing appetite, and controlling body weight (Tricia Tan etc., J Clin Endocrinol Metab,2017,102 (7): 2364-2372). From a 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 new generation of diabetes drug development mainly seeks 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).
Currently, the following methods are mainly used for designing and developing the medicines: 1. based on the modification and development of human endogenous polypeptide Oxyntomodulin (OXYNTMODULIN, OXM) with GLP-1/GCG dual activity. OXM is a polypeptide naturally found in humans with dual GLP-1 and GCG activities (Diabetes, 2005, 54. 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 (LY 2944876) is another PEG-modified OXM analog (Chakradhar, shraddha. "All in one: research company drugs for diabetes and obesity." Nature Medicine, vol.22, no.7,2016: 694-5). PSA-OXM is an OXM analog modified with polysialic acid (Vorobiev I et al, biochimie,2013, 95 (2): 264-70). However, 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 (increten) sequence, and obtains 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 BDA0003757568040000021
Etc., molecular polysaccharides for Treatment of Diabetes and obesitiy, 24:51-62,2016). Matthias H.
Figure BDA0003757568040000022
The review article of et al describes in detail the various hybrid peptide forms currently in clinical or preclinical form. As reported by Alessandro Pocai et al, an OXM-based Dual-effect GLP-1R/GCGR agonist (Glucaon-Like Peptide 1/Glucaon Receptor Dual agonist responses in Mice, diabetes;58 (10): 2258-2266, 2009), or a GCG-based Dual-effect GLP-1R/GCGR agonist (US 9018164B 2) reported by Richard D. DiMarchi et al, or even a triple-effect GLP-1R/GCG/GIPR agonist (US 9150632). Most of these multispecific hybrid peptides are based on GLP-1 or GCG, and have improved activity and resistance to proteolysis by sequence mutations, 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, and the addition of a fatty acid chain or polyethylene glycol (PEG) modification to increase half-life, with the clinically expected dosing cycle being 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 native 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-Ser 2-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 microorganism, diabetes.
Although the research and development of molecules with multiple GLP-1R, GCGR and GIPR agonistic 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 stable 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. Taspogliptine, a GLP-1receptor agonist developed by Roche and Yipu in combination, reaches 49% of antibody production rate, and all clinical phase III studies have been suspended (JuLIO ROSENSTOCK et al, the fact of Taspogliptin, a Weekly GLP-1receptor agonist, versus with-Daily Exenatide for Type 2, DIABETES CARE, 36. PHIL AMBERY et al (THE ENDOCRINOLOGIST, SPRING,2017, 12-13) screened more than 500 structures on THE basis of THE sequence of GCG, and only obtained a candidate peptide MEDI0382. 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 (10): 4293-4303) introduce 9 mutation sites on the basis of the structure of Exendin-4, the mutation rate reaches about 23%, and fatty acid chain modification is carried out, so that the hybrid peptide with high GLP-1 and GCG dual activities is obtained; 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 prior technical scheme often introduces more mutation sites, and often introduces unnatural amino acids and other modifications to obtain the polypeptide with 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 agonist peptides or GLP-1R/GCG/GIPR triple-effect agonist 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 a good blood sugar-reducing and weight-losing effect; however, WO2015155139A1 and WO2015155140A1 are prepared into GLP-1R/GCGR double-effect agonist peptides in order to avoid the risk of hypoglycemia caused by GIPR agonist activity, and have better hypoglycemic and weight-losing effects. Seth et al also believe that the introduction of GIP activity does not enhance the effect of GLP-1 on glycemic control (A. Seth et al, co-administration of a localized GIPR agonist with a GLP-1 expression no additional functional benefit on HbA1c; "over GLP1 expression in db/db mice, 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 biology of life-treating hypoglycobiology, molecular Metabolism, 3. 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. Instead, the GCGR agonistic activity was enhanced as reported by Jonathan W Day et al (Jonathan W Day et al, A new glucagon and GLP-1 co-aginst eliminates obesites in rodents, nature Chemical Biology, 5, 749-757, 2009) by performing different mutations at position 16 of GCG, which is in complete contradiction to the alanine scan results of Joseph channel. 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 retaining GCG activity (only 1/3 of the activity is retained when mutation is made to Ala), but Brian Finan et al report (Finan B et al, A ratio all designed monomeric peptide mutation residues and nucleotides in cadences. Nat. Med.2015;21, 27-36.) that 2S → Aib, 2S → dSer, 2S → G, 2S → dAla substitution mutations are respectively made on the second position amino acid of GCG, and the relative agonistic activity of GCGR is increased to 200% -640% when mutation at other positions is combined. In our studies it has also been found that the effect of a combination of mutations which is beneficial for increasing GLP-1, GCG or GIP activity is often completely inconsistent with that of a single site mutation. 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, which results in a loss of about 90% of GCGR agonistic activity (Alessandro Pocai et al, gluconon-Like Peptide 1/Gluconon Receptor Dual agonist peptides Mice, diabetes;58 (10): 2258-2266, 2009, henderson SJ et al, robust anti-activity and metallic effects of Dual GLP-1/Gluconon Receptor peptides in mutants and humans, diabetes probes Metab, 2016).
As also reported by Joseph R.Channne and Richard D.DiMarchi et al, the addition of a small C-terminal peptide cex of Exendin-4 (SEQ ID NO.67, GPSSGAPPPS) at the C-terminus of Gluguaon increased GLP-1R agonist activity from 0.7% to 1.6%, by about 2-fold (Optimization of the Native Gluguaon Sequence for Medicinal purpose purpos, J Diabetes Sci Technol.4 (6): 1331, 2010 and US 9018164B 2), and also lost about 50% GCG activity. Evers A et al also reported that (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) the GLP-1R agonistic activity decreased by about 2/3 on the contrary, but the agonistic activity of GCG was more than 90% lost after adding cex sequence to the C-terminal of GCG analogue (Table 2, peptides 7and 8 in the article). Thus, for a small peptide of 30 amino acids length such as GLP-1,glucagon, the change in sequence is extremely sensitive to changes in its activity; in the case of dual active polypeptides, the changes are more complex due to the agonism involved at the two different receptors, and it is not at all predictable what consequences of GLP-1R and GCGR agonistic activity will be after any one amino acid change.
The complexity of downstream signaling at 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 including 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, and the physiological effects of the activation of the different channels are different, even the relationship between the activity and the physiological function of the channels is unknown. For example, by introducing different mutations or different amino acid sequences into the GLP-1 sequence, structures of different preferential agonism (biased-agonist) can be obtained, resulting in different physiological effects (Marlies V. Et al, J Am Chem Soc., 138 (45): 14970-14979, 2016 Hongkai Zhang et al, nat Commun.,6 8918, 2015.
Thus, although it is theoretically very clinically meaningful to design a polypeptide having high activities of GLP-1, glucagon and GIP at the same time, it is actually very difficult. If a multi-active hybrid peptide with balanced activity, but with less introduction of mutation sites and unnatural amino acids as possible, which is close to a natural sequence as possible, is designed, the potential immunogenicity is lower, the stability is improved, and the blood sugar control and the weight control are good, the multi-active hybrid peptide is very clinically significant.
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 which exhibits GLP-1R/GCGR/GIPR triple receptor agonistic activity.
Native GCG has approximately 1% GLP-1R agonist activity relative to native GLP-1, but no GIPR agonist 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 absent, 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 end of the glucagon analogue is subjected to amidation modification.
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 glucagon analog has the structural formula:
HSQGTFTSD-Y-SKYLD-X 16 -X 17 -AAQDFVQWLMN-X 29 -X z -NH 2 (SEQ ID NO. 4) means that the C-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 Endocrinol Metab,95,1851-1855,2010 a Hadji-Georgopoulos, jclin Endocrinol Metab,56,648-652, 1983). 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 in the literature that antagonism of the GIPR is also a desirable approach for reducing daily feeding, reducing body weight, increasing insulin sensitivity and energy expenditure (Irwin et al, diabetologia 2007,50,1532-1540, althrage et al, J Biol Chem,2008,283 (26): 18365-18376. In a mouse animal model test, compared with a comparative analogue with lower GIP activity or even no GIP activity, the glucagon analogue with higher GIP activity provided by the invention has more remarkable effects on daily food intake control, weight loss and insulin sensitivity improvement of obese mice.
The glucagon analogues of the invention have better enzyme-resistant stability, including Neutral Endopeptidase (NEP) and dipeptidyl peptidase-4 (DPP-4); has longer half-life and sustained action time in vivo compared with natural glucagon, GLP-1, GIP.
In a second aspect of the invention, there is provided an isolated polynucleotide encoding a glucagon analogue as described above.
In a third aspect of the invention, there is provided a recombinant expression vector comprising the isolated polynucleotide as described above.
In a fourth aspect of the invention, there is provided a host cell comprising the recombinant expression vector or the isolated polynucleotide having exogenous sequences 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 analogue by using 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 described above 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, and the like.
Metabolic syndrome is associated with 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 dysfunction, 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 such as reducing appetite, reducing food intake, reducing fat levels in the body of the patient, increasing 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 aforementioned glucagon analog to the subject.
In one embodiment, the glucagon analogs are used in the present invention for the treatment of 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 invention, there is provided the 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.
Those skilled in the art will appreciate that modifications may be made to the glucagon analogs of the present invention in order to increase their half-life or stability, for example, polyethylene glycol or derivatives thereof, hydroxyethyl starch derivatives or fatty acids may be covalently attached 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, has a longer half-life and duration of action in vivo than native glucagon, GLP-1, GIP. In view of the above, the GCG analogs reported so far generally employ (1) a single-molecule hybrid peptide-crosslinked fatty acid, PEG, FC, or the like, and are administered at a frequency of once a day or more (mathhias H).
Figure BDA0003757568040000091
Etc., molecular weights for Treatment of Diabetes and obesitiy, 24:51-62,2016); or (2) mutating the second-position Ser of natural GCG into D-Ser and other unnatural amino acids to resist DPP-IV degradation (Novel DPP IV-resistant C-terminated glucose and amino acids in high-fat-fed microorganism treated with sodium glucose and GLP-1receptor activation, diabetes 57 1927-1936, 2014), and the administration is performed twice a day. 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 pH 7.4 aqueous solution.
FIG. 4 is a schematic view of: HPLC profile of polypeptide No. C002 in aqueous pH 7.4 solution.
FIG. 5 is a schematic view of: 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 of several glucagon analogs are shown to have detectable agonistic activity towards GLP-1R.
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 glucagon analogues and the control stimulated the cAMP content produced by the GIPR 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 cAMP content stimulated by GIPR production for the glucagon analogs and controls at different concentration gradients.
FIG. 9H: the glucagon analogues and the control stimulated the cAMP content produced by the GIPR 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: compare the weight loss in DIO mice.
FIG. 13: the graph compares 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 reduces 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:
native GLP-1, glucagon, or Oxyntomodulin (Oxyntomodulin) are not truly clinical drugs due to too poor serum stability and too short in vivo half-life. But also onlyExenatide (Exenatide, trade name) of 39 amino acids
Figure BDA0003757568040000111
) But successfully marketed due to the improved stability. In one embodiment 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 the treatment of metabolic-related diseases, such as drugs in the fields of diabetes, obesity, etc., are extremely high. The glucagon analogue obtained by the invention has lower immunogenicity under the condition of introducing no more than 3 mutation sites, and reaches the ideal activity and stability standard, 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 inhibition, 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 disorders, and thus the efficiency of glucose uptake by 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) above 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. 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 progoglucagon-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 (Thiarakan G, tan T, bloom S. Expanding therapeutics in the treatment of diabetes 2011: beyond GLP-1.Trends Pharmacol Sci.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-1receptor assays for the diabetes of biological Chem Lett 20123 (14): 4011-8.Tomlinson B, hu M, zhang Y, chan P, liu ZM.Ann. overview of new GLP-1 receptors for type 2diabetes of expert Opin Drugs 2016 (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). In overweight and obese patients, subcutaneous administration of natural oxyntomodulin reduced body weight by 1.7 kg over four weeks. Oxyntomodulin has also been shown to reduce food intake and increase energy expenditure in humans (Subcutaneous oxypodulin recovery body weight in overhead and object subjects: a double-blind, randomized, controlled trial, wynne K et al, diabetes, 54. But again 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-1receptor (GLP-1R) and a glucagon receptor (GCGR) are generally based on oxyntomodulin, mutations (oxyntomodulin analogues) are made in order to improve the short-acting property and enzymolysis defect of the oxyntomodulin, and a method of mutating a second-position serine Ser into alpha-amino isobutyric acid (Aib) or D-Ser is adopted mostly to resist the enzymolysis of DPP-IV by introducing non-natural amino acid. Although the oxyntomodulin analogue shows primary blood sugar reducing and fat reducing effects, the action mechanism is still uncertain, and an oxyntomodulin receptor is not found, and the oxyntomodulin can be combined with the 2 receptors to play a role only through mouse knockout of GCGR or GLP-1R 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, 689-697) and these effects are stable and sustained in rodents. Glucagon has many physiological effects, such as by stimulating glycogenolysis and gluconeogenesis, increasing blood glucose levels 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 the same or a similar characteristic response as 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 double-effect agonist comprises a protein or polypeptide which can stimulate GLP-1R and GCGR simultaneously. Oxyntomodulin-based Dual-effect agonists as reported by Alessandro Pocai et al (Alessandro Pocai et al, glucaon-Like Peptide 1/Glucaon Receptor Dual agonist responses in Mice, diabetes;58 (10): 2258-2266, 2009), or Glucaon-based Dual-effect agonists as reported by Richard D.DiMarchi et al (US 9018164B 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) means the concentration of a drug or substance required to stimulate 50% of its corresponding biological response. A lower EC50 value indicates a stronger stimulation or stimulation of the drug or substance, e.g. more intuitively it may appear that a stronger intracellular signal is elicited, and thus a better 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. Which 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 in 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 remove LDL from the artery, acts as a scavenger, and removes LDL from the artery and 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 underlying or genetic diseases are 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. The G protein 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) dimethylaminomethylmorpholinomethylene) ] 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 embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. 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 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, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.
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 are described in particular in Sambrook et al, molecular CLONING: a LABORATORY MANUAL, second edition, cold Spring Harbor LABORATORY Press,1989and 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; METHOD 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 analogs
Using prior art techniques, e.g. Prior documents: (
Figure BDA0003757568040000171
V. et al, beilstein j. Org. Chem., 10; palomo, j.m., RSC adv.,4(2014) (ii) a The polypeptides to which this patent relates are prepared by solid phase synthesis and modification of polypeptides in behredt, r. et al, j.pept.sci., 22.
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 (0.25 mmol/g,1 g) was swollen in NMP (10 mL) before use, added to a solid phase synthesis apparatus, piperidine/DMF (20%, 10 mL) was added to the resin to react for 30min for Fmoc deprotection, drained, washed with DMF (5 × 10 mL), and drained. The first Fmoc-amino acid solution (0.2M, NMP/DMF/DCM, 1. piperidine/DMF (20%, 10 mL) was then added for 30min for Fmoc deprotection, suction dried, washed with DMF (5X 10 mL), suction dried. The above steps of Fmoc-amino acid addition for reaction and piperidine/DMF deprotection for Fmoc-deprotection were repeated until the final histidine coupling was completed.
Resin was washed with EtOH (3X 10 mL) and Et 2 O (3X 10 mL) was washed 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,40 mL), 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. The crude peptide was purified by preparative reverse phase HPLC using a preparative liquid chromatograph model Waliana SD-1 equipped with a C-18 column and a partial trap, 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%, the resulting peptides being 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 (0.4 mmol/g,1 g), 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 assigned monomeric peptide derivatives depletion objects and diabetes in rodents. Nat. Med.2015; 21-36.) or Chhabr et al (Chhabr et al, apparatus of New variant 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. The mass spectrometry conditions were as follows:
the instrument comprises: waters ZQ 2000
Mass spectrum (Probe): ESI
Nebulizer Flow rate (Nebulizer Gas Flow): 1.5L/min
CDL：-20.0v
CDL temperature: 250 deg.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 BDA0003757568040000181
Figure BDA0003757568040000191
Figure BDA0003757568040000201
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 having the structure shown in formula I below:
Figure BDA0003757568040000202
TABLE 2
Figure BDA0003757568040000203
Figure BDA0003757568040000211
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 polypeptide to be tested (glucagon analogue) and the control were prepared in 20mM phosphate buffer PB or acetate buffer at a pH corresponding to a final concentration of 0.2mg/ml and sterile filtered using 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 samples not incubated 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 t 7) × 100 ]/peptide peak area t0.
Stability is expressed as "residual peptide content".
Detection method
Detection wavelength: 214nm;
a chromatographic column: column temperature 40 deg.C, phenomenex Luna C8 (2) 5 μm (150X 4.6 mm);
mobile phase: h 2 O +0.1% of TFA ACN +0.1% of TFA (flow rate 1.0 ml/min);
gradient: 95 (0 min) to 0;
and (3) analysis of experimental results: 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 BDA0003757568040000221
Figure BDA0003757568040000231
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 BDA0003757568040000232
the corresponding integrated data of fig. 2:
Figure BDA0003757568040000233
raw data corresponding to fig. 3:
Figure BDA0003757568040000241
raw data corresponding to fig. 4:
Figure BDA0003757568040000242
raw data for fig. 5:
Figure BDA0003757568040000243
raw data corresponding to fig. 6:
Figure BDA0003757568040000251
raw data for fig. 7:
Figure BDA0003757568040000252
example 3: serum stability
(1) The corresponding polypeptide in Table 1 was prepared in 1.0mg/ml concentration using 5mM Tris-HCl, pH8.5,0.02% TWEEN80 solution, sterile filtered (0.22 μm, millipore SLGP033 RB), diluted 10-fold with rat serum, mixed well and dispensed into sterile centrifuge tubes;
(2) Freezing 3 tubes of the samples at-20 ℃ respectively as a reference, placing the rest in a constant temperature box at 37 ℃, and sampling at different time points to detect the 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 fig. 8A and 8B.
TABLE 4
Figure BDA0003757568040000261
Figure BDA0003757568040000271
Note: n.d. indicates below the lower limit of detection
Example 4: cell viability assay
(one) 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 proportion of 1 to 10 in a molar ratio, and stably-transformed expression strains are screened.
Culturing the cells in a 9-cm cell culture dish with a DMEM/F12 medium containing 10% FBS and 300. Mu.g/ml G418, when the degree of confluence is about 90%, discarding the culture supernatant, after adding 2ml of trypsin for digestion for 3min, adding 2ml of DMEM/F12 medium containing 10% FBS and 300. Mu.g/ml G418 for neutralization, transferring to a 15ml centrifuge tube, after centrifuging at 1000rpm for 5min, discarding the supernatant, adding 2ml of DMEM/F12 medium containing 10% FBS and 300. Mu.g/ml G418 for resuspension, and counting. Diluting the cells 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, the medium was changed to DMEM/F12 medium containing 0.2% FBS. After discarding the supernatant 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 luciferae 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 cell strain is the same as the above. FIGS. 9C and 9D show the results of GCGR agonistic activity assays.
TABLE 5
Figure BDA0003757568040000281
Figure BDA0003757568040000291
(III) GIPR agonistic 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 antibody enzyme weights-lower and ligands-protective effects in high-fat-glucose-induced peptide activity, aisling M.Lynch et al, diabelogia, 57 1927-1936, 2014) rat BRIN-BD11 cells were used for the determination of insulin release by active protein stimulation, with a slight modification that 1.0X 106 cells per well were added in 24 well plates (Orange Scientific, brannel' Alleud, belgium), the supernatant was centrifuged after 37 ℃ and 1.0 mM NaCl per well was added (115 mM NaCl, 4.7mM, caCl 1.7.7.7 mM) overnight 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: immunogenicity experiments in mice
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 (30 nmol/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. The enzyme label plate is coated with the corresponding polypeptide,mouse sera were run as 1:50;1:200,1:1000,1: diluting with 5000 gradient, adding enzyme label plate, and detecting with goat anti-mouse antibody. Serum was used as negative control before each mouse administration, and OD was tested 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 BDA0003757568040000301
Example 7: glucose Tolerance Test (IPGTT) in Normal ICR mice
Normal ICR mice were divided into 27 groups of 6 mice each. After fasting overnight, tail blood was collected (scored as t = 0min blood glucose sample), vehicle control (acetate buffer, 20mM acetic acid, 250mM mannitol, pH5.0) and glucagon analogues of the invention in Table 1 (30 nmol/kg in PBS buffer) and vehicle control, liraglutide (R) (Trade name
Figure BDA0003757568040000302
Figure BDA0003757568040000303
40nmol/kg, diluted in PBS buffer). Glucose (2 g/kg body weight) was intraperitoneally injected 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 the body weight was about 45g for the test. DIO mice were randomly divided into groups of 6 mice each, with no difference in basal body weight, and each group of mice was subcutaneously injected with glucagon-like peptide dailyEqual volume of substance (30 nmol/kg in PBS) or PBS, control group liraglutide (trade name)
Figure BDA0003757568040000311
30 nmol/kg), 2 times daily, weighing to 30 days daily.
Figures 12A-C are daily changes in DIO mouse body weight after administration of each glucagon analog, and the final percent weight loss is shown in figure 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 (30 nmol/kg in PBS) or PBS, respectively, and a control group of liraglutide (trade name: liraglutide)
Figure BDA0003757568040000312
30nmol/kg, diluted in PBS) 2 times daily and the fatty-acidified GCG analogue (30 nmol/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.

Claims (10)

1. A glucagon analogue, the structure of the glucagon analogue contains a structure shown as a formula I or a formula II, the structure shown as the formula I is as follows:
HSQGTFTSD-X 10 -SKYLD-X 16 -X 17 -AA-X 20 -X 21 -F-X 23 -QWLMN-X 29 -X z ，
the structure shown in formula II is:
HSQGTFTSD-X 10 -SKYLD-X 16 -X 17 -AA-X 20 -X 21 -F-X 23 -QWLMN-X 29 -X z -NH 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 Is selected from any one of Q or R; x 21 Selected from either D or E; x 23 Selected from either V or I; x 29 Is T or absent, X z Selected from any one of GGPSSGAPPPS, GGPSSGAPPS, GPSSGAPPPS, GPSSGAPPS, PSSGAPPPS, SSGAPPPS, or SSGAPPS;
the sequence of the glucagon analogue is shown in any one of SEQ ID NO 6-11, SEQ ID NO 14-35, SEQ ID NO 44-53 and SEQ ID NO 60-64.
2. An isolated polynucleotide encoding the glucagon analog of claim 1.
3. A recombinant expression vector comprising the isolated polynucleotide of claim 2.
4. A host cell comprising the recombinant expression vector of claim 3 or the isolated polynucleotide of claim 2 integrated into the genome.
5. The method of preparing a glucagon analog of claim 1, selected from the group consisting of:
(1) Synthesizing the glucagon analog by a chemical synthesis method;
(2) Culturing the host cell of claim 4 under suitable conditions to allow expression of said glucagon analog, followed by isolation and purification to obtain said glucagon analog.
6. A composition comprising a culture of the glucagon analog of claim 1 or the host cell of claim 4, and a pharmaceutically acceptable carrier.
7. A fusion protein comprising the glucagon analog of claim 1 in its structure.
8. The fusion protein of claim 7, wherein the structure of the fusion protein further comprises a long-acting unit, preferably the long-acting unit is selected from the group consisting of albumin, transferrin, and immunoglobulin and fragments.
9. Use of a glucagon analogue or fusion protein according to any one of claims 1, 7 or 8 in the manufacture of a medicament for the treatment of a metabolic-related disorder.
10. A modified polypeptide comprising the glucagon analog of claim 1, wherein said glucagon analog is modified with a fatty acid, polyethylene glycol, albumin, transferrin, or an immunoglobulin and fragment.
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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
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EP3842061A1 (en) * 2019-12-23 2021-06-30 Merck Sharp & Dohme Corp. Stapled triazole co-agonists of the glucagon and glp-1 receptors
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CA2913805A1 (en) * 2005-11-07 2007-05-18 Indiana University Research And Technology Corporation Glucagon analogs exhibiting physiological solubility and stability
WO2010096052A1 (en) * 2009-02-19 2010-08-26 Merck Sharp & Dohme Corp. Oxyntomodulin analogs
BRPI1014508A2 (en) * 2009-06-16 2016-04-05 Univ Indiana Res & Tech Corp glucagon peptide analogs, dimer of two glucagon peptides, pharmaceutical composition derived from them and methods for their use "
RS56173B1 (en) * 2011-06-22 2017-11-30 Univ Indiana Res & Tech Corp Glucagon/glp-1 receptor co-agonists
PL2864350T3 (en) * 2012-06-21 2019-01-31 Indiana University Research And Technology Corporation Analogs of glucagon exhibiting gip receptor activity
AR094181A1 (en) * 2012-12-21 2015-07-15 Sanofi Sa GLP1 / GIP OR TRIGONAL DUAL AGONISTS OF GLP1 / GIP / GLUCAGON
TW201625669A (en) * 2014-04-07 2016-07-16 賽諾菲公司 Peptidic dual GLP-1/glucagon receptor agonists derived from Exendin-4
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