CN109836488A - A kind of glucagon analogue for treating metabolic disease - Google Patents

A kind of glucagon analogue for treating metabolic disease Download PDF

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CN109836488A
CN109836488A CN201711194175.0A CN201711194175A CN109836488A CN 109836488 A CN109836488 A CN 109836488A CN 201711194175 A CN201711194175 A CN 201711194175A CN 109836488 A CN109836488 A CN 109836488A
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ser
glucagon
artificial sequence
glp
glucagon analogue
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CN109836488B (en
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黄岩山
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Zhejiang Doer Biologics Technology Co Ltd
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Zhejiang Doer Biologics Technology Co Ltd
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Priority to PCT/CN2018/116166 priority patent/WO2019101035A1/en
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Abstract

The present invention relates to field of biological medicine, more particularly to a kind of glucagon analogue for treating metabolic disease, structural formula are as follows: H-X2‑X3‑GTFTSD‑X10‑SKYLD‑X16‑X17‑AAQ‑DFVQWLMN‑X29‑XzOr H-S-Q-GTFTSD-Y-SKYLD-X16‑X17‑AAQ‑DFVQWLMN‑X29‑Xz‑NH2.Glucagon analogue of the invention, with tri- receptor agonist activity of GLP-1/GCG/GIP, and better resistance to enzyme stability, including to neutral endopeptidase (NEP) and dipeptidyl peptidase-4 (DPP-4), with natural glucagon, GLP-1, GIP, which are compared, has longer Half-life in vivo and continuous action time.

Description

A kind of glucagon analogue for treating metabolic disease
Technical field
The present invention relates to field of biological medicine, more particularly to a kind of glucagon analogue for treating metabolic disease and Preparation method and use.
Background technique
Diabetes are a kind of serious chronic diseases, when pancreas can not produce enough insulin or human body can not be effectively When using generated insulin, it just will appear diabetes.The protide diabetes medicament listed at present is mainly GLP-1 receptor (GLP-1R) agonist, as Du Lalu peptide (Dulaglutide, trade name:), albiglutide (Albiglutide, Trade name), Liraglutide (Liraglutide, trade nameAndIt is respectively used to treat Fat and diabetes), Exenatide (Exenatide, trade name), sharp hila peptide (Lixisenatide, trade name) and the possible Suo Malu peptide (Semaglutide) etc. that will be listed.Du Lalu peptide, albiglutide, Liraglutide And Suo Malu peptide is all the analog of natural glucagon sample peptide -1 (GLP-1), after GLP-1 series jump respectively with FC segment, human albumin and the fatty acid fusion or crosslinking of IgG, obtains activity height and stable GLP-1R agonist.And Ai Sai That peptide (Exenatide, i.e. Exendin-4) is then a kind of 39 for deriving from lizard (Heloderma suspectum) glandula Aminoacid small peptide.Although Exendin-4 is the potent agonist of GLP-1R, but activity is all than natural GLP-1 and GLP-1 analog It is high.Although the agonist of these GLP-1R can be effectively reduced blood glucose, appetite is controlled, the effect for loss of weight is not It is too significant.Wherein Liraglutide (trade nameAlthough) by granted for treating obesity, actually its weight Mitigate probably only 5.6 kilograms.It is currently used for fat drug loss of weight generally in 5-10% or so (compared with placebos), i.e., whole The ratio of average loss of weight is no more than 10% (Rudolph L.Leibel etc., Biologic Responses of patient's weight on body to Weight Loss and Weight Regain:Report From an American Diabetes Association Research Symposium, Diabetes, 64 (7): 2299-2309,2015).
Bariatric surgery (Bariatric surgery) can significantly improve obesity and treatment diabetes, however it is applied Not extensively, it because of the considerations of most patient is for operation risk and long-term sequelae, is reluctant to receive this operation (Obesity and Diabetes, New Surgical and Nonsurgical Approaches, Springer publishing house, 2015).(the Obesity and the study found that patient's duodenin (Incretin) secretion through surgical treatment of obesity operation can increase sharply Diabetes, New Surgical and Nonsurgical Approaches, Springer publishing house, 2015).It is preclinical And clinical research is, it was also found that be transfused GLP-1/ glucagon (Glucagon, GCG) (Tricia M.Tan to patient simultaneously Etc., DIABETES, VOL.62:1131-1138,2013) or GLP-1, oxyntomodulin (Oxyntomodulin, OXM) and PYY, for promoting energetic supersession, appetite-suppressing controls weight (Tricia Tan etc., J Clin 2017,102 (7): Endocrinol Metab 2364-2372) has positive effect.It, can be simply for clinic needs These polypeptides are directly mixed for clinic.But due to the internal stability of these different types of polypeptides and degradation speed Difference causes final drug effect in vivo uncontrollable, is really difficult to use after simply mixing these polypeptides as compound medicine. Therefore, the diabetes medicament exploitation of a new generation mainly is tried to concentrate on these agonist activities in one molecule at present, such as GLP-1R/GIPR and GLP-1R/GCGR dual agonist or even GLP-1R/GIPR/GCGR triple effect agonist (Chakradhar, Shraddha.All in one:researchers create combination drugs for diabetes and Obesity.Nature Medicine, 22 (7): 694-695,2016).
Current this kind of drug designs and develops the mode of being mainly the following: 1. based on GLP-1/GCG double activity Human endogenous's polypeptide oxyntomodulin (Oxyntomodulin, OXM) modification exploitation.OXM is that a kind of human body naturally has The polypeptide (Diabetes, 2005,54:2390-2395) of GLP-1 and GCG double activity.But the activity of OXM is not high (to be had About 10% GCG activity and about 1% GLP-1 activity), and stability and half-life period are all very poor in vivo, therefore OXM nothing itself Method is directly used in clinic, generally requires by introducing unnatural amino acid, various modifications etc. improve its activity in vivo and stabilization Property.If the Mod-6030 of OPKO Biologics is exactly the long-acting OXM (Oren that N-terminal has carried out that degradable PEG is modified Hershkovitz:Presentation Number:SAT-787.The Endocrine Society's 95th Annual Meeting and Expo,June 15–18,2013-San Francisco).TT401 (LY2944876) is then another PEG OXM analog (Chakradhar, Shraddha. " All in the one:researchers create of modification combination drugs for diabetes and obesity."Nature Medicine,vol.22,no.7,2016: 694-5).PSA-OXM then be with polysialic acids (polysialic acid) modification OXM analog (Vorobiev I etc., Biochimie, 2013,95 (2): 264-70).But the reasons such as the low activity of OXM, stability difference are limited to, clinical effectiveness is simultaneously Bad, most of research has been abandoned.2. using the homology in duodenin (incretin) sequence, in OXM, GLP- By multiple mutation, modification in the structure basiss such as 1 and GCG, or even unnatural amino acid is introduced to obtain the stabilization of multiple activities Hybrid peptide (Matthias H.Deng Unimolecular Polypharmacy for Treatment of Diabetes and Obesity, 24:51-62,2016).Matthias H.Deng survey article at large introduce It is currently in clinical or preclinical various hybrid peptide forms.Pair based on OXM of such as Alessandro Pocai report Imitate GLP-1R/GCGR agonist (Glucagon-Like Peptide 1/Glucagon Receptor Dual Agonism Reverses Obesity in Mice, Diabetes；58 (10): 2258-2266,2009) or Richard The economic benefits and social benefits GLP-1R/GCGR agonist (US9018164B2) based on GCG of the reports such as the D.DiMarchi even GLP- of triple effect 1R/GCG/GIPR agonist (US9150632).The hybrid peptide of these polyspecifics is mostly based on GLP-1 or GCG, passes through sequence Mutation for D type amino acid (such as D-Ser) or introduces to improve activity and resistance protease hydrolytic, such as by L-type amino acid mutation Unnatural amino acid Aib improves internal stability, while carrying out fatty acid chain or polyethylene glycol (PEG) modification etc. to extend half It declines the phase, the expected dosage period of clinic is once a day (fatty acid modifying) or weekly (PEG modification).In addition, Aisling M.Lynch etc. is reported the second Ser of natural GCG being sported D-Ser, and introduces the C-terminal of Exendin-4 in C-terminal The GCG analog (D-Ser2-glucagon-exe) of peptide, and effect experiment has been carried out in DIO body, it is administered twice daily (Novel DPP IV-resistant C-terminally extended glucagon analogue exhibits weight-lowering and diabetes-protective effects in high-fat-fed mice mediated Through glucagon and GLP-1 receptor activation, Diabetologia:57:1927-1936, 2014), weight loss effect is obvious.
It is to have very much clinical landscapes although researching and developing the molecule with the multiple agonist activity of GLP-1R, GCGR, GIPR , but an ideal this kind of drug is really obtained, it is actually very difficult.
It is safety issue first, especially Immunogenicity.Hypoglycemic weight-reducing class drug needs to be used for a long time, to safety Property require it is high.A polypeptide with GLP-1, GCG and GIP high activity and stable in vivo is obtained in order to design, it is existing Technical solution all often all introduce more mutational site, and often introduce unnatural amino acid and other modification.This A little mutation and the introducing of unnatural amino acid, both increase the risk of potential immunogenicity.Have under normal circumstances with source of people sequence There is higher homology, immunogenicity risk is with regard to opposite lower in human body.The GLP-1 of Roche and the general raw R & D Cooperation of benefit by Body agonist antidiabetic drug Taspoglutide (only introduces 2 unnatural amino acid Aib), and antibody tormation rate has reached 49%, At present had timed out all third stages research (JULIO ROSENSTOCK etc., The Fate of Taspoglutide, a Weekly GLP-1 Receptor Agonist,Versus wice-Daily Exenatide for Type 2, DIABETES CARE,36:498-504,2013).PHIL AMBERY etc. (THE ENDOCRINOLOGIST, SPRING, 2017: A structure more than 500 12-13) has been screened in the sequence basis of GCG, just obtains a candidate peptide MEDI0382.Wherein, in order to Higher GLP-1 and GCG double activity and internal stability are kept, compared with GCG, MEDI0382 has introduced 9 mutational sites, Mutation rate has reached about 30%；Equally, (the J Med Chem.2017May 25 such as Andreas Evers；60(10):4293- 4303) 9 mutational sites are introduced in the structure basis of Exendin-4, mutation rate has reached about 23%, and has carried out fat Sour chain modification, just obtains while having the hybrid peptide of higher GLP-1 Yu GCG double activity；(the Brian such as Brian Finan Finan etc., Nat Med.21:27-36,2015) tri- active peptide of GLP-1/GCG/GIP of design is joined in the C-terminal of GCG GPSSGAPPPS sequence, and 7 mutating acids are introduced, including second sports unnatural amino acid Aib.Therefore, existing Some technical solutions often all introduce more mutational site, and often introduce unnatural amino acid and other modifications, It can obtain while have the polypeptide of GLP-1, GCG and GIP high activity.These mutation, modification and the introducing of unnatural amino acid, all Increase the risk of potential immunogenicity.And for treating diabetes, the drug of fat this kind of disease, safety is of crucial importance 's.Therefore, one kind is developed without unnatural amino acid, and includes the high activity multiple-effect GLP-1/ of mutating acid few as far as possible The multiple agonist of GCG, is significantly.
On the other hand, existing research is to the suitable ratio how combined between the activity and activity of these duodenins Example, does not there is public opinion also always.As PCT application WO2015155139A1, WO2015155140A1 and WO201515541A1 disclose The GLP-1R/GCGR economic benefits and social benefits excitement peptide or GLP-1R/GCG/GIPR triple effect excitement peptide being transformed based on Exendin-4.Wherein WO201515541A1 discloses a kind of hybrid peptide with GLP-1R/GCGR/GIPR triple effect agonist activity, research think this three The peptide for imitating agonist activity has good hypoglycemic fat-reducing effect；But WO2015155139A1 and WO2015155140A1 be then for Risk of hypoglycemia caused by GIPR agonist activity is avoided, is prepared into GLP-1R/GCGR economic benefits and social benefits excitement peptide, is had instead better Hypoglycemic fat-reducing effect.The researchs such as A.Seth also think that GLP-1 can not be enhanced to glycemic control effect by introducing GIP activity (A.Seth etc., Co-administration of a lipidated GIPR agonist with a GLP-1analogue Provides no additional benefit on HbA1c%over GLP1 analogue in db/db mice, EASD virtual meeting, 2015).
Again, sequence very high homology this kind of for GLP-1, Exendin-4, GCG, receptor belong to GPCR family, peptide chain It is right after single locus mutation or several site simultaneous mutations for length only has the small peptide of 30-40 amino acid or so The activity change of isoacceptor is not very difficult to prediction, therefore is also pole to obtain ideal multiple agonist activity hybrid peptide Its difficulty.Such as Joseph Chabenne etc. reports (Joseph Chabenne etc., A glucagon analog chemically stabilized for immediate treatment of life-threatening Hypoglycemia, Molecular Metabolism, 3:293-300,2014) alanine scanning (Ala is being carried out to GCG Scan), after each site of GCG is independently replaced by alanine, opposite residual activity retains span from 0.2%-100%, and Indicate GCG the 1st, 2,3,4,6-12,14,15,22,23,25-27,29 mutation all GCGR agonist activity can be made substantially to weaken (table 4 in article).However we can also see in others report taken in these above-mentioned sites it is single or several Site is mutated simultaneously, and when with other amino acid substitutions, active variation is not always the result one with alanine scanning It causes.Such as Jonathan W Day (Jonathan W Day etc., A new glucagon and GLP-1 co-agonist Eliminates obesity in rodents, Nature Chemical Biology, 5:749-757,2009) report, it is right The different mutation such as 16 progress 16S → G, 16S → T, 16S → H, 16S → E of GCG, GCGR agonist activity are to improve instead , the complete contradiction of alanine scanning result of this and Joseph Chabenne.Secondly, Joseph Chabenne research is thought 23 are replaced with alanine, be will lead to GCGR agonist activity and are almost lost and (only retain 1.1%)；But Jonathan W Day etc. is mutated into Ile for 23, and there is no decline for GCG activity.For another example alanine scanning result thinks second S to guarantor GCG activity is stayed to be very important (activity only remains 1/3 when sporting Ala), but Brian Finan etc. reports (Finan B etc., A rationally designed monomeric peptide triagonist corrects obesity and diabetes in rodents.Nat Med.2015；21:27-36.), to GCG second amino acid carry out respectively 2S → Aib, 2S → dSer, 2S → G, 2S → dAla substitution mutation, after the mutation in other sites of recombinant, the opposite agonist activity of GCGR is instead Rise to 200% -640%.In our study, it was also found that it is some be conducive to improve GLP-1, GCG or GIP it is active It is completely inconsistent when many times its effect and single locus are mutated when mutation combination introduces.Also, GLP-1, This kind of polypeptide of Exendin-4, GCG or GIP, its biological activity can all be influenced by increasing or decreasing amino acid in N and C-terminal.Such as N One or two amino acid is removed at end, and the agonist activity of GLP-1, GCG etc. will completely lose.As Oxyntomodulin only compares The C-terminal of Glucagon more this 8 amino acid of KRNRNNIA, GCGR agonist activities just lose 90% or so (Alessandro Pocai etc., Glucagon-Like Peptide 1/Glucagon Receptor Dual Agonism Reverses Obesity in Mice, Diabetes；58 (10): 2258-2266,2009；Henderson SJ etc., Robust anti-obesity and metabolic effects of a dual GLP-1/Glucagon receptor peptide Agonist in rodents and non-human primates, Diabetes Obes Metab, 2016).
For another example Joseph R.Chabenne and Richard D.DiMarchi etc. were once reported, were increased in the C-terminal of Glucagon After the C-terminal small peptide cex (SEQ ID NO.67, GPSSGAPPPS) for adding one section of Exendin-4, so that its GLP-1R agonist activity 1.6% is increased to from 0.7%, improves about 2 times or so (Optimization of the Native Glucagon Sequence For Medicinal Purposes, J Diabetes Sci Technol.4 (6): 1322-1331,2010 and patent US9018164B2 about 50% GCG activity), and is also had lost.Evers A etc. also reports (Evers A, Design of Novel Exendin-Based Dual Glucagon-like Peptide-1(GLP-1)/Glucagon Receptor Agonists, J Med Chem.；60 (10): 4293-4303.2017) after the C-terminal of GCG analog adds cex sequence, GLP-1R agonist activity has dropped 2/3 or so instead, but the agonist activity of GCG is even more the (table in article that has lost 90% or more 2, peptide 7 and 8).Therefore, for GLP-1, the small peptide of this kind of 30 amino acid lengths of Glucagon, the change of sequence is to its activity Variation is extremely sensitive；And for double activity polypeptide, due to being related to the excitement to two not isoacceptors, variation is just more Which type of consequence is complexity can be at all to GLP-1R and GCGR agonist activity after any one unpredictable amino acid change.
It is preferably multiple to also increase design one for the complexity of the GPCR receptor downstream signal transduction such as GCGR, GLP-1R The difficulty of active hybrid peptide.There are a plurality of signal transduction channels, including G-protein (G α s, G α intracellular for the receptor of GCGR, GLP-1R I, G α q etc.) and to inhibit the downstream signals factor such as albumen (β-arrestin-1 and β-arrestin-2) to form a plurality of different Signal transduction channel, its physiological effect of the activation in different channels are different or even some channel activities and physiologic function Relationship be not illustrated.Such as different mutation or different amino acid sequences are introduced in GLP-1 sequence, it can obtain not With the structure of skewed popularity exciting (biased-agonist), to generate different physiological effects (Marlies V. etc., J Am Chem Soc., 138 (45): 14970-14979,2016；Hongkai Zhang etc., Nat Commun., 6:8918,2015).
Therefore, although design obtains one while having the active polypeptide of high GLP-1, Glucagon and GIP in theory Be have very much clinical meaning, but actually be also it is very difficult.If a kind of active balance can be designed, but again to the greatest extent Amount introduces mutational site and unnatural amino acid less, makes its potential immunogenicity lower close to native sequences as far as possible, and steady Qualitative raising, the multiple activities hybrid peptide with good glycemic control and body weight control, is to have very much clinical meaning.
Summary of the invention
In view of the foregoing deficiencies of prior art, the purpose of the present invention is to provide a kind of glucagon analogue, The glucagon analogue shows tri- receptor agonist activity of GLP-1R/GCGR/GIPR.
Natural GCG has the GLP-1R agonist activity relative to natural GLP-1 about 1%, but there is no GIPR excitements to live Property.And glucagon analogue of the invention, it can express tri- receptor agonist activity of GLP-1R/GCGR/GIPR.
In order to achieve the above objects and other related objects, the first aspect of the present invention provides a kind of glucagon analogue (GCG analog) contains the structure as shown in Formulas I or Formula II, structure shown in Formulas I in the structure of the glucagon analogue Are as follows:
HSQGTFTSD-X10-SKYLD-X16-X17-AA-X20-X21-F-X23-QWLMN-X29-Xz(SEQ ID NO.1), formula Structure shown in II are as follows:
HSQGTFTSD-X10-SKYLD-X16-X17-AA-X20-X21-F-X23-QWLMN-X29-Xz-NH2(SEQ ID NO.2), wherein X10Any, X selected from Y, K or L16Selected from any of S, E or A, X17Selected from any of Q, E, A or R, X20It is selected from Q, R's or K is any；X21Selected from any of D, L or E；X23Selected from any of V or I；X29For T or missing, XzIt is selected from GGPSSGAPPPS(SEQ ID NO.65)、GGPSSGAPPS(SEQ ID NO.66)、GPSSGAPPPS(SEQ ID NO.67)、 GPSSGAPPS(SEQ ID NO.68)、PSSGAPPPS(SEQ ID NO.69)、PSSGAPPS(SEQ ID NO.70)、 SSGAPPPS's (SEQ ID NO.71) or SSGAPPS (SEQ ID NO.72) is any.
Further, when the structural formula of the glucagon analogue are as follows:
HSQGTFTSD-X10-SKYLD-X16-X17-AA-X20-X21-F-X23-QWLMN-X29-Xz-NH2(SEQ ID NO.2) When, refer to that the C-terminal of the glucagon analogue has carried out amidation modification.
As cited by some embodiments of the present invention, the amino acid sequence of glucagon analogue of the present invention such as SEQ ID NO.6-28 and SEQ ID NO.47-53's is any shown.
Further, in a preferred embodiment, the structural formula of the glucagon analogue are as follows:
HSQGTFTSDYSKYLD-X16-X17-AAQ-DFVQWLMN-X29-Xz(SEQ ID NO.3)
Or HSQGTFTSDYSKYLD-X16-X17-AAQ-DFVQWLMN-X29-Xz-NH2(SEQ ID NO.4)
Wherein, X16Any, X selected from S or E17Any, X selected from Q or E29For T or missing, XzSelected from GGPSSGAPPPS (SEQ ID NO.65)、GGPSSGAPPS(SEQ ID NO.66)、GPSSGAPPPS(SEQ ID NO.67)、GPSSGAPPS (SEQ ID NO.68)、PSSGAPPPS(SEQ ID NO.69)、PSSGAPPS(SEQ ID NO.70)、SSGAPPPS(SEQ ID NO.71) or SSGAPPS (SEQ ID NO.72) it is any.
Further, when the structural formula of the glucagon analogue are as follows:
HSQGTFTSD-Y-SKYLD-X16-X17-AAQDFVQWLMN-X29-Xz-NH2When (SEQ ID NO.4), refer to described The C-terminal of glucagon analogue carries out amidation modification.
Above-mentioned glucagon analogue have be similar to or better than natural glucagon GCGR agonist activity, and tool There are the GLP-1R agonist activity being similar to or better than natural GLP-1, and additional increased GIPR agonist activity.
It is of the invention in one embodiment, preferred glucagon analogue natural glucagon basis GPSSGAPPPS is added in upper C-terminal, is at least only mutated 2-3 amino acid and does not introduce unnatural amino acid, repairs without any Decorations can retain or improve GLP-1R and GCGR agonist activity, but also have additional increased GIPR agonist activity, product sheet Body has good stability.Less mutational site, and without subsequent modification, natural structure is maintained as far as possible, is reduced Potential immunogenicity risk.
For existing document report in treating diabetes, high-caliber GIP can cause frequent Hypoglycemic symptoms (T McLaughlin et al., J Clin Endocrinol Metab, 95,1851-1855,2010；A Hadji- Georgopoulos,Jclin Endocrinol Metab,56,648-652,1983).But it is tested in mouse model In, the glucagon analogue provided by the invention with GIPR agonist activity can smoothly control blood glucose, have no low blood Sugared symptom.Separately there is document report to claim, in order to reduce it is daily ingest, lose weight, improve insulin sensitivity and energy consumption, it is short of money Anti- GIPR is also desirable method (Irwin etc., Diabetologia 2007,50,1532-1540；Althage etc., J Biol Chem,2008,283(26):18365–18376).And in mouse model test, GIP activity provided by the invention is higher Glucagon analogue, it is lower or even without the active comparison analog of GIP relative to GIP activity, to the day of obesity mice Normal control of ingesting, weight loss and insulin sensitivity raising have more significant effect.
Glucagon analogue of the present invention has a better resistance to enzyme stability, including to neutral endopeptidase (NEP) and Dipeptidyl peptidase-4 (DPP-4)；With natural glucagon, GLP-1, GIP, which are compared, to be had longer Half-life in vivo and continues Action time.
The second aspect of the present invention provides a kind of isolated polynucleotides, the aforementioned pancreas of isolated polynucleotide encoding Glucagons analog.
The third aspect of the present invention provides a kind of recombinant expression carrier, includes aforementioned isolated polynucleotides.
The fourth aspect of the present invention, provides a kind of host cell, and the cell contains aforementioned recombinant expression carrier or gene The aforementioned isolated polynucleotides of external source are integrated in group.
The fifth aspect of the present invention provides the preparation method of aforementioned glucagon analogue, selected from following any:
(1) glucagon analogue is synthesized using chemical synthesis process；
(2) foregoing host cell is cultivated under suitable conditions, is allowed to express the glucagon analogue, then be divided From and purifying obtain the glucagon analogue.
Specifically, glucagon analogue of the invention can be prepared by standard peptide synthesis methods, for example, passing through Standard solid-phase or liquid phase process gradually or by segment assemble, and separate and purify final peptide compounds product, or pass through weight Group and synthetic method any combination.Glucagon class of the invention is preferably synthesized by solid phase or liquid phase peptide symthesis method Like object.
The sixth aspect of the present invention provides aforementioned glucagon analogue in the drug of preparation treatment metabolism related diseases In purposes.
Glucagon analogue provided by the invention can be used for treating the relevant metabolic syndrome of diabetes, such as blood lipid Imbalance, including triglycerides is excessively high, low HDL cholesterol and high LDL cholesterol；Insulin resistance or poor glucose tolerance etc..
It is related that metabolic syndrome and coronary heart disease and vascular plaque accumulate relevant other illnesss risk increases, for example, apoplexy with It is sick (ASCVD) to become athero- artereosclerotic cardiovascular for peripheral artery disease.Patient with metabolic syndrome can be from early stage Insulin resistant state develop into full ripe type-II diabetes, and the risk of ASCVD further increases.It is not only restricted to Any specific theory, the relationship between insulin resistance, metabolic syndrome and vascular diseases can be related to one or more common Pathogenesis, the vasodilation obstacle including insulin stimulating, the insulin resistance correlation caused by oxidation stress enhancing can It is reduced with property and fat cell derives hormone (such as adiponectin) abnormal (Lteif, Mather, Can.J.Cardiol.20 (supplementary issue B): 66B-76B, 2004).
Glucagon analogue of the invention can also be used to treat obesity.In certain aspects, pancreas of the invention is high Blood glucose element analog is by reducing appetite, reducing the mechanism such as food intake, reduction patient's body fat level, raising energy consumption To treat obesity.
In some potential embodiments, glucagon analogue of the invention can be used for treating non-alcoholic fatty Hepatopathy (NAFLD).NAFLD refers to wide spectrum liver diseases, and range becomes from simple fatty liver (steatosis) to non-alcoholic fatty Property hepatitis (NASH) to cirrhosis (the irreversible advanced stage cicatrization of liver).It is thin that all stadium of NAFLD show liver Accumulation of fat in born of the same parents.Simple fatty liver is some type of fat, abnormal accumulation of the triglycerides in liver cell, but without hair Scorching or cicatrization.In NASH, accumulation of fat and different degrees of liver inflammation (hepatitis) and cicatrization (fibrosis) phase It closes.Inflammatory cell can destroy liver cell (necrosis of liver cells).In term " steatosis hepatitis " and " steatosis necrosis " In, steatosis refers to fatty infiltration, and hepatitis refers to the inflammation in liver, and " necrosis " refers to the liver cell through destroying. NASH can eventually lead to liver cicatrization (fibrosis) and result then in irreversible advanced stage cicatrization (cirrhosis), by NASH Caused cirrhosis is last and most serious the stadium in NAFLD spectrum.
The seventh aspect of the present invention provides a kind of method for treating metabolism related diseases, comprising steps of before applying to object State glucagon analogue.
The glucagon analogue is used to treat fat, Metabolic syndrome by the present invention in one of the embodiments, Disease and non-alcoholic hepatitis etc..
Researcher of the invention has found glucagon analogue of the invention in neutral pH or micro- weakly acidic With enough water solubilitys and with improved chemical stability under pH.IPGTT reality has been carried out in one of the embodiments, It tests.The mouse of glucagon analogue of the present invention be applied after injectable dextrose monohydrate, show extremely stable blood glucose fluctuation.This Outside, glucagon analogue of the invention is after the application of DIO mouse, induction of the significant decrease of weight.Blood lipid is related simultaneously Various indexs be decreased obviously.
The present invention further provides a kind of promotion weight loss or the methods for preventing weight gain, are included in object and apply With the glucagon analogue.
The eighth aspect of the present invention provides a kind of composition, thin containing aforementioned glucagon analogue or aforementioned host The culture and pharmaceutically acceptable carrier of born of the same parents.
The ninth aspect of the present invention provides aforementioned glucagon analogue and is preparing the purposes in fusion protein.
The tenth aspect of the present invention provides a kind of fusion protein, contains the high blood of aforementioned pancreas in the structure of the fusion protein Sugared element analog.
Further, long-acting unit is also contained in the structure of the fusion protein.
Further, the long-acting unit, which is selected from, is covalently attached fatty acid, polyethylene glycol or derivatives thereof, and albumin turns Ferritin and immune globulin bletilla segment.
The eleventh aspect of the present invention provides a kind of polypeptide being modified, contains aforementioned glucagon class in structure Like object.
Further, the glucagon analogue is modified by fatty acid, polyethylene glycol or derivatives thereof；It is described The polypeptide being modified combined with covalent or non-covalent fashion and albumin, transferrins and immune globulin bletilla segment.
As known to those skilled in the art, in order to increase glucagon analogue of the present invention half-life period or stability can be with It is modified, such as polyethylene glycol or derivatives thereof, hydroxyethyl starch derivative or fatty acid can be covalently attached to Glucagon analogue of the invention.It, can be of the invention in order to improve stability in a specific embodiment Glucagon analogue expected will not influence introduces lysine residue on receptor combination/activation position, is covalently attached to γ- Glutamic acid spacer and on epsilon-amino be added palmitinic acid modified.
Compared with prior art, the invention has the following beneficial effects:
Glucagon analogue of the invention has tri- receptor agonist activity of GLP-1/GCG/GIP, and preferably resistance to Enzyme stability, including the resistance to neutral endopeptidase (NEP) and dipeptidyl peptidase-4 (DPP-4)；Therefore with the high blood of natural pancreas Sugared element, GLP-1, GIP, which are compared, has longer Half-life in vivo and continuous action time.In conclusion current existing report GCG analog generallys use (1) unimolecule hybrid peptide cross-linked fatty acid, PEG or FC etc., implements frequency once a day or above (Matthias H. is administeredDeng Unimolecular Polypharmacy for Treatment of Diabetes And Obesity, 24:51-62,2016)；Or the second Ser of natural GCG is sported the non-natural aminos such as D-Ser by (2) Acid resists DPP-IV degradation (Novel DPP IV-resistant C-terminally extended glucagon analogue exhibits weight-lowering and diabetes-protective effects in high- Fat-fed mice mediated through glucagon and GLP-1 receptor activation, Diabetologia:57:1927-1936,2014), it is taken twice daily.Keep natural amino acid composition and by twice daily The multiple activities polypeptide of administration has not been reported yet.And the present invention is then to provide a kind of sufficiently stable, high activity multiple-effect GCG Analog, without cross-linked fatty acid, PEG albumin or immunoglobulin Fc segments, and it is not necessary that second Ser is sported non-day Right amino acid, therefore can farthest lower potential immunogenicity risk, and omit cumbersome chemical modification/crosslinking step Suddenly, simplify preparation process, improve product consistency.
Detailed description of the invention
Fig. 1: to number HPLC spectrogram of the polypeptide for being C381 in 7.4 aqueous solution of pH.
Fig. 2: to number HPLC spectrogram of the polypeptide for being C493 in pH4.5 aqueous solution.
Fig. 3: to number HPLC spectrogram of the polypeptide for being C816 in 7.4 aqueous solution of pH.
Fig. 4: to number HPLC spectrogram of the polypeptide for being C002 in 7.4 aqueous solution of pH.
Fig. 5: to number HPLC spectrogram of the polypeptide for being C611 in 4.5 aqueous solution of pH.
Fig. 6: to number HPLC spectrogram of the polypeptide for being C611 in 7.4 aqueous solution of pH.
Fig. 7: for HPLC spectrogram of the C239 in 7.4 aqueous solution of pH.
Fig. 8 A: curve graph is changed over time for residual activity.
Fig. 8 B: curve graph is changed over time for residual activity.
Fig. 9 A: GLP-1R agonist activity is detected for illustrative several glucagon analogues and is schemed.
Fig. 9 B: GLP-1R agonist activity is detected for illustrative several glucagon analogues and is schemed.
Fig. 9 C: GCGR agonist activity is detected for illustrative several glucagon analogues and is schemed.
Fig. 9 D: GCGR agonist activity is detected for illustrative several glucagon analogues and is schemed.
Fig. 9 E: the cAMP content generated for glucagon analogue under various concentration gradient and control stimulation GIPR.
Fig. 9 F: the cAMP content generated for glucagon analogue under various concentration gradient and control stimulation GIPR.
Fig. 9 G: the cAMP content generated for glucagon analogue under various concentration gradient and control stimulation GIPR.
Fig. 9 H: the cAMP content generated for glucagon analogue under various concentration gradient and control stimulation GIPR.
Figure 10: for cell in vitro insulin secretion measurement result.
Figure 11 A: change of blood sugar curve graph is tested for the IPGTT of normal ICR mouse.
Figure 11 B: change of blood sugar curve graph is tested for the IPGTT of normal ICR mouse.
Figure 11 C: for Area under the curve of blood glucose (AUC) comparison result.
Figure 12 A: for the chart of changes of weight (%) and time (day) relationship in Diet-Induced Obesity (DIO) mouse.
Figure 12 B: for the chart of changes of weight (%) and time (day) relationship in Diet-Induced Obesity (DIO) mouse.
Figure 12 C: for the chart of changes of weight (%) and time (day) relationship in Diet-Induced Obesity (DIO) mouse.
Figure 12 D: compare figure for the weight loss situation in DIO mouse.
Figure 13: compare figure for the weight loss situation in DIO mouse.
Figure 14: for C495 mass spectral analysis figure.
Figure 15: for C382 mass spectral analysis figure.
Specific embodiment
Unless defining additionally below, all technologies mentioned by the present invention and scientific words are with of the art The normally understood meaning of technical staff.
Glucagon analogue:
18 arginine (R) are sported alanine (A) by glucagon analogue provided by the invention.18 the third ammonia The mutation of acid can make GCGR agonist activity decline about 30% (Joseph Chabenne etc., A Glucagon analog chemically stabilized for immediate treatment of life-threatening Hypoglycemia, Molecular Metabolism, 3:293-300,2014).However, largely being combined by the present inventor It is found after screening, carries out specific amino acid mutation, such as the specific amino acid mutation group with 16 and 17 in some specific sites After conjunction, and CEX or similar sequence is added in C-terminal, even if 18 are mutated into A, GCGR activity, which also has no, to be substantially reduced.It is heavier It wants, 18 A mutation are obviously improved GLP-1 the and GIPR agonist activity of glucagon analogue, to make the present invention Glucagon analogue become effective tri-specific active peptide.
Tri-specific active peptide of the invention has the effect of excitement GLP-1R, GCGR and GIPR, and it is each it is active relative to GLP-1, GCG and GIP, activity preservation rate are high.Three most active peptides all introduce on the basis of natural polypeptides more at present A amino acid mutation, or even unnatural amino acid could become stable triple effect agonist.It is sent out in screening process of the invention Existing, multidigit point introduces mutation and is easier to obtain the higher hybrid polypeptide of GLP-1R, GCGR and GIPR activity really, in addition non-natural The introducing of amino acid is also easier to obtain the high polypeptide of stability.However external activity and the height of stability can only become One precondition of clinical medicine, it is also necessary to pay close attention to safety.Excessive mutational site or unnatural amino acid are introduced, is easy Bring higher immunogenicity risk.
Serum stability experiment:
Natural GLP-1, glucagon Glucagon or oxyntomodulin (Oxyntomodulin) are all because of serum stable The too poor and too short Half-life in vivo of property and can not really be used as clinical medicine.And equally there was only the small peptide Ai Sai of 39 amino acid That peptide (Exenatide, trade name) but can successfully be listed because stability improves.It is real in one of them of the invention It applies in example, preferred glucagon analogue presents very high stability.
Immunogenicity experiments:
In example 6 in accordance with the invention, natural source of people Glucagon (C001) is extremely low in the intracorporal immunogenicity of rat.Pancreas When glucagons analog is not more than 3 amino acid relative to natural Glucagon mutation, antibody titer is respectively less than < 1:200, and As the amino acid quantity of mutation increases, antibody titer is also increased as, and shows that potential immunogenicity risk increases.For controlling The drug of metabolism related diseases is treated, such as diabetes, the drug in the fields such as obesity, the requirement of safety is high.And it is of the invention The glucagon analogue of acquisition has lower immunogenicity in the case where introducing no more than 3 mutational site, and reaches Ideal activity and stability criterion are arrived, this is that never someone reported.
Pharmacodynamic study in animal body:
In one embodiment of the invention, preferred glucagon analogue has good reduction blood glucose, inhibits Adipose tissue is formed, and the effect of weight is lowered.Although GIP and GLP-1 and Glucagon belong to duodenin (Incretin) family, but it is not widely deployed into the trend of drug.To find out its cause, one is due to two type of part Diabetic loses sensibility to GIP, and Another reason is in rodent, and GIPR excitement appearance potentially causes fat fat Phenomenon (Miyawaki, K. etc., Inhibition of gastric inhibitory polypeptide signaling Prevents obesity.Nat.Med.8,738-742,2002).However in an embodiment of the present invention, preferred GIPR swashs The dynamic higher glucagon analogue of activity obviously has more significant weight loss effect.
In another animal body of the invention in embodiment 9, preferred GCG analog and same amino acid sequence and warp Similar weight loss effect is presented in the corresponding GCG analog of fatty acid modifying.
Term:
Term " diabetes " include a patients with type Ⅰ DM, type-II diabetes, gestational diabetes mellitus and cause hyperglycemia its His symptom.The term is for referring to that pancreas can not produce enough insulin or the cell of body fails suitably due to metabolic disorder Insulin is responded, therefore histocyte absorbs glucose efficiency and declines the symptom for causing glucose to accumulate in blood.
One patients with type Ⅰ DM is also referred to as insulin-dependent diabetes mellitus and Juvenile onset patients with type Ⅰ DM, is drawn by β cytoclasis It rises, typically results in absolute insulin shortage.
Type-II diabetes are also referred to as adult-onset diabetes and adult-onset diabetes, generally anti-with insulin Property it is related.
Term " obesity " means the excess of adipose tissue, when caloric intake is more than energy consumption, excessive calorie storage In fat, then lead to obesity.Herein body mass index square of height (rice) (BMI=weight (kilogram) divided by) is more than 25 Individual be considered as obesity.
Term " receptor stimulating agent " can be defined as in conjunction with receptor and causing polypeptide, the egg of the usual response of native ligand White or other small molecules.Duodenin is the insulin secretion by enhancing glucose stimulation come the gastrointestinal hormone of regulating and controlling blood sugar (Drucker.D J,Nauck,MA,Lancet 368:1696-705,2006).There are two types of known intestines are hypoglycemic so far Element: glucagon-like-peptide-1 (GLP-1) and glucose-dependent-insulinotropic polypeptide (GIP).Preceding Proglucagon (preproglucagon) be 158 amino acid composition Precursor Peptide, in the tissue by otherness process and formed a variety of Relevant Proglucagon derived peptide in structure, including glucagon (Glucagon), glucagon-like-peptide-1 (GLP- 1), glucagon-like-peptide-2 (GLP-2) and oxyntomodulin (Oxyntomodulin, OXM).GIP is by 133 amino The mature peptide for 42 amino acid that the precursor (pre-pro-GIP) of acid is processed by proteolysis, these molecules participate in more Kind biological function, including glucose homeostasis, insulin secretion, gastric emptying and intestines growth and food intake are adjusted.
Glucagon-like-peptide-1 (GLP-1) is that a kind of intestines for 30 or 31 amino acid secreted from intestines L- cell promote pancreas islet Plain hormone has two kinds of active forms of GLP-1 (7-36) and GLP-1 (7-37).GLP-1 is discharged into circulation after dining, and is led to It crosses activation GLP-1 receptor and plays bioactivity.GLP-1 has many biological actions, the rush pancreas islet including glucose dependency Element secretion, glucagon suppression generate, and delay gastric emptying and appetite-suppressing (Tharakan G, Tan T, Bloom S.Emerging therapies in the treatment of‘diabesity’:beyond GLP-1.Trends Pharmacol Sci 2011；32 (1): 8-15.) etc..Natural GLP-1 is due to can be neutral by dipeptidyl peptidase-4 (DPP-4) Endopeptidase (NEP), the fast degradations such as plasma kallikrein or fibrinolysin thus limit its treatment potentiality.Due to natural GLP-1 only has about 2 minutes ultrashort half-life period in vivo, therefore, occurs by utilizing chemical modification and/or dosage form To improve effect to treat method (Lorenz M, Evers A, the Wagner M.Recent of diabetes and obesity progress and future options in the development of GLP-1 receptor agonists for the treatment of diabesity.Bioorg Med Chem Lett 2013；23(14):4011-8.Tomlinson B,Hu M,Zhang Y,Chan P,Liu ZM.An overview of new GLP-1receptor agonists for type 2 diabetes.Expert Opin Investig Drugs 2016；25(2):145-58).
Oxyntomodulin (Oxyntomodulin) is the small peptide of 37 amino acid, and it is complete that it includes glucagons 29 amino acid sequences.Oxyntomodulin is the dual agonists of GLP-1R and GCGR, after dining by intestines L- cell with GLP-1 secretes together.Similar with glucagon, oxyntomodulin generates significant weight in people and rodent and subtracts Gently.The anti-obesity activity of oxyntomodulin is compared in obesity mice with the selective GLP-1R agonist of equimolar dosage Compared with.It has been found that oxyntomodulin has anti-high-blood-sugar function compared with the GLP-1R agonist of selectivity, it can be significant Lose weight and have Lipid-lowering activities (The glucagon receptor is involved in mediating the Body weight-lowering effects of oxyntomodulin, Kosinski JR etc., Obesity (Silver Spring), 20): 1566-71,2012).In overweight and obese patient, the natural oxyntomodulin of subcutaneous administration is in surrounding Reduce 1.7 kilograms of weight.Oxyntomodulin is also proved to that the food intake of the mankind can be reduced and increases energy consumption (Subcutaneous oxyntomodulin reduces body weight in overweight and obese Subjects:a double-blind, randomized, controlled trial, Wynne K etc., Diabetes, 54: 2390-5,2005；Oxyntomodulin increases energy expenditure in addition to decreasing energy intake in overweight and obese humans:a 14andomized controlled trial；Wynne K etc., Int J Obes (Lond), 30:1729-36,2006).But also due to molecule Less than normal and DPP-IV degradation is measured, oxyntomodulin has shorter half-life period.GLP-1 receptor (GLP-1R) and pancreas are high at present The dual agonist of blood glucose element receptor (GCGR) is generally all based on oxyntomodulin, and secretes sour adjusting to improve stomach Element it is short-acting and enzymatic hydrolysis defect and done and be mutated (Oxyntomodulin analogs), and mostly use second serine Ser α-aminoacid (Aib) or the method for D-Ser are sported, resists the enzymatic hydrolysis of DPP-IV by introducing unnatural amino acid. Although Oxyntomodulin analogs show preliminary hypoglycemic and fat reducing effect, but its mechanism of action is still inaccurate, stomach It secretes acid and adjusts the never discovery of plain receptor, tested at present merely by the GCGR or GLP-1R mouse knocked out or test cell line Card oxyntomodulin can work in conjunction with this 2 kinds of receptors.
Glucagon (Glucagon) is the peptide of 29 amino acid, corresponds to the position 53-81 of preceding Proglucagon Amino acid, sequence (C.G.Fanelli etc., Nutrition, Metabolism& as shown in SEQ ID NO.5 Cardiovascular Diseases(2006)16,S28-S34).Glucagon receptor activation has been displayed in rodent With increase energy consumption in both people and reduce food intake (Habegger K.M. et al., The metabolic actions Of glucagon revisited, Nat.Rev.Endocrinol.2010,6,689-697) and these effects in rodent It is stable and lasting in animal.Glucagon has many physiological effects, such as by stimulating decomposition of glycogen and gluconeogenesis, Increase the blood glucose level under hypoglycaem ic condition, adjust liver ketogenesis, adjusts bile acid biosynthesis and by vagal full abdomen effect It answers.Glucagon has been used to Acute hypoglycemia, glucagon receptor activation reduce food intake and promote animal and The lipolysis and weight loss of people.
Glucose dependency pancreotropic hormone sample peptide (GIP) is the polypeptide with 42 amino acid, after food intake It is discharged from small intestine K cell, main function is gastric acid secretion inhibiting and the insulin secretion for reinforcing glucose stimulation, therefore is known as pressing down Stomach peptide (gastric inhibitory peptide)/glucose-dependent-insulinotropic polypeptide (glucose-dependent insulinotropic peptide)。
" GLP-1 receptor (GLP-1R) agonist " can be defined as in conjunction with GLP-1R and can cause and natural GLP-1 Polypeptide, albumen or other small molecules of same or similar characteristic reaction.GLP-1R agonist by activating completely or partially GLP-1R then causes a series of intracellular downstream signaling pathways to react, generates corresponding cell activity: as β cell is secreted Insulin；Typical GLP-1R agonist includes natural GLP-1 and its mutant, analog, such as Exenatide, Liraglutide (Liraglutide) etc..
" glucagon receptor (GCGR) agonist " can be defined as in conjunction with GCGR and can cause high with natural pancreas Polypeptide, albumen or other small molecules of the same or like characteristic reaction of blood glucose element (glucagon).GCGR agonist has passed through GCGR is fully or partially activated, then causes a series of intracellular downstream signaling pathways to react, generates corresponding cell activity: such as Hepatocyte Glycogen decomposition, gluconeogenesis, fatty acid oxidation and ketogenesis etc..
GLP-1R/GCGR dual agonist: GLP-1R/GCGR dual agonist of the invention includes energy excitement GLP- simultaneously The albumen or polypeptide of 1R and GCGR.The dual agonist based on Oxyntomodulin of such as Alessandro Pocai report (Alessandro Pocai etc., Glucagon-Like Peptide 1/Glucagon Receptor Dual Agonism Reverses Obesity in Mice, Diabetes；58 (10): 2258-2266,2009) or Richard The dual agonist (US9018164B2) based on glucagon of the reports such as D.DiMarchi.
GLP-1R/GCGR/GIPR triple effect agonist: GLP-1R/GCGR/GIPR triple effect agonist of the invention includes can be same The albumen or polypeptide of Shi Jidong GLP-1R, GCGR and GIPR, or be " tri-specific agonist ".
Tri-specific active peptide: referring to through preferred tri-specific active peptide while there is GLP-1R/ in the present invention The polypeptide of GCGR/GIPR agonist activity, or be " three way activity peptide ".
EC50(concentration for 50%of maximal effect) refers to that a certain drug or substance are piercing Swash concentration required when the 50% of its corresponding biologically.EC50 value is lower, shows the stimulation of the drug or substance or swashs Kinetic force is stronger, for example, more intuitively can behave as that caused Intracellular signals are stronger, thus the ability for inducing certain hormone to generate It is better.
Low-density lipoprotein (LDL): belonging to one kind of plasma lipoprotein, is the main carriers of Blood Cholesterol.It inclines To in by cholesterol deposition on arterial wall.Leucocyte attempts to digest low-density lipoprotein, but in this process by them Become toxin.More and more leucocytes are attracted to changed region, cause arterial wall inflammation.With pushing away for time It moves, these plaque deposition objects can accumulate on arterial wall, so that channel becomes very narrow and lacking toughness.If too many spot Block accumulation, then artery can block completely.When the compound (LDL-C) that LDL and cholesterol are formed generates too much on arterial wall When patch, blood cannot flow freely over artery.Patch may collapse suddenly in the artery at any time, lead to blood vessel blockage, most Cause heart disease eventually.
High-density protein (HDL): help to remove eparterial LDL, play the role of street cleaner, by LDL from artery It walks clearly and returns to liver.
Triglycerides (TG): being another type of fat, for storing the energy of hyperphagia.It is high-caliber in blood Triglycerides is related with atherosclerosis.High triglyceride can be by overweight and fat, and body lacks movement, and smoking is excessive Alcohol consumption and Hi CHO (60% more than total calorie) intake cause.Sometimes underlying diseases or genetic disease are The reason of high triglyceride.People with high triglyceride usually has a high total cholesterol level, including high LDL cholesterol and Low HDL cholesterol, many has a heart disease or the people of diabetes also has high triglyceride horizontal.
GPCR:G G-protein linked receptor (G Protein-Coupled Receptor), it is the weight in cellular signal transduction Protein is wanted, topological conformation is the receptor of 7 cross-films.When the ligand outside film acts on this receptor, inside the film of this receptor Divide and be combined with each other with G-protein, activated G protein.G-protein can transmit extracellular information by two kinds of approach: first way is Transmembrane ion channel is opened, extraneous ion is allowed to enter；The second way is activation second messenger, such as cAMP, IP3/ DAG etc.. Calcium ion is typically considered cAMP, IP3The third messenger in the downstream /DAG.
Abbreviation
[(1- (cyano -2- ethyoxyl -2- oxo ethyleneimino oxygroup) dimethylaminomorpholine is for methylene by COMU:1- Base)] first ammonium hexafluorophosphate
DCM: methylene chloride
DMF:N, dinethylformamide
DIPEA: diisopropylethylamine
EtOH: ethyl alcohol
Et2O: ether
HATU:2- (7- aoxidizes benzotriazole)-N, N, N', N'- tetramethylurea hexafluorophosphoric acid ester
MeCN；Acetonitrile
NMP:N- methyl pyrrolidone
TFA: trifluoroacetic acid
TIS: tri isopropyl silane
Illustrate embodiments of the present invention below by way of specific specific example, those skilled in the art can be by this specification Other advantages and efficacy of the present invention can be easily understood for disclosed content.The present invention can also pass through in addition different specific realities The mode of applying is embodied or practiced, the various details in this specification can also based on different viewpoints and application, without departing from Various modifications or alterations are carried out under spirit of the invention.
Before further describing the specific embodiments of the present invention, it should be appreciated that protection scope of the present invention is not limited to down State specific specific embodiment；It is also understood that term used in the embodiment of the present invention is specific specific in order to describe Embodiment, rather than limiting the scope of protection of the present invention.
Unless otherwise defined, all technical and scientific terms and those skilled in the art of the present technique used in the present invention are usual The meaning of understanding is identical.In addition to specific method, equipment, material used in the embodiment, according to those skilled in the art Grasp and record of the invention to the prior art, can also use and method described in the embodiment of the present invention, equipment, material Any method, equipment and the material of the similar or equivalent prior art realizes the present invention.
Unless otherwise stated, disclosed in this invention experimental method, detection method, preparation method be all made of this technology neck Molecular biology, biochemistry, chromatin Structure and the analysis of domain routine, analytical chemistry, cell culture, recombinant DNA technology and The routine techniques of related fields.These technologies have perfect explanation in the prior art, and for details, reference can be made to Sambrook etc. MOLECULAR CLONING:A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001；Ausubel etc., 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；METHODS IN ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic Press, San Diego, 1999；With METHODS IN MOLECULAR BIOLOGY, Vol.119, Chromatin Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999 etc..
Embodiment 1: glucagon analogue be typically prepared and purification process
Using the prior art, such as existing literature (V. et al., Beilstein J.Org.Chem., 10:1197- 1212(2014)；Palomo, J.M., RSC Adv., 4:32658-32672 (2014)；Behrendt, R. et al., J.Pept.Sci., (2015) 22:4-27) in Solid-phase synthesis peptides and method of modifying it is each more involved in this patent to prepare Peptide.
Specifically, available standards Fmoc method carries out Solid phase peptide synthesis on CEM Liberty peptide synthesizer.
Before the use by Rink Amide TentaGel S Ram resin (0.25mmol/g, 1g) in NMP (10mL) Swelling, is added in synthesis in solid state device, and piperidines/DMF (20%, 10mL) reaction 30min is added in resin and carries out de- Fmoc guarantor Shield, drains, washs (5 × 10mL) with DMF, drain.Be added first Fmoc- amino acid solution (0.2M, NMP/DMF/DCM, 1: 1:1,5mL) and COMU/NMP (0.5M, 2mL) and DIPEA/DMF (2.0M；1mL), 1h is reacted at room temperature, ninhydrin detects resin It is colorless and transparent, it drains, is washed with DMF (5 × 10mL).Then piperidines/DMF (20%, 10mL) reaction 30min is added to be taken off Fmoc protection, drains, washs (5 × 10mL) with DMF, drain.Repeat above-mentioned addition Fmoc- amino acid carry out reaction and piperidines/ DMF carries out taking off fmoc-protected step until completing the coupling of the last one histidine.
By resin EtOH (3 × 10mL) and Et2O (3 × 10mL) is washed and is dried at room temperature for constant weight.Resin is added TFA/TIS/ phenol/EDT/ water (82.5/5/5/2.5/5, v/v, 40mL) ice bath reacts 2h, under thick peptide is cut from resin Come, filtering repeats the step three times.Filtrate is merged, most of TFA is removed under reduced pressure, is precipitated with ether, is centrifuged, precipitating uses second Ether washs three times, dries to constant weight obtain thick peptide at room temperature.Use the Varian SD-1 equipped with C-18 column and fraction collector Type preparative liquid chromatograph, with mobile phase A (0.1%TFA, aqueous solution) and Mobile phase B (0.1%TFA, 90%MeCN, it is water-soluble Liquid) gradient elution, by preparative reversed-phase HPLC by thick peptide purification to purity be greater than 97%, gained peptide is listed in table 1.Wherein, C The polypeptide of end amide ending is synthesized using the above method；Remaining polypeptide using Wangle Resin (0.4mmol/g, 1g) into Row synthesis in solid state is directly added into Fmoc- amino acid after resin swelling and carries out coupling reaction, takes off Fmoc protection, and polypeptide cutting is pure Change, operating procedure is identical as C-terminal amide ending Peptide systhesis step.Peptide systhesis and purifying with fatty acid modifying are conventional Technology, and can be found in (Finan B etc., A rationally designed the monomeric peptide such as Finan B triagonist corrects obesity and diabetes in rodents.Nat Med.2015；21:27-36.) or (Chhabr etc., Appraisal of New Variants of Dde Amine Protecting the Group for such as Chhabr Solid Phase Peptide Synthesis.Tetrahedron Lett.1998,39 (12), 1603-1606) article institute It states.
It is analyzed by peptide after purification by LC/MS, analyzes result such as table 2.Wherein, Figure 14,15 are respectively illustrative Number is C495 and the glucagon analogue mass spectral analysis figure of C382.Mass Spectrometry Conditions are as follows:
Instrument: Waters ZQ 2000
Mass spectrum (Probe): ESI
Sprayer flow velocity (Nebulizer Gas Flow): 1.5L/min
CDL:-20.0v
CDL temperature: 250 DEG C
It heats deblocking temperature (Block Temp): 200 DEG C
Mass spectrum voltage (Probe Bias) :+4.5kv
Detector (Detector): 1.5kv
Flow rate of mobile phase (T.Flow): 0.2ml/min
Buffer concentration (B.Conc.): 50%H2O/50%ACN
Table 1
Note: the X in table is aminoisobutyric acid, and K ' indicates that this position is lysine residue and is covalently attached to fatty acid, should Fatty acid structure is as shown in following formula I:
Table 2
Embodiment 2: stability study
The purpose of the present embodiment is to study the various glucagon analogues that prepare of embodiment 1 in aqueous solution Chemical stability.
By candidate polypeptide (glucagon analogue) and reference substance, it is formulated in the phosphate buffer of the 20mM of corresponding pH In PB or acetate buffer solution, the final concentration of 0.2mg/ml of polypeptide and with sterilizing filter (0.22 μm, Millipore SLGP033RB) it is filtered degerming.The polypeptide solution of preparation is placed 7 days at 40 DEG C.Then it is centrifuged 20 minutes in 4500rpm And use RP-HPLC-UV analysis supernatant (t7).The amount of the remaining complete peptide of measurement, and the sample of the non-isothermal holding of parallel analysis (t0).The peak area for comparing the target compound in t0 and t7 obtains " remaining peptide % " according to following equalities:
Remaining peptide content %=[(peptide peak area t7) × 100]/peptide peak area t0.
Stability is expressed as " remaining peptide content ".
Detection method
Detection wavelength: 214nm；
Chromatographic column: 40 DEG C of column temperature, (2) 5 μm of Phenomenex Luna C8 (150 × 4.6mm)；
Mobile phase: H2O+0.1%TFA:ACN+0.1%TFA (flow velocity 1.0ml/ minutes)；
Gradient: 95:5 (0 minute) to 0:100 (30 minutes)；
Analysis of experimental results: from 3 experimental data of table it can be concluded that currently preferred glucagon analogue (polypeptide) Higher stability is all had in neutral and slightly acidic water solution.
Table 3
Fig. 1-7 illustratively shows the liquid phase HPLC analysis of spectra of several glucagon analogues such as C381.Fig. 1 is corresponding Integration data:
The corresponding integration data of Fig. 2:
The corresponding initial data of Fig. 3:
The corresponding initial data of Fig. 4:
The corresponding initial data of Fig. 5:
The corresponding initial data of Fig. 6:
The corresponding initial data of Fig. 7:
Embodiment 3: serum stability
(1) corresponding polypeptide 5mM Tris-HCl in table 1, pH8.5,0.02%TWEEN80 solution are configured to concentration and are The solution of 1.0mg/ml after aseptic filtration (0.22 μm, Millipore SLGP033RB), dilutes 10 times with rat blood serum, mixes It is even, it is dispensed into sterile centrifugation tube；
(2) above-mentioned sample respectively takes 3 pipes to be frozen in -20 DEG C as control, remaining sets 37 DEG C of insulating boxs, takes in different time points Sample detection activity；
(3) using method shown in embodiment 4, polypeptide GCGR agonist activity is detected.
Relative activity: with 0 hour activity value for 100%, the value that subsequent point in time measures obtains by comparison.Experiment Interpretation of result: from table 4 and Fig. 8 A and 8B it can be concluded that serum stability.
Table 4
Note: N.D. indicates to be lower than Monitoring lower-cut
Embodiment 4: cytoactive detection
(1) GLP-1R agonist activity measures:
The detection of GLP-1R agonist activity uses luciferase reporter gene detection method (Jonathan W Day etc.: Nat Chem Biol.2009Oct；5(10):749-57).By source of people GLP-1R gene cloning to mammalian cell expression plasmid In pCDNA3.1, it is built into recombinant expression plasmid pCDNA3.1-GLP-1R, while luciferase (luciferase) full-length gene It is cloned into pCRE plasmid and obtains pCRE-Luc recombinant plasmid.PcDNA3.1-GLP-1R and pCRE-Luc plasmid 1:10 in molar ratio Ratio transfection CHO cell, screening is steady to turn expression strain.
With the DMEM/F12 culture medium culture cell containing 10%FBS and 300 μ g/ml G418 in 9-cm Tissue Culture Dish, Etc. convergence degrees to 90% or so when, discard culture supernatant, after 2ml pancreatin digestion 3min is added, 2ml be added and contains 10%FBS and 300 The DMEM/F12 culture medium of μ g/ml G418 neutralizes, and is transferred in 15ml centrifuge tube, after 1000rpm is centrifuged 5min, discards supernatant, 2ml is added to be resuspended containing the DMEM/F12 culture medium of 10%FBS and 300 μ g/ml G418, counts.With the DMEM/ containing 10%FBS F12 culture medium diluting cells are to 1 × 105/Ml, every hole spreads 100 μ l in 96 orifice plates, i.e., and 1 × 104/ hole changes into after adherent containing 0.2% The DMEM/F12 culture medium culture of FBS.Be layered on 96 orifice plates cell discard supernatant after, by the recombinant protein of purifying use contain 0.1% The DMEM/F12 culture medium of FBS is diluted to a series of prescribed concentrations, is added in cell culture well, 100 holes μ l/, after stimulating 6h Detection.It is examined according to lucifersae reporter kit (Ray Biotech, Cat:68-LuciR-S200) specification It surveys.Fig. 9 A, 9B are GLP-1R agonist activity testing result.
(2) GCGR agonist activity detection method:
The detection of GCGR agonist activity equally also uses luciferase reporter gene detection method.Extremely by source of people GCGR gene cloning In mammalian cell expression plasmid pcDNA3.1, be built into recombinant expression plasmid pCDNA3.1-GCGR, transfect HEK 293T and The screening building of stable cell strain is same as above.Fig. 9 C, 9D are GCGR agonist activity testing result.
Table 5
(3) GIPR agonist activity detection method:
PcDNA3.1-GIPR plasmid transfection Chinese hamster ovary celI simultaneously screens positive stable cell strain.It is connect in 96 porocyte culture plates Kind about 200,000 cells/well, overnight incubation, after being washed with Hanks balanced salt solution, testing protein is diluted to a series of fingers The 3-isobutyl-1-methylxanthine (IBMX) for determining concentration with 200 μM is added in cell jointly, after 37 DEG C of culture 20min, is abandoned Culture supernatant is gone, is added after lysate lytic cell and is contained with cAMP Parameter assay kit referring to specification measurement cAMP It measures (R&D company, the U.S., article No.: SKGE002B).As a result as shown in Fig. 9 E-H.
Embodiment 5: the insulin secretion experiment of glucose stimulation
Method (A novel DPP IV-resistant C- of the present embodiment referring to Aisling M.Lynch et al. terminally extended Glucagon analogue exhibits weight-lowering and diabetes- protective effects in high-fat-fed mice mediated through Glucagon and GLP-1 Receptor activation, Aisling M.Lynch etc., Diabetologia, 57:1927-1936,2014) use rat BRIN-BD11 cell stimulates caused insulin releasing for measuring activated protein, but is modified slightly, i.e., in 24 orifice plates 1.0 × 106 cells are added in every hole in (Orange Scientific, Brainel ' Alleud, Belgium), and 37 DEG C were cultivated Centrifugation goes supernatant, then every hole that 1.0ml KRB (115mM NaCl, 4.7mM KCl, 1.28mM CaCl is added after night2、1.2mM MgSO4、1.2mM KH2PO4、25mM HEPES、10mM NaHCO3, NaOH adjust pH to 7.4), 0.1% (wt/vol.) BSA and 1.1mM glucose.Cell is placed in 37 DEG C of cultures after forty minutes, and centrifugation removes supernatant and is substituted for the fresh KRB solution of 1.0ml and ladder Spend the activated protein of concentration.After twenty minutes, centrifugation removal buffer is simultaneously stayed overnight in -20 DEG C of storages for 37 DEG C of cultures, remakes immune put Penetrate detection insulin content.The results are shown in Figure 10.
Embodiment 6: Mouse immunogenicity experiment
7 week old Balb/c mouse, every group 6, each caudal vein blood sampling obtains 50ul serum as blank control before administration. It is daily to inject corresponding glucagon analogue (30nmol/kg, in PBS buffer solution), continuous injection 28 days.To the 45th day eye Socket of the eye blood sampling, solidification separation serum.Its antibody titer is measured using Salmonella method.Corresponding polypeptide coated elisa plate, by mouse blood 1:50 is pressed clearly；ELISA Plate is added in 1:200,1:1000,1:5000 gradient dilution, and sheep anti mouse secondary antibody is detection antibody.Each mouse is given Serum is negative control before medicine, under the premise of identical dilution, test specimen OD450It is worth average value and is greater than negative control sera OD450The result judgement of 2.1 times of average value of value is positive (+), otherwise is determined as negative (-), and result is positive highest dilution As antibody titer.
Table 6
Embodiment 7: the dextrose tolerance test (IPGTT) of normal ICR mouse
Normal ICR mouse is divided into 27 groups, every group 6.It being fasted overnight, (being denoted as t=0 minutes blood glucose samples) is taken a blood sample in tail portion, It is subcutaneously injected in solvent body control (acetate buffer, 20mM acetic acid, 250mM mannitol, pH5.0) and table of the present invention 1 Glucagon analogue (30nmol/kg, PBS buffer solution in) and Vehicle controls body, Liraglutide (Trade name 40nmol/kg is diluted in PBS buffer solution).Glucose (2 g kgs of weight) is injected intraperitoneally after 15 minutes, and at t=30 points Clock, t=45 minutes, t=60 minutes, t=120 minutes measurement blood glucose levels.During the experiment animal still fasting to prevent The only interference of food intake.Concrete outcome is referring to Figure 11 A-C.
Embodiment 8: the loss of weight experiment in Diet-Induced Obesity (DIO) mouse
The preparation of DIO mouse model: about 7 week old male C57BL/6J male mices give high lipid food (60%kcal from Fat) continue to raise about 16 weeks (totally 23 weeks), be tested when to weight being about 45g.DIO mouse is randomly divided into group, and every group 6, Basal body mass indifference, every group of mouse be subcutaneously injected respectively daily each glucagon analogue (in 30nmol/kg, PBS) or PBS is isometric, control group Liraglutide (trade name30nmol/kg), daily administration 2 times are weighed into 30 days daily.
Figure 12 A-C is every diurnal variation of DIO mouse weight after the administration of each glucagon analogue, final weight loss Percentage is shown in Figure 12 D.
Embodiment 9: the loss of weight experiment in Diet-Induced Obesity (DIO) mouse
DIO mouse is randomly divided into group, and every group 6, basal body mass indifference, subcutaneous injection is each respectively daily for every group of mouse GCG analog (in 30nmol/kg, PBS) or PBS, control group Liraglutide (trade name30nmol/kg is dilute Release in PBS) daily administration 2 times, the GCG analog (in 30nmol/kg, PBS) of fatty acid is administered once daily, and is claimed daily Weight was to 30 days.
Figure 13 is the weight loss percentage that DIO mouse is final after each glucagon analogue is administered.GCG as shown in the figure Corresponding GCG analog analog C381, C464 and C493 identical as amino acid sequence and through fatty acid modifying presents similar Weight loss effect, and C225, C163 are then acted on without obvious loss of weight without fatty acid.
In conclusion the present invention effectively overcomes various shortcoming in the prior art and obtaining one group has potential clinic to answer The triple effect agonist of the value.
The above-described embodiments merely illustrate the principles and effects of the present invention, and is not intended to limit the present invention.It is any ripe The personage for knowing this technology all without departing from the spirit and scope of the present invention, carries out modifications and changes to above-described embodiment.Cause This, institute is complete without departing from the spirit and technical ideas disclosed in the present invention by those of ordinary skill in the art such as At all equivalent modifications or change, should be covered by the claims of the present invention.
Sequence table
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35
<210> 8
<211> 39
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 8
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 (16)

1. a kind of glucagon analogue, containing as shown in Formulas I or Formula II in the structure of the glucagon analogue Structure, structure shown in Formulas I are as follows: HSQGTFTSD-X10-SKYLD-X16-X17-AA-X20-X21-F-X23-QWLMN-X29-Xz, Formula II Shown structure are as follows:
HSQGTFTSD-X10-SKYLD-X16-X17-AA-X20-X21-F-X23-QWLMN-X29-Xz-NH2,
Wherein, X10Any, X selected from Y, K or L16Selected from any of S, E or A, X17Selected from any of Q, E, A or R, X20Selected from Q, R's or K is any；X21Selected from any of D, L or E；X23Selected from any of V or I；X29For T or missing, XzIt is selected from GGPSSGAPPPS, GGPSSGAPPS, GPSSGAPPPS, GPSSGAPPS, PSSGAPPPS, PSSGAPPS, SSGAPPPS or SSGAPPS's is any.
2. glucagon analogue according to claim 1, which is characterized in that the glucagon analogue contains The structure as shown in formula III or formula IV, structure shown in formula III are as follows:
HSQGTFTSDYSKYLD-X16-X17-AAQ-DFVQWLMN-X29-Xz,
Structure shown in formula IV are as follows:
HSQGTFTSDYSKYLD-X16-X17-AAQ-DFVQWLMN-X29-Xz-NH2,
Wherein, X16Any, X selected from S or E17Any, X selected from Q or E29For T or missing, XzSelected from GGPSSGAPPPS, GGPSSGAPPS, GPSSGAPPPS, GPSSGAPPS, PSSGAPPPS, PSSGAPPS, SSGAPPPS or SSGAPPS's is any.
3. -2 described in any item glucagon analogues according to claim 1, which is characterized in that the glucagon Analog has tri- receptor agonist activity of GLP-1/GCG/GIP.
4. a kind of isolated polynucleotides, isolated polynucleotide encoding pancreas as described in any one of claim 1-3 is high Blood glucose element analog.
5. a kind of recombinant expression carrier includes the polynucleotides separated as claimed in claim 4.
6. a kind of host cell, the cell contains to be integrated with outside in recombinant expression carrier or genome as claimed in claim 5 The polynucleotides separated as claimed in claim 4 in source.
7. the preparation method of glucagon analogue as described in any one of claim 1-3, which is characterized in that selected from following It is any:
(1) glucagon analogue is synthesized using chemical synthesis process；
(2) host cell as claimed in claim 6 is cultivated under suitable conditions, is allowed to express the glucagon similar Object then separates and purifies the acquisition glucagon analogue.
8. glucagon analogue is in the drug of preparation treatment metabolism related diseases as described in any one of claim 1-3 Purposes.
9. a kind of promote weight loss or the method that prevents weight gain, be included in object application such as claim 1-3 it Described in any item glucagon analogues.
10. a kind of composition, containing the glucagon analogue as described in any one of claim 1-3 or such as claim 6 The culture and pharmaceutically acceptable carrier of the host cell.
11. glucagon analogue is preparing the purposes in fusion protein as described in any one of claim 1-3.
12. a kind of fusion protein, the glucagon analogue as described in any one of claim 1-3 is contained in structure.
13. fusion protein according to claim 12, which is characterized in that also containing long-acting in the structure of the fusion protein Unit.
14. fusion protein according to claim 13, which is characterized in that the long-acting unit is selected from albumin, turns iron egg White and immune globulin bletilla segment.
It is similar containing the glucagon as described in any one of claim 1-3 in structure 15. a kind of polypeptide being modified Object.
16. the polypeptide according to claim 15 being modified, which is characterized in that the glucagon analogue is by rouge Fat acid, polyethylene glycol, albumin, transferrins or immune globulin bletilla segment are modified.
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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111349155A (en) * 2018-12-24 2020-06-30 杭州和泽医药科技有限公司 Glucagon analogue and preparation method and application thereof
CN112898404A (en) * 2019-12-03 2021-06-04 天津药物研究院有限公司 Long-acting modified glucagon peptide analogue or salt thereof and application thereof
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CN111349155A (en) * 2018-12-24 2020-06-30 杭州和泽医药科技有限公司 Glucagon analogue and preparation method and application thereof
CN112898404A (en) * 2019-12-03 2021-06-04 天津药物研究院有限公司 Long-acting modified glucagon peptide analogue or salt thereof and application thereof
WO2021129894A1 (en) * 2019-12-23 2021-07-01 万新医药科技(苏州)有限公司 Triple agonist for glucagon-like peptide-1 receptor, glucagon receptor, and gastric inhibitory polypeptide receptor
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
CN113493503A (en) * 2020-04-08 2021-10-12 浙江道尔生物科技有限公司 Incretin analogue and preparation method and application thereof
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CN114685642A (en) * 2020-12-29 2022-07-01 浙江和泽医药科技股份有限公司 Pharmaceutically acceptable salt of incretin analogue, preparation method and application
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