CN109384839A - Glucagon-like peptide-1 analogs and application thereof - Google Patents
Glucagon-like peptide-1 analogs and application thereof Download PDFInfo
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- CN109384839A CN109384839A CN201710660343.4A CN201710660343A CN109384839A CN 109384839 A CN109384839 A CN 109384839A CN 201710660343 A CN201710660343 A CN 201710660343A CN 109384839 A CN109384839 A CN 109384839A
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- glucagon
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- 235000009582 asparagine Nutrition 0.000 description 1
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- QRUDEWIWKLJBPS-UHFFFAOYSA-N benzotriazole Chemical compound C1=CC=C2N[N][N]C2=C1 QRUDEWIWKLJBPS-UHFFFAOYSA-N 0.000 description 1
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- BBBFJLBPOGFECG-VJVYQDLKSA-N calcitonin Chemical compound N([C@H](C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N1[C@@H](CCC1)C(N)=O)C(C)C)C(=O)[C@@H]1CSSC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(=O)N1 BBBFJLBPOGFECG-VJVYQDLKSA-N 0.000 description 1
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- BGRWYRAHAFMIBJ-UHFFFAOYSA-N diisopropylcarbodiimide Natural products CC(C)NC(=O)NC(C)C BGRWYRAHAFMIBJ-UHFFFAOYSA-N 0.000 description 1
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- 125000000524 functional group Chemical group 0.000 description 1
- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 1
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- 229940088597 hormone Drugs 0.000 description 1
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- 125000003588 lysine group Chemical group [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- 125000003099 maleoyl group Chemical group C(\C=C/C(=O)*)(=O)* 0.000 description 1
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- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 description 1
- 230000035515 penetration Effects 0.000 description 1
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- 229910052698 phosphorus Inorganic materials 0.000 description 1
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- 229920000642 polymer Polymers 0.000 description 1
- 210000004896 polypeptide structure Anatomy 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000002953 preparative HPLC Methods 0.000 description 1
- GCYXWQUSHADNBF-AAEALURTSA-N preproglucagon 78-108 Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(N)=O)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC=1N=CNC=1)[C@@H](C)O)[C@@H](C)O)C(C)C)C1=CC=CC=C1 GCYXWQUSHADNBF-AAEALURTSA-N 0.000 description 1
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- 239000007974 sodium acetate buffer Substances 0.000 description 1
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
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- 125000002653 sulfanylmethyl group Chemical group [H]SC([H])([H])[*] 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 125000004213 tert-butoxy group Chemical group [H]C([H])([H])C(O*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 125000005931 tert-butyloxycarbonyl group Chemical group [H]C([H])([H])C(OC(*)=O)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 125000003396 thiol group Chemical group [H]S* 0.000 description 1
- ZGYICYBLPGRURT-UHFFFAOYSA-N tri(propan-2-yl)silicon Chemical compound CC(C)[Si](C(C)C)C(C)C ZGYICYBLPGRURT-UHFFFAOYSA-N 0.000 description 1
- 125000002221 trityl group Chemical group [H]C1=C([H])C([H])=C([H])C([H])=C1C([*])(C1=C(C(=C(C(=C1[H])[H])[H])[H])[H])C1=C([H])C([H])=C([H])C([H])=C1[H] 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/605—Glucagons
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biophysics (AREA)
- Gastroenterology & Hepatology (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Toxicology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Endocrinology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
The present invention provides a kind of Precursor Peptide of glucagon-like peptide-1 analogs or its pharmaceutically acceptable salt, the Precursor Peptide has amino acid sequence shown in following general formula I；I HX of general formula1X2GTFTSDVSSYLEEX3AAX4EFIX5WLVKX6X7X8X9；Wherein, the X1、X2、X3、X4、X5、X6、X7、X8And X9Indicate arbitrary amino acid.The present invention also provides a kind of glucagon-like peptide-1 analogs or its pharmaceutically acceptable salts.Glucagon-like peptide-1 analogs of the invention are conjugated by the polyethylene glycol based on polypeptide and specific structure after endogenous GLP-1 (7-36/37) sequence optimisation and are obtained, with stronger hypoglycemic activity, dosage can be reduced, and Half-life in vivo significantly extends, it is expected to improve Clinical Compliance, and precursor polypeptide sequence provided by the invention and endogenous GLP-1 (7-36/37) very high homology, immunogenicity can be reduced, therefore there are the preferably potentiality as drug development application.
Description
Technical field
The invention belongs to pharmaceutical technology fields, and in particular to a kind of glucagon-like peptide-1 analogs and its pharmaceutical
On the way.
Background technique
Glucagon-like peptide 1 (GLP-1) is a kind of Entero hormone, mainly thin in the L of end barnyard, ileum and colon
Born of the same parents' synthesis has meal and is discharged into circulation in reacting.GLP-1 (7-36,7-37) is the chief active form of GLP-1 in body circulation, is led to
Cross complicated mechanism control blood glucose, the secretion including insulin and glucagon, the emptying of stomach and the tune of periphery insulin
Section.GLP-1 (7-36,7-37) hypoglycemic effect is glucose dependency, can avoid hypoglycemia, and have and inhibit pancreas
The apoptosis of island beta cell promotes the effect of hyperplasia of pancreatic islet beta cell, and progression of the disease can be reversed.But the blood plasma of natural GLP-1 partly declines
Phase is only 1-2 minutes, and metabolism unstability limits its application as drug.
It is the main path being metabolized in polypeptides matter body that enzyme degradation and kidney, which are removed,.Studies have shown that intracorporal dipeptides kassinin kinin
Receptor-binding activity position N-terminal His-Ala segment in enzyme (DPPIV) specific recognition and GLP-1 structure of degrading and make its quickly
Inactivation, while other proteolytic enzymes such as endopeptidase also assists in degradation process in polypeptide body.Kidney is eliminating the objects such as peptide, albumen
It plays an important role in matter, blood plasma middle-molecular-weihydroxyethyl is less than 5KD and effective radius is less thanFree fraction molecule it is easily small by kidney
Ball filtration, and peptide hormone (such as calcitonin, GLP-1) is degraded by the metabolic enzyme in cortex renis in kidney circulation, is further arranged
It lets out in urine.Research report kidney is responsible for removing at least 80% Exendin-4 (CN1372570).
Metabolic stability is improved, extends plasma half-life, so that improving clinical application compliance is the drug based on GLP-1
The technical goal of development field.The structure of enzyme degradation critical sites in existing patented technology for source of people GLP-1 sequence changes
Making (CN00806548.9, CN99814187.9, CN200410017667.9 etc.) only considered enzyme one elimination factor of degradation, reach
It is long-acting less than ideal；And by introducing fatty acyl group in parent peptide chain structure, it improves with plasma protein binding force to keep away
Exempt from the skill that polypeptide quickly removes (CN201210513145.2, CN200810124641.2, CN20118000352.1 etc.) in vivo
Though art can extend half-life period (Liraglutide such as listed is by daily single) to a certain extent, compliance still needs
It improves, and since plasma protein binding rate therein is high, leads to drug efficacy delay, also as introducing aliphatic chain in peptide chain causes
Its dissolubility reduces, and needs in preparation using organic solvent.
Polyethylene glycol (PEG) change technology is the relatively applicable long-actingization technology in one, field of current protein/polypeptide class administration.
It generallys use linear straight chain or branched polyethylene glycol modifies protein/polypeptide, protein/polypeptide can be improved
Stable in physicochemical property reduces immunogenicity, improves proteolytic degradation ability, reduces metabolism of the kidney scavenging effect to drug,
Extend the Half-life in vivo of drug significantly, while improving drug solubility and enhancing the penetration power of cell membrane.In general,
High molecular weight (>=20KD) PEG modification is more advantageous to the Half-life in vivo for extending polypeptide or protein medicaments, but usually each PEG
Molecule only has 1 active end group and precursor activated molecule coupling labeled, and molecule drugloading rate is limited, and it is macromolecule modified after polypeptide with
The combination of receptor usually weakens, and influences drug effect, and therefore, the activity intensity of Precursor Peptide is to determine final modified outcome patent medicine
An important factor for property.In addition, shadow of the physicochemical property of Precursor Peptide to the efficiency of modification reaction, the yield of final product and quality
Sound is larger, therefore, selects suitable GLP-1 analog molecule as macromolecule modified Precursor Peptide, is to realize target molecule
Long-acting chemical drug effect an important factor for.
Summary of the invention
For the limitation of the prior art, the object of the present invention is to provide a kind of glucagon-like peptide-1 analogs,
It is more specifically described similar for the high molecular polymer of polypeptide precursor of the endogenous GLP-1 (7-36/37) after sequence optimisation
The polyethylene glycol conjugate of object.
The present invention is surprised to find that a kind of GLP-1 analog obtained by Structural Transformation, the excitement to GLP-1 receptor
Activity significantly increases, and dissolubility and stability are obviously improved, and is used as macromolecule modified Precursor Peptide, no
Improve only the bioactivity of target product, and also improve macromolecule modified reaction efficiency, final product yield and
Quality.
Glucagon-like peptide-1 analogs of the invention have stronger hypoglycemic activity, can reduce dosage, and
And Half-life in vivo significantly extends, and is expected to improve Clinical Compliance, and glucagon-like-peptide-1 provided by the invention is similar
The precursor polypeptide sequence and endogenous GLP-1 (7-36/37) very high homology of object can reduce immunogenicity, therefore have more preferable
As drug development application potentiality.
On the one hand, the present invention provides a kind of Precursor Peptide of glucagon-like peptide-1 analogs or its is pharmaceutically acceptable
Salt, the Precursor Peptide have following general formula I shown in amino acid sequence；
I HX of general formula1X2GTFTSDVSSYLEEX3AAX4EFIX5WLVKX6X7X8X9；
Wherein, the X1、X2、X3、X4、X5、X6、X7、X8And X9Indicate arbitrary amino acid.
Preferably, any in the precursor polypeptide sequence of the glucagon-like peptide-1 analogs such as SEQ ID NO:1-56
Shown in item；
On the other hand, the present invention provides a kind of glucagon-like peptide-1 analogs or its pharmaceutically acceptable salt,
For above-mentioned Precursor Peptide or the polyethylene glycol conjugate of its pharmaceutically acceptable salt；
Preferably, the average molecular weight range of the polyethylene glycol is 5-50KDa, more preferably 20-50KDa；Into one
Step is preferably 40-50KDa；
Preferably, the polyethylene glycol is straight chain type, branching type polyethylene glycol；
Preferably, the polyethylene glycol is selected from the branching type polyethylene glycol that average molecular weight range is 40-50KDa, then excellent
Selection of land is the two branching type polyethylene glycol of 40-50KDa.
Preferably, it is similar by the activated group on exposed terminated groups to be conjugated in glucagon-like-peptide-1 for the polyethylene glycol
Object or its pharmaceutically acceptable salt, the activated group are selected from dimaleoyl imino, sulfydryl, succinimido, aldehyde radical, halogen
Plain isoreactivity functional group, preferably dimaleoyl imino；
Preferably, the polyethylene glycol is conjugated in the lysine side-chain of SEQ ID NO:1-56 any bar by activated group
Or cysteine side chain；It is highly preferred that the polyethylene glycol is conjugated in SEQ ID NO:9-12,17-20 by activated group
Any bar.
It is a further object to provide a kind of pharmaceutical composition, described pharmaceutical composition includes any of the above-described institute
The Precursor Peptide stated or its pharmaceutically acceptable salt or glucagon-like peptide-1 analogs or its is pharmaceutically acceptable
Salt, it is preferable that described pharmaceutical composition also includes pharmaceutically acceptable carrier and/or auxiliary material；It is highly preferred that the carrier
And/or auxiliary material is selected from one of water-soluble filler, pH adjusting agent, stabilizer, water for injection or osmotic pressure regulator or more
Kind；
Preferably, the water-soluble filler is selected from mannitol, D-40, sorbierite, polyethylene glycol, grape
One of sugar, lactose or galactolipin etc. are a variety of；The pH adjusting agent includes but is not limited to citric acid, phosphoric acid, lactic acid, winestone
The organic or inorganic acids such as acid, hydrochloric acid and potassium hydroxide, sodium hydroxide, ammonium hydroxide, sodium carbonate, potassium carbonate, ammonium carbonate, carbon
One of the physiologically acceptable inorganic bases or salt or a variety of such as potassium hydrogen phthalate, sodium bicarbonate or bicarbonate ammonium salt；The stabilization
Agent is selected from EDTA-2Na, sodium thiosulfate, sodium pyrosulfite, sodium sulfite, dipotassium hydrogen phosphate, sodium bicarbonate, sodium carbonate, smart ammonia
Acid, lysine, glutamic acid, aspartic acid, polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, carboxyl/hydroxylated cellulose or
In its derivative such as HPC, HPC-SL, HPC-L or HPMC, cyclodextrin, lauryl sodium sulfate or trishydroxymethylaminomethane
It is one or more；The osmotic pressure regulator is sodium chloride and/or potassium chloride.
Further object of the present invention be to provide the glucagon-like peptide-1 analogs Precursor Peptide or its pharmaceutically
Acceptable salt, the glucagon-like peptide-1 analogs or its pharmaceutically acceptable salt are in preparation for treating glycosuria
Disease, the purposes in fat, metabolic syndrome pharmaceutical composition and the pharmaceutical composition are in preparation for treating glycosuria
Purposes in disease, fat, metabolic syndrome drug.
The detailed description of summary of the invention
Polypeptide sequence
The Precursor Peptide of glucagon-like peptide-1 analogs of the present invention is manually repairing for GLP-1 (7-36/37)
Decorations form.The native sequences of GLP-1 (7-36/37) are as follows:
HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRNH2/G.Dipeptides kininase (DPPIV) in blood is by N-terminal
The hydrolysis excision of His-Ala dipeptide fragment, remaining GLP-1 (9-36/37) sequence lose or weaken bioactivity, reality of the invention
It applies in scheme to X in endogenous sequence8Or X9Carry out amino acid substitution appropriate, the embodiment of the present invention 3 the experimental results showed that this
Taken measure is invented it is possible to prevente effectively from the enzyme of polypeptide is degraded and inactivated.
In general, the polypeptide ligand for acting on Type B GCGR receptor be with alpha-helix secondary structure in conjunction with receptor, because
This, the binding force of polypeptide and receptor is helped to improve conducive to the Structural Transformation for forming alpha-helix, enhances the bioactivity of polypeptide.
In the present embodiment, amino acid has been carried out to the appropriate site in source of people GLP-1 (7-36/37) sequence to replace
It changes, such as G22/E、G35/ Aib, to enhance the bioactivity of Precursor Peptide.It is in the embodiment of the present invention that precursor provided by the invention is more
Peptide and Gly2、Cys37GLP-1 (7-37) (sequence SEQ IDNO:57
) and Gly HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRC2、Aib35、Ala38、Cys39GLP-1(7-37)
(sequence SEQ ID NO:58
HGEGTFTSDVSSYLEGQAAKEFIAWLVKAibRGAC test) is compared, the results showed that, by these changes
Polypeptide sequence obtained by changing in vitro significantly increases the agonist activity of GLP-1 receptor, shows more in animal experiment in vivo
Strong hypoglycemic activity and better internal stability.
Meanwhile for modification polyethylene group is conjugated convenient for fixed point, the appropriate site of sequence is introduced or is replaced half Guang ammonia
Sour residue (A30/C、C37、C38), or replaced by appropriate, a lysine residue (K is only remained in sequence34/R、K26/R)。
The result of study of the embodiment of the present invention shows that these structure changes do not have an impact the activity of polypeptide.In the embodiment of the present invention 5
Display the experimental results showed that, the polyethyleneglycol modified body of preferred polypeptide provided by the invention is living to the excitement of GLP-1 receptor
Property be significantly stronger than other relatively weak Precursor Peptide modifiers of activity, and the hypoglycemic drug effect result of study in embodiment 8
Prompt the polyethylene glycol conjugate of preferred polypeptide that there is better drug effect and long-term effect, dosage is reduced, therefore more druggability is latent
Power.
It is commonly used for efficiency of the physicochemical property to modification reaction of macromolecule modified Precursor Peptide, the yield of final product
And the influence of quality is bigger.Precursor Peptide (the usually buffer solution of proper pH value) dissolubility in modification reaction system
The step of difference, polypeptide aggregation object are precipitated, and will lead to reaction efficiency reduction, need to increase removal remaining polypeptides in post-processing, such as from
The heart, molecular sieve chromatography separation etc., or even directly using organic solvent such as DMSO etc. as reaction dissolvent, increase post-processing difficulty and prolong
Long manufacturing cycle.Be found surprisingly that in embodiment of the present invention by aforementioned structure convert provided by polypeptide, dissolubility and steady
It is qualitative to be obviously improved, it is more suitable for macromolecule modified Precursor Peptide.Such as embodiment 4, in used reaction system before
Body polypeptide preferably dissolves, and reaction efficiency is high, noresidue polypeptide or other impurities in final product, and subsequent purification processing is easy,
The yield (average 75-80%) and quality (purity >=98%) of target product reach expected horizontal.
Pegylation
Peptide hormone mainly removes approach metabolism by enzyme degradation and kidney in body, and wherein kidney removing accounts for leading, is
Influence the principal element of polypeptide drug Half-life in vivo.Structural modification in polypeptide sequence on some amino acid residue sides is special
It is not that the conjugations of the macro-radicals such as alkylation or polyethylene glycol can delay kidney to remove, effectively extends biological half-life.
In specific embodiment of the invention, it is Cys or Lys that site is contained at least one in provided polypeptide sequence, with
Just in the side chain thiol of Cys or the ε-NH of Lys side chain2Polyethylene glycol (PEG) group is covalently conjugated in upper fixed point.The polyethylene glycol
Average molecular weight be 5-50KD；It is highly preferred that the average molecular weight of the polyethylene glycol is 20-50KDa；Further preferably
Ground, the average molecular weight of the polyethylene glycol are 40-45KDa；
Polyethylene glycol for modification be usually include the straight chain an of activated group or the structure of branching type, wherein dividing
Branch type polyethylene glycol include two branches and four branching types, the activated group be the free sulfhydryl groups referred to polypeptide structure or
The group that amino covalence combines, such as aldehyde, amino, ester, mercaptan, alpha-halogenate acetyl group, dimaleoyl imino or diazanyl.
Preferably two branching type polyethylene glycol in embodiment of the present invention, and preferably comprised 1 or 2 activation function
Group, the preferred dimaleoyl imino of active function groups.
Polyethylene glycol of the present invention be it is available through a variety of ways, including commercial sources obtain or according to this
Known method is voluntarily prepared in field.Polyethylene glycol in embodiment of the present invention for modification is preferably but not limited to selected from such as
Flowering structure:
PEG of the present invention is modified can be realized by any method well known in the art, including via acylation, reproducibility
Alkanisation, Michael addition, mercaptan alkanisation pass through the active group of peg moiety (such as aldehyde, amino, ester, mercaptan, alpha-halogenate second
Acyl group, dimaleoyl imino or diazanyl) other chemo-selective conjugation methods.In specific embodiment of the invention, lead to
Michael addition reaction is crossed, i.e., with the thiol side chain of the PEG modification Cys of maleimide activation, is keyed by thioether poly-
Glycation polypeptide.
Purposes
For the limitation of the existing drug therapies such as current treatment diabetes, obesity, metabolic syndrome, the present invention provides one
Kind novel method for the treatment of, the method are related to giving comprising Precursor Peptide of the invention or its pharmaceutically acceptable salt or pancreas
The pharmaceutical composition of -1 analog of glucagon-like peptide or its pharmaceutically acceptable salt.Comprising Precursor Peptide of the invention or its
The pharmaceutical composition of pharmaceutically acceptable salt or glucagon-like peptide-1 analogs or its pharmaceutically acceptable salt is having
Effect ground has long-term effect while reducing blood glucose, and compliance, more clinical application potentiality can be improved.
Pharmaceutical composition
In another aspect, described pharmaceutical composition includes above-mentioned Precursor Peptide the present invention also provides a kind of pharmaceutical composition
Or its pharmaceutically acceptable salt or glucagon-like peptide-1 analogs or its pharmaceutically acceptable salt.
Preferably, described pharmaceutical composition also includes pharmaceutically acceptable carrier and/or auxiliary material.
It is highly preferred that the carrier and/or auxiliary material be selected from water-soluble filler, pH adjusting agent, stabilizer, water for injection or
One of osmotic pressure regulator is a variety of.
Preferably, the water-soluble filler is selected from mannitol, D-40, sorbierite, polyethylene glycol, grape
One of sugar, lactose or galactolipin etc. are a variety of；The pH adjusting agent includes but is not limited to citric acid, phosphoric acid, lactic acid, winestone
The organic or inorganic acids such as acid, hydrochloric acid and potassium hydroxide, sodium hydroxide, ammonium hydroxide, sodium carbonate, potassium carbonate, ammonium carbonate, carbon
One of the physiologically acceptable inorganic bases or salt or a variety of such as potassium hydrogen phthalate, sodium bicarbonate or bicarbonate ammonium salt；The stabilization
Agent is selected from EDTA-2Na, sodium thiosulfate, sodium pyrosulfite, sodium sulfite, dipotassium hydrogen phosphate, sodium bicarbonate, sodium carbonate, smart ammonia
Acid, lysine, glutamic acid, aspartic acid, polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, carboxyl/hydroxylated cellulose or
In its derivative such as HPC, HPC-SL, HPC-L or HPMC, cyclodextrin, lauryl sodium sulfate or trishydroxymethylaminomethane
It is one or more；The osmotic pressure regulator is sodium chloride and/or potassium chloride.
Yet another aspect, the present invention provides above-mentioned Precursor Peptide or its pharmaceutically acceptable salts or glucagon
- 1 analog of sample peptide or its pharmaceutically acceptable salt are being prepared for treating diabetes, obesity, the medicine group of metabolic syndrome
Close the purposes in object.
Preferably, composition of the present invention can in the form of vein, muscle or subcutaneous injection agent or oral, rectum,
Nasal-cavity administration.Dosage range can be 5 μ g-10mg/ time, this depending on treatment object, administration mode, indication and other because
Element etc..
Synthesis
It is by methods known in the art in the basic peptide chain of the Precursor Peptide with logical structure shown in formula I provided by the invention
It is prepared:
1) synthesis gradually or by segment is assembled by conventional solid or liquid phase process；
2) expression encodes the nucleic acid construct of polypeptide in host cell, and recycles expression from host cell cultures and produce
Object；
3) the cell free in vitro expression of the nucleic acid construct of coding polypeptide is influenced, and recycles expression product；
Or pass through method 1), 2) any combination or 3) obtain peptide fragment, connect these segments then to obtain target
Peptide.
Preferably, target peptide is prepared using Fmoc solid phase synthesis process.
Preferably, the pegylation of target polypeptides is completed by the following method: by PEG and this hair through overactivation
Bright polypeptide pH5.0-7.0 reaction, wherein the molar ratio of PEG and peptide is 1-10, and the reaction time is 0.5-12 hours, reaction temperature
Degree is 4-37 DEG C.
After conjugation reaction, target product can be separated by appropriate method well known in the art.Applicable method packet
Include but be not limited to ultrafiltration, dialysis or chromatography etc..
Activity rating
It is tested in embodiment of the present invention using normal mouse glucose load, using Liraglutide as positive control drug, evaluation
It is of the invention that the hypoglycemic activity and long-term effect of Precursor Peptide are provided.
The glucagon is had rated using ob/ob diabetic mice in another embodiment of the invention
The hypoglycemic effect of -1 analog of peptide and influence to weight, the results showed that glucagon-like-peptide-1 provided by the invention is similar
Object has significant hypoglycemic effect, and administration frequency is reduced, and has apparent application advantage.
Detailed description of the invention
Fig. 1 is Precursor Peptide, the glucagon-like peptide-1 analogs of glucagon-like peptide-1 analogs of the invention
And the HPLC chromatogram of the analog modification group, wherein figure A is before glucagon-like peptide-1 analogs of the invention
Body polypeptide SEQ ID NO:9, figure B are glucagon-like peptide-1 analogs SEQ ID NO:9- II, figure C is II (m of structure
PEG)2The HPLC chromatogram of-Mal (40KDa)；
The MALDI-TOF that Fig. 2 is glucagon-like peptide-1 analogs SEQ ID NO:9- II of the invention schemes；
Fig. 3 be glucagon-like peptide-1 analogs SEQ ID NO:9- II and SEQ ID NO:58- II of the invention (see
Embodiment 2) hypoglycemic effect and long-term effect compare.
Specific embodiment
The present invention is further illustrated combined with specific embodiments below.The present embodiment is only to explain the present invention, unexpectedly
Taste limit the invention in any way content.
The explanation of amino acid abbreviations:
Gly: glycine (G)
Ala: alanine (A)
Val: valine (V)
Leu: leucine (L)
Phe: phenylalanine (F)
Trp: tryptophan (W)
Ser: serine (S)
Thr: threonine (T)
Glu: glutamic acid (E)
Gln: glutamine (Q)
Asp: aspartic acid (D)
Asn: asparagine (N)
Tyr: phenylalanine (Y)
Arg: arginine (R)
Lys: lysine (K)
His: histidine (H)
Aib: α-aminoacid
The explanation of reagent abbreviation
Boc: tert-butoxycarbonyl
Tert-Bu: tert-butyl
DCM: methylene chloride
DIC: diisopropylcarbodiimide
Fmoc:9- fluorenes methoxycarbonyl
HoBt:1- hydroxybenzotriazole
HBTU:2- (1H- benzotriazole -1- base) -1,1,3,3- tetramethyl-urea hexafluorophosphoric acid ester
HATU:O- (7- azepine benzo triazol-1-yl)-N, N, N ', N '-tetramethyl-urea hexafluorophosphoric acid ester
Mtt:4- methyltrityl
NMP:N- methyl pyrrolidone
DMF: dimethylformamide
Pbf:2,2,4,6,7- pentamethyl Dihydrobenzofuranes
Trt: trityl group
EDT: dithioglycol
TFA: trifluoroacetic acid
TIS: tri isopropyl silane
FBS: fetal calf serum
Embodiment 1 prepares glucagon-like peptide-1 analogs Precursor Peptide
Precursor Peptide shown in general formula I is prepared by following step
1) it synthesizes: using Fmoc strategy, with 336 type Peptide synthesizer of CS (CS Bio), gradually closing in accordance with the following steps
At:
A) it is coupled to obtain Fmoc- by resin solid phase carrier and fmoc-protected C-terminal amino acid in the presence of activator systems
Amino acid-resin；Wherein, the synthesis end C- amidation polypeptide uses amino resins, such as Rink Amide AM, Rink Amide,
Rink MBHA etc..
B) extension of peptide chain: by solid-phase synthesis according to peptide sequence amino acid sequence connect amino acid, obtain the end N- and
Peptide-resin conjugate of side chain protection；Band side chain amino acid takes following safeguard measure: tryptophan Boc, and glutamic acid is used
OtBu, lysine Boc, glutamine Trt, tyrosine tBu, serine Trt or tBu, aspartic acid OtBu, Soviet Union
Propylhomoserin tBu, cysteine Trt, histidine Trt or Boc, arginine are protected with Pbf.The coupling activator used is
HOBT/HBTU/DIEA and HOBT/HATU/DIEA, ninhydrin method detect reaction efficiency.
C) on resin polypeptide cracking: TFA/EDT/TIS/H2O (92.5:2.5:2.5:2.5v/v) solution is set at room temperature
React 90min, deprotection and deresination.Filtrate is filtered to obtain, precipitates thick polypeptide with excess diethyl ether, precipitating is collected in centrifugation, then with less
Ether washing precipitating is measured, drying under vacuum obtains crude product polypeptide.Deprotection base and resin simultaneously, obtain crude product glucagon
Sample peptide -1 and glucagon fragment analogue chimeric polyeptides；
2) it purifies: crude product polypeptide being dissolved in water or 10-15% acetonitrile (10-50mg/ml), two sulphur of 50-100mM are added
Base threitol DTT or beta -mercaptoethanol are denaturalized, using preparative HPLC method, C18 chromatographic column, acetonitrile-water-trifluoroacetic acid
System isolates and purifies, and is concentrated, freeze-drying, obtains the free sterling polypeptide of sulfydryl.
Precursor Peptide shown in following SEQ ID NO:1-56 is in the above way prepared.
SEQ ID NO:1HAPGTFTSDVSSYLEEQAAKEFIAWLVKAibRGC-NH2
SEQ ID NO:2HAPGTFTSDVSSYLEEQAAKEFIAWLVKAibRC-NH2
SEQ ID NO:3HAPGTFTSDVSSYLEEQAAKEFICWLVKAibRG-NH2
SEQ ID NO:4HAPGTFTSDVSSYLEEQAAKEFICWLVKAibR-NH2
SEQ ID NO:5HAPGTFTSDVSSYLEERAAKEFIAWLVKAibRGC-NH2
SEQ ID NO:6HAPGTFTSDVSSYLEERAAKEFIAWLVKAibRC-NH2
SEQ ID NO:7HAPGTFTSDVSSYLEERAAKEFIAWLVKGC-NH2
SEQ ID NO:8HAPGTFTSDVSSYLEERAAKEFICWLVKAibRG-NH2
SEQ ID NO:9HGEGTFTSDVSSYLEEQAAKEFIAWLVKAibRGC-NH2
SEQ ID NO:10 HGEGTFTSDVSSYLEEQAAKEFIAWLVKAibRC-NH2
SEQ ID NO:11 HGEGTFTSDVSSYLEEQAAKEFICWLVKAibRG-NH2
SEQ ID NO:12 HGEGTFTSDVSSYLEEQAAKEFICWLVKAibR-NH2
SEQ ID NO:13HGEGTFTSDVSSYLEERAAKEFIAWLVKAibRGC-NH2
SEQ ID NO:14HGEGTFTSDVSSYLEERAAKEFIAWLVKAibRC-NH2
SEQ ID NO:15 HGEGTFTSDVSSYLEERAAKEFICWLVKAibRG-NH2
SEQ ID NO:16 HGEGTFTSDVSSYLEERAAKEFICWLVKAibR-NH2
SEQ ID NO:17 H(d-A)EGTFTSDVSSYLEEQAAKEFICWLVKAibRG-NH2
SEQ ID NO:18 H(d-A)EGTFTSDVSSYLEEQAAKEFICWLVKAibR-NH2
SEQ ID NO:19 H(d-A)EGTFTSDVSSYLEEQAAKEFIAWLVKAibRGC-NH2
SEQ ID NO:20 H(d-A)EGTFTSDVSSYLEEQAAKEFIAWLVKAibRC-NH2
SEQ ID NO:21 H(d-A)EGTFTSDVSSYLEERAAKEFIAWLVKAibRGC-NH2
SEQ ID NO:22 H(d-A)EGTFTSDVSSYLEERAAKEFIAWLVKAibRC-NH2
SEQ ID NO:23 H(d-A)EGTFTSDVSSYLEERAAKEFICWLVKAibRG-NH2
SEQ ID NO:24 H(d-A)EGTFTSDVSSYLEERAAKEFICWLVKAibR-NH2
SEQ ID NO:25 HAPGTFTSDVSSYLEEQAAKEFIAWLVRAibRG-NH2
SEQ ID NO:26 HAPGTFTSDVSSYLEEQAAKEFIAWLVRAibR-NH2
SEQ ID NO:27 HAPGTFTSDVSSYLEERAAKEFIAWLVRAibRG-NH2
SEQ ID NO:28 HAPGTFTSDVSSYLEERAAKEFIAWLVRAibR-NH2
SEQ ID NO:29 HAPGTFTSDVSSYLEERAAKEFIAWLVRG-NH2
SEQ ID NO:30 HGEGTFTSDVSSYLEEQAAKEFIAWLVRAibRG-NH2
SEQ ID NO:31 HGEGTFTSDVSSYLEEQAAKEFIAWLVRAibG-NH2
SEQ ID NO:32 HGEGTFTSDVSSYLEERAAKEFIAWLVRAibRG-NH2
SEQ ID NO:33 HGEGTFTSDVSSYLEERAAKEFIAWLVRAibG-NH2
SEQ ID NO:34 H(d-A)EGTFTSDVSSYLEEQAAKEFIAWLVRAibRG-NH2
SEQ ID NO:35 H(d-A)EGTFTSDVSSYLEEQAAKEFIAWLVRAibG-NH2
SEQ ID NO:36 HAPGTFTSDVSSYLEEQAAREFIAWLVKAibRG-NH2
SEQ ID NO:37 HAPGTFTSDVSSYLEEQAAREFIAWLVKAibR-NH2
SEQ ID NO:38 HAPGTFTSDVSSYLEERAAREFIAWLVKAibRG-NH2
SEQ ID NO:39 HAPGTFTSDVSSYLEERAAREFIAWLVKAibR-NH2
SEQ ID NO:40 HAPGTFTSDVSSYLEERAAREFIAWLVKG-NH2
SEQ ID NO:41 HGEGTFTSDVSSYLEEQAAREFIAWLVKAibRG-NH2
SEQ ID NO:42 HGEGTFTSDVSSYLEEQAAREFIAWLVKAibR-NH2
SEQ ID NO:43 H(d-A)EGTFTSDVSSYLEEQAAREFIAWLVKAibRG-NH2
SEQ ID NO:44 H(d-A)EGTFTSDVSSYLEEQAAREFIAWLVKAibR-NH2
SEQ ID NO:45HAPGTFTSDVSSYLEEQAAREFIAWLVRAibKG-NH2
SEQ ID NO:46HAPGTFTSDVSSYLEEQAAREFIAWLVRAibK-NH2
SEQ ID NO:47HAPGTFTSDVSSYLEERAAREFIAWLVRAibKG-NH2
SEQ ID NO:48HAPGTFTSDVSSYLEERAAREFIAWLVRAibK-NH2
SEQ ID NO:49HGEGTFTSDVSSYLEEQAAREFIAWLVRAibKG-NH2
SEQ ID NO:50HGEGTFTSDVSSYLEEQAAREFIAWLVRAibK-NH2
SEQ ID NO:51HGEGTFTSDVSSYLEERAAREFIAWLVRAibKG-NH2
SEQ ID NO:52HGEGTFTSDVSSYLEERAAREFIAWLVRAibK-NH2
SEQ ID NO:53H(d-A)EGTFTSDVSSYLEEQAAREFIAWLVRAibKG-NH2
SEQ ID NO:54H(d-A)EGTFTSDVSSYLEEQAAREFIAWLVRAibK-NH2
SEQ ID NO:55H(d-A)EGTFTSDVSSYLEERAAREFIAWLVRAibKG-NH2
SEQ ID NO:56H(d-A)EGTFTSDVSSYLEERAAREFIAWLVRAibK-NH2
The dissolubility of 2 glucagon-like peptide-1 analogs Precursor Peptide of embodiment, stability experiment
Carried out the evaluation test of dissolubility and stability of solution to preferred polypeptide provided by the invention, at the same with to GLP-
1 former sequence only do simple transformation polypeptide sequence (Gly2, Cys37GLP-1 (7-37): SEQ ID NO:
Sequence 57HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRC) and in patent (CN201610211144.0) being related to
(Gly2, Aib35, Ala38, Cys39GLP-1 (7-37): SEQ ID NO:58
HGEGTFTSDVSSYLEGQAAKEFIAWLVKAibRGAC it) compares, finds in technical solution of the present invention
Structural Transformation efficiently solves the dissolubility and stability of solution of Precursor Peptide.
Sample:
SEQ ID NO:57、SEQ ID NO:58、
SEQ ID NO:1,9,11,15,17,24, self-control, purity >=98%.
Dissolubility:
The above-mentioned sample solution that aimed concn is 2mg/ml is prepared, the 20mMPBS of pH 6.0,7.4 is appropriate in room-temperature dissolution
Sample, 4000r centrifugation, takes supernatant to detect.Acetonitrile-water (30%) solution of the polypeptide sample of debita spissitudo is prepared as test
Use reference substance solution.
HPLC-UV measurement:
Chromatographic column: 3.6 μm of Aeriswidepore XB-C18,4.6 × 150mm；
Mobile phase: A:0.05%TFA/H2O；B:0.05%TFA/ acetonitrile
Detection wavelength: 214nm
Stability of solution:
Weigh appropriate above-mentioned sample, with the 20mM sodium-acetate buffer of pH4.2, pH 6.0,7.4 20mMPBS in room temperature
Dissolution, is configured to 2mg/ml in 25 DEG C of placement 8hr, measures polypeptide amount in solution according to the above method, calculates compared with primary quantity residual
Allowance percentage.It the results are shown in Table 1.
1 polypeptide sample dissolubility of table, Detection of Stability result
The hypoglycemic effect of the Precursor Peptide of 3 glucagon-like peptide-1 analogs of embodiment is evaluated
Using the normal mouse glucose load model evaluation hypoglycemic effect of part of polypeptide provided by the invention.
Given the test agent:
SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:4,9,11,15,17,19 are made by oneself, purity >=98%
Method:
Animal (n=8) overnight fasting before testing, subcutaneous injection physiological saline (10mL/kg) is as a control group；It is subcutaneous single
Secondary injection Liraglutide (200 μ g/kg) is used as positive control drug group；Blood glucose is measured before administration, gives positive drug and given the test agent
Glucose (4.5g/kg) is injected intraperitoneally simultaneously and after 2,4 hours in (200 μ g/kg), administration, and tail point takes hematometry to 30min after sugar
Blood glucose value, calculate blood glucose inhibiting rate (%) relative to positive control drug.It the results are shown in Table 2.
2 polypeptide of table is to normal mouse glucose load model hypoglycemic effect
Note: blood glucose inhibiting rate is the percentage relative to positive control drug in table
Conclusion: it is naked that the blood glucose inhibiting rate data in You Shangbiao 2 relative to positive drug can be seen that the precursor that the present invention designs
Peptide still shows stronger hypoglycemic effect after administration 2-4 hours, and not optimized sequence Gly2, Cys37GLP-1(7-37)
Only activity is shown at once in administration.Illustrate to be significantly better than by the Precursor Peptide activity in vivo that optimizes of the present invention and stability without
The sequence of optimization.
The preparation of 4 glucagon-like peptide-1 analogs of embodiment
1) it connects:
Maleoyl functionalization: the polypeptide as shown in SEQ ID NO:3 is dissolved in the 50mM phosphorus of the pH6 containing 5mM EDTA
In sour sodium buffer solution, concentration 2mg/mL.The solid PEG- maleimide of 1.2-1.5 times of mole is added, stirs molten
Solution, in room temperature reaction 0.5-2hr.Reaction is monitored with HPLC, is terminated and is reacted with 5mM beta -mercaptoethanol, is set pure after room temperature 30min
Change.
Iodoacetyl functionalization: by the mPEG (1:1) of peptide precursor and iodoacetyl functionalization in 7M urea/50mM Tris
It is polyethyleneglycol modified that reaction completion in 45 minutes is stirred at room temperature in buffer (pH7.5-8.5), between the Cys on PEG and peptide chain
Form covalent thioether key.
2) it purifies: preparative ion-exchange chromatography is used, with SP SepharoseHP filler, with 0-500mM sodium chloride
PH6 phosphate buffer linear gradient elution.Efflux collects PEG- polypeptide flow point with HPLC and SDS- electrophoresis detection,
It is concentrated by ultrafiltration, is freeze-dried and obtains.The result is shown in Figure 1, wherein figure A is the precursor of glucagon-like peptide-1 analogs of the invention
Polypeptide SEQ ID NO:9, figure B are glucagon-like peptide-1 analogs SEQ ID NO:9- II, figure C is structure II (mPEG)2-
The HPLC chromatogram of Mal (40KDa).
3) sterling polypeptide carries out full molecular weight scanning through MALDI-TOF, determines average molecular weight.As a result see that Fig. 2, Fig. 2 are
The MALDI-TOF of glucagon-like peptide-1 analogs SEQ ID NO:9- II of the invention schemes.
Polypeptide carbowax modifier shown in following table is prepared for using the above method.
3 polypeptide carbowax modifier of table
The Precursor Peptide and glucagon-like peptide-1 analogs pair of 5 glucagon-like peptide-1 analogs of embodiment
The agonism of GLP-1 receptor
Given the test agent:
GLP-1、SEQ ID NO:57、SEQ ID NO:58、SEQ ID NO:4、SEQ ID NO:9、SEQ ID NO:11、
SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19 and SEQ ID NO:9- II, SEQ ID NO:11- II, SEQ
ID NO:57- II, SEQ ID NO:58- II self-control, purity >=98%
Method: untested compound serum-free medium (containing 0.1%BSA, 0.5mM IBMX) is made into the 2 of working concentration
Times.Cell serum-free medium is suspended and (contains 0.1%BSA, 0.5mM IBMX), and counted by vitellophag (HEK293)
Number is added 384 orifice plates according to 2000c cell/5 holes μ l/, 5 μ l untested compounds is then added, room temperature is protected from light 30min.Instead
After answering, cAMP detection substrate is added, room temperature is protected from light 60min.After reaction, multi-functional micro- in Envision2104
The detection of orifice plate microplate reader.The activity ratio (%Response) under each concentration conditions of each sample is calculated.Pass through %Response
To the logarithm X of sample concentration, carries out Nonlinear Quasi and EC50 value is calculated.
As a result 4 be see the table below.
The agonist activity of table 4GLP-1 analogue polypeptide and PEG modified body to GLP-1 receptor
As can be seen from the above table, the polypeptide of structure is improved through technical solution of the present invention, it is significant to the agonist activity of receptor
The GLP-1 analog simply changed is only made with other higher than endogenous GLP-1；After PEG modification to the activity intensity of receptor under
About 1/the 10 of Precursor Peptide are down to, therefore, Precursor Peptide activity is stronger more advantageous to the activation plays of target modified outcome.
The hypoglycemic effect and long-term effect of 6 straight chain type PEG modified polypeptide of embodiment are evaluated
The hypoglycemic effect and long-term effect of polypeptide of the present invention are had rated using normal mouse glucosieloading test.
Animal (n=8) overnight fasting before testing, subcutaneous injection physiological saline (10mL/kg) is as a control group；Subcutaneous note
Liraglutide (100nmol/kg, one time a day) is penetrated as positive control drug group；Remaining test component is not SEQ ID NO:1
(mPEG20,40KD), SEQ ID NO:9-mal-mPEG20KD, SEQ ID NO:11-mal-mPEG20KD, dosage
100nmol/kg.Dosage 100nmol/kg.
Blood glucose is measured before administration, gives given the test agent, glucose is injected intraperitoneally in different time sections after administration respectively
After (4.5g/kg), tail point takes hematometry to calculate blood glucose inhibiting rate to the blood glucose value of 30min after sugar.
It the results are shown in Table 5.
Conclusion: trying 4 samples and show different degrees of hypoglycemic activity and long-term effect, and it is right to be superior to the positive
According to medicine, while showing that increasing molecular weight helps to extend drug effect.The modifier hypoglycemic effect of SEQ ID NO:9 and 11 is without obvious
Difference illustrates that the modification of 30 and the end C- in the sequence can be achieved to keep activity and long-actingization.
The hypoglycemic effect (n=8) of 5. straight chain type PEG modified polypeptide of table
7 branching type PEG modified polypeptide hypoglycemic effect of embodiment and long-term effect
Given the test agent: the SEQ ID NO:1- I that is prepared in embodiment 4, SEQ ID NO:9- II, SEQ ID NO:11- II,
SEQ ID NO:15- II, SEQ ID NO:17- II, evaluation method: with embodiment 6.As a result 6 be see the table below.
6 branching type PEG modified polypeptide hypoglycemic effect of table and long-term effect
Conclusion: positive drug Liraglutide drug effect only continued to 28 hours as can be seen from Table 6, and tested group of sample medicine
Effect continued to 148 hours, it is contemplated that the difference in genera of mouse and body metabolism, it can be determined that reaches expected long-actingization.
Influence of the different polypeptide precursors of embodiment 8 to PEG modified outcome drug effect
Compare the hypoglycemic effect and long-term effect of SEQ ID NO:9- II Yu SEQ ID NO:58- II.
Given the test agent is self-control, purity >=98%.
Positive control drug is Liraglutide.
Given the test agent SEQ ID NO:9- II sets 250,25,2.5nmol/kg (be respectively designated as in figure sample b-1, b-2,
B-3) dosage group, positive drug and Gly2, Aib35, Ala38, Cys39GLP-1 (7-37)-II (is denoted as sample a) only to set in figure
25nmol/kg dosage.
As a result method is shown in Fig. 3 with embodiment 6.As can be seen from the figure given the test agent SEQ ID NO:9- II is in this experiment
Drug effect can steadily continue to 120hr or more under set high dose, and drug effect and long-term effect are in dosage correlation.Positive drug Li Lalu
Peptide shows drug effect in 12hr under 25nmol/kg dosage, and 24 hours drug effects decay.The SEQ ID under 25nmol/kg dosage
II drug effect of NO:9- can still maintain 76hr, and SEQ ID NO:58- II has substantially weakened in 52hr blood glucose inhibiting rate, illustrate pair
The optimization of precursor polypeptide sequence helps to improve the drug effect of final product.
9 PEG modifier of embodiment acts on the blood glucose-control of ob/ob mouse
Ob/ob model mice, male, 6 week old, the purchase of Beijing biotech inc Hua Bukang, credit number:
SCXK (capital) 2014-0004.
Animal quarantine is raised after two weeks, and fasting measures fasting blood-glucose, is grouped at random according to fasting blood-glucose, is divided into 5 groups, every group
8, respectively SEQ ID NO:9- II, SEQ ID NO:11- II, II group of SEQ ID NO:17-, positive control drug Liraglutide
Group and model control group, subcutaneous administration, drug dose are 100nmol/kg, positive control drug Liraglutide daily administration 2 times,
Other three samples are administered once for every 4 days, successive administration 21 days, monitor fasting blood-glucose weekly, grape is given in fasting abdominal cavity after 21 days
Sugared 2.5g/kg measures blood glucose value before to sugar and to 0.5h, 3 and 6h after sugar, calculates area under blood sugar concentration-time graph
(AUC).It the results are shown in Table 7 and 8.
7 PEG modified polypeptide successive administration of table 21 days, measures weekly fasting blood sugar
* (N=8,100nmol/kg)
8 PEG modified polypeptide successive administration of table blood sugar concentration-timetable after 21 days (N=8)
Compared with model control group:*P < 0.05,**P<0.01.
As can be seen from the above table, given the test agent, that is, long-actingization provided by the invention polypeptide conjugate is in diabetes animal model
It shows and is acted on the comparable blood glucose-control of positive drug Liraglutide, but administration frequency reduces (vs 4 days 1 time 2 times a day), tool
There is apparent application advantage.In view of the Difference of Metabolism of mouse and people, test data speculates according to the present invention, can expire in clinic
The administration frequency of foot 1 times a week, is remarkably improved compliance.
Although present invention has been a degree of descriptions, it will be apparent that, do not departing from the spirit and scope of the present invention
Under the conditions of, the appropriate variation of each condition can be carried out.It is appreciated that the present invention is not limited to the embodiments, and it is attributed to right
It is required that range comprising the equivalent replacement of each factor.
Sequence table
<110>Tianjin Inst. of Materia Medica Co., Ltd
<120>glucagon-like peptide-1 analogs and application thereof
<130> DIC17110002
<160> 58
<170> PatentIn version 3.3
<210> 1
<211> 32
<212> PRT
<213>artificial sequence
<220>
<223>Precursor Peptide
<220>
<221> misc_feature
<222> (29)..(29)
<223>Xaa indicates Aib, α-aminoacid
<220>
<221> misc_feature
<222> (32)..(32)
<223>C-terminal of Cys connects NH2
<400> 1
His Ala Pro Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Xaa Arg Gly Cys
20 25 30
<210> 2
<211> 31
<212> PRT
<213>artificial sequence
<220>
<223>Precursor Peptide
<220>
<221> misc_feature
<222> (29)..(29)
<223>Xaa indicates Aib, α-aminoacid
<220>
<221> misc_feature
<222> (31)..(31)
<223>C-terminal of Gly connects NH2
<400> 2
His Ala Pro Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Xaa Arg Cys
20 25 30
<210> 3
<211> 31
<212> PRT
<213>artificial sequence
<220>
<223>Precursor Peptide
<220>
<221> misc_feature
<222> (29)..(29)
<223>Xaa indicates Aib, α-aminoacid
<220>
<221> misc_feature
<222> (31)..(31)
<223>C-terminal of Gly connects NH2
<400> 3
His Ala Pro Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Cys Trp Leu Val Lys Xaa Arg Gly
20 25 30
<210> 4
<211> 30
<212> PRT
<213>artificial sequence
<220>
<223>Precursor Peptide
<220>
<221> misc_feature
<222> (29)..(29)
<223>Xaa indicates Aib, α-aminoacid
<220>
<221> misc_feature
<222> (30)..(30)
<223>C-terminal of Arg connects NH2
<400> 4
His Ala Pro Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Cys Trp Leu Val Lys Xaa Arg
20 25 30
<210> 5
<211> 32
<212> PRT
<213>artificial sequence
<220>
<223>Precursor Peptide
<220>
<221> misc_feature
<222> (29)..(29)
<223>Xaa indicates Aib, α-aminoacid
<220>
<221> misc_feature
<222> (32)..(32)
<223>C-terminal of Cys connects NH2
<400> 5
His Ala Pro Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Arg Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Xaa Arg Gly Cys
20 25 30
<210> 6
<211> 31
<212> PRT
<213>artificial sequence
<220>
<223>Precursor Peptide
<220>
<221> misc_feature
<222> (29)..(29)
<223>Xaa indicates Aib, α-aminoacid
<220>
<221> misc_feature
<222> (31)..(31)
<223>C-terminal of Cys connects NH2
<400> 6
His Ala Pro Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Arg Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Xaa Arg Cys
20 25 30
<210> 7
<211> 30
<212> PRT
<213>artificial sequence
<220>
<223>Precursor Peptide
<220>
<221> misc_feature
<222> (30)..(30)
<223>C-terminal of Cys connects NH2
<400> 7
His Ala Pro Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Arg Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Cys
20 25 30
<210> 8
<211> 31
<212> PRT
<213>artificial sequence
<220>
<223>Precursor Peptide
<220>
<221> misc_feature
<222> (29)..(29)
<223>Xaa indicates Aib, α-aminoacid
<220>
<221> misc_feature
<222> (31)..(31)
<223>C-terminal of Gly connects NH2
<400> 8
His Ala Pro Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Arg Ala Ala Lys Glu Phe Ile Cys Trp Leu Val Lys Xaa Arg Gly
20 25 30
<210> 9
<211> 32
<212> PRT
<213>artificial sequence
<220>
<223>Precursor Peptide
<220>
<221> misc_feature
<222> (29)..(29)
<223>Xaa indicates Aib, α-aminoacid
<220>
<221> misc_feature
<222> (32)..(32)
<223>C-terminal of Cys connects NH2
<400> 9
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Xaa Arg Gly Cys
20 25 30
<210> 10
<211> 31
<212> PRT
<213>artificial sequence
<220>
<223>Precursor Peptide
<220>
<221> misc_feature
<222> (29)..(29)
<223>Xaa indicates Aib, α-aminoacid
<220>
<221> misc_feature
<222> (31)..(31)
<223>C-terminal of Cys connects NH2
<400> 10
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Xaa Arg Cys
20 25 30
<210> 11
<211> 31
<212> PRT
<213>artificial sequence
<220>
<223>Precursor Peptide
<220>
<221> misc_feature
<222> (29)..(29)
<223>Xaa indicates Aib, α-aminoacid
<220>
<221> misc_feature
<222> (31)..(31)
<223>C-terminal of Gly connects NH2
<220>
<221> misc_feature
<222> (31)..(31)
<223>C-terminal of Gly connects NH2
<400> 11
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Cys Trp Leu Val Lys Xaa Arg Gly
20 25 30
<210> 12
<211> 30
<212> PRT
<213>artificial sequence
<220>
<223>Precursor Peptide
<220>
<221> misc_feature
<222> (29)..(29)
<223>Xaa indicates Aib, α-aminoacid
<220>
<221> misc_feature
<222> (30)..(30)
<223>C-terminal of Arg connects NH2
<400> 12
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Cys Trp Leu Val Lys Xaa Arg
20 25 30
<210> 13
<211> 32
<212> PRT
<213>artificial sequence
<220>
<223>Precursor Peptide
<220>
<221> misc_feature
<222> (29)..(29)
<223>Xaa indicates Aib, α-aminoacid
<220>
<221> misc_feature
<222> (32)..(32)
<223>C-terminal of Cys connects NH
<400> 13
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Arg Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Xaa Arg Gly Cys
20 25 30
<210> 14
<211> 31
<212> PRT
<213>artificial sequence
<220>
<223>Precursor Peptide
<220>
<221> misc_feature
<222> (29)..(29)
<223>Xaa indicates Aib, α-aminoacid
<220>
<221> misc_feature
<222> (31)..(31)
<223>C-terminal of Cys connects NH2
<400> 14
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Arg Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Xaa Arg Cys
20 25 30
<210> 15
<211> 31
<212> PRT
<213>artificial sequence
<220>
<223>Precursor Peptide
<220>
<221> misc_feature
<222> (29)..(29)
<223>Xaa indicates Aib, α-aminoacid
<220>
<221> misc_feature
<222> (31)..(31)
<223>C-terminal of Gly connects NH2
<400> 15
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Arg Ala Ala Lys Glu Phe Ile Cys Trp Leu Val Lys Xaa Arg Gly
20 25 30
<210> 16
<211> 30
<212> PRT
<213>artificial sequence
<220>
<223>Precursor Peptide
<220>
<221> misc_feature
<222> (29)..(29)
<223>Xaa indicates Aib, α-aminoacid
<220>
<221> misc_feature
<222> (30)..(30)
<223>C-terminal of Arg connects NH2
<400> 16
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Arg Ala Ala Lys Glu Phe Ile Cys Trp Leu Val Lys Xaa Arg
20 25 30
<210> 17
<211> 31
<212> PRT
<213>artificial sequence
<220>
<223>Precursor Peptide
<220>
<221> misc_feature
<222> (2)..(2)
<223>D type amino acid
<220>
<221> misc_feature
<222> (29)..(29)
<223>Xaa indicates Aib, α-aminoacid
<220>
<221> misc_feature
<222> (31)..(31)
<223>C-terminal of Gly connects NH2
<400> 17
His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Cys Trp Leu Val Lys Xaa Arg Gly
20 25 30
<210> 18
<211> 30
<212> PRT
<213>artificial sequence
<220>
<223>Precursor Peptide
<220>
<221> misc_feature
<222> (2)..(2)
<223>D type amino acid
<220>
<221> misc_feature
<222> (29)..(29)
<223>Xaa indicates Aib, α-aminoacid
<220>
<221> misc_feature
<222> (30)..(30)
<223>C-terminal of Arg connects NH2
<400> 18
His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Cys Trp Leu Val Lys Xaa Arg
20 25 30
<210> 19
<211> 32
<212> PRT
<213>artificial sequence
<220>
<223>Precursor Peptide
<220>
<221> misc_feature
<222> (2)..(2)
<223>D type amino acid
<220>
<221> misc_feature
<222> (29)..(29)
<223>Xaa indicates Aib, α-aminoacid
<220>
<221> misc_feature
<222> (32)..(32)
<223>C-terminal of Cys connects NH2
<400> 19
His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Xaa Arg Gly Cys
20 25 30
<210> 20
<211> 31
<212> PRT
<213>artificial sequence
<220>
<223>Precursor Peptide
<220>
<221> misc_feature
<222> (2)..(2)
<223>D type amino acid
<220>
<221> misc_feature
<222> (29)..(29)
<223>Xaa indicates Aib, α-aminoacid
<220>
<221> misc_feature
<222> (31)..(31)
<223>C-terminal of Cys connects NH2
<400> 20
His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Xaa Arg Cys
20 25 30
<210> 21
<211> 32
<212> PRT
<213>artificial sequence
<220>
<223>Precursor Peptide
<220>
<221> misc_feature
<222> (2)..(2)
<223>D type amino acid
<220>
<221> misc_feature
<222> (29)..(29)
<223>Xaa indicates Aib, α-aminoacid
<220>
<221> misc_feature
<222> (32)..(32)
<223>C-terminal of Cys connects NH2
<400> 21
His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Arg Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Xaa Arg Gly Cys
20 25 30
<210> 22
<211> 31
<212> PRT
<213>artificial sequence
<220>
<223>Precursor Peptide
<220>
<221> misc_feature
<222> (2)..(2)
<223>D type amino acid
<220>
<221> misc_feature
<222> (29)..(29)
<223>Xaa indicates Aib, α-aminoacid
<220>
<221> misc_feature
<222> (31)..(31)
<223>C-terminal of Cys connects NH2
<400> 22
His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Arg Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Xaa Arg Cys
20 25 30
<210> 23
<211> 31
<212> PRT
<213>artificial sequence
<220>
<223>Precursor Peptide
<220>
<221> misc_feature
<222> (2)..(2)
<223>D type amino acid
<220>
<221> misc_feature
<222> (29)..(29)
<223>Xaa indicates Aib, α-aminoacid
<220>
<221> misc_feature
<222> (31)..(31)
<223>C-terminal of Gly connects NH2
<400> 23
His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Arg Ala Ala Lys Glu Phe Ile Cys Trp Leu Val Lys Xaa Arg Gly
20 25 30
<210> 24
<211> 30
<212> PRT
<213>artificial sequence
<220>
<223>Precursor Peptide
<220>
<221> misc_feature
<222> (2)..(2)
<223>D type amino acid
<220>
<221> misc_feature
<222> (29)..(29)
<223>Xaa indicates Aib, α-aminoacid
<220>
<221> misc_feature
<222> (30)..(30)
<223>C-terminal of Arg connects NH2
<400> 24
His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Arg Ala Ala Lys Glu Phe Ile Cys Trp Leu Val Lys Xaa Arg
20 25 30
<210> 25
<211> 31
<212> PRT
<213>artificial sequence
<220>
<223>Precursor Peptide
<220>
<221> misc_feature
<222> (29)..(29)
<223>Xaa indicates Aib, α-aminoacid
<220>
<221> misc_feature
<222> (31)..(31)
<223>C-terminal of Gly connects NH2
<400> 25
His Ala Pro Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Xaa Arg Gly
20 25 30
<210> 26
<211> 30
<212> PRT
<213>artificial sequence
<220>
<223>Precursor Peptide
<220>
<221> misc_feature
<222> (29)..(29)
<223>Xaa indicates Aib, α-aminoacid
<220>
<221> misc_feature
<222> (30)..(30)
<223>C-terminal of Arg connects NH2
<400> 26
His Ala Pro Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Xaa Arg
20 25 30
<210> 27
<211> 31
<212> PRT
<213>artificial sequence
<220>
<223>Precursor Peptide
<220>
<221> misc_feature
<222> (29)..(29)
<223>Xaa indicates Aib, α-aminoacid
<220>
<221> misc_feature
<222> (31)..(31)
<223>C-terminal of Gly connects NH2
<220>
<221> misc_feature
<222> (31)..(31)
<223>C-terminal of Gly connects NH2
<400> 27
His Ala Pro Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Arg Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Xaa Arg Gly
20 25 30
<210> 28
<211> 30
<212> PRT
<213>artificial sequence
<220>
<223>Precursor Peptide
<220>
<221> misc_feature
<222> (29)..(29)
<223>Xaa indicates Aib, α-aminoacid
<220>
<221> misc_feature
<222> (30)..(30)
<223>C-terminal of Arg connects NH2
<400> 28
His Ala Pro Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Arg Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Xaa Arg
20 25 30
<210> 29
<211> 29
<212> PRT
<213>artificial sequence
<220>
<223>Precursor Peptide
<220>
<221> misc_feature
<222> (29)..(29)
<223>C-terminal of Gly connects NH2
<400> 29
His Ala Pro Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Arg Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly
20 25
<210> 30
<211> 31
<212> PRT
<213>artificial sequence
<220>
<223>Precursor Peptide
<220>
<221> misc_feature
<222> (29)..(29)
<223>Xaa indicates Aib, α-aminoacid
<220>
<221> misc_feature
<222> (31)..(31)
<223>C-terminal of Gly connects NH2
<400> 30
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Xaa Arg Gly
20 25 30
<210> 31
<211> 30
<212> PRT
<213>artificial sequence
<220>
<223>Precursor Peptide
<220>
<221> misc_feature
<222> (29)..(29)
<223>Xaa indicates Aib, α-aminoacid
<220>
<221> misc_feature
<222> (30)..(30)
<223>C-terminal of Gly connects NH2
<400> 31
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Xaa Gly
20 25 30
<210> 32
<211> 31
<212> PRT
<213>artificial sequence
<220>
<223>Precursor Peptide
<220>
<221> misc_feature
<222> (29)..(29)
<223>Xaa indicates Aib, α-aminoacid
<220>
<221> misc_feature
<222> (31)..(31)
<223>C-terminal of Gly connects NH2
<400> 32
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Arg Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Xaa Arg Gly
20 25 30
<210> 33
<211> 30
<212> PRT
<213>artificial sequence
<220>
<223>Precursor Peptide
<220>
<221> misc_feature
<222> (29)..(29)
<223>Xaa indicates Aib, α-aminoacid
<220>
<221> misc_feature
<222> (30)..(30)
<223>C-terminal of Gly connects NH2
<400> 33
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Arg Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Xaa Gly
20 25 30
<210> 34
<211> 31
<212> PRT
<213>artificial sequence
<220>
<223>Precursor Peptide
<220>
<221> misc_feature
<222> (2)..(2)
<223>D type amino acid
<220>
<221> misc_feature
<222> (29)..(29)
<223>Xaa indicates Aib, α-aminoacid
<220>
<221> misc_feature
<222> (31)..(31)
<223>C-terminal of Gly connects NH2
<400> 34
His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Xaa Arg Gly
20 25 30
<210> 35
<211> 30
<212> PRT
<213>artificial sequence
<220>
<223>Precursor Peptide
<220>
<221> misc_feature
<222> (2)..(2)
<223>D type amino acid
<220>
<221> misc_feature
<222> (29)..(29)
<223>Xaa indicates Aib, α-aminoacid
<220>
<221> misc_feature
<222> (30)..(30)
<223>C-terminal of Gly connects NH2
<400> 35
His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Xaa Gly
20 25 30
<210> 36
<211> 31
<212> PRT
<213>artificial sequence
<220>
<223>Precursor Peptide
<220>
<221> misc_feature
<222> (29)..(29)
<223>Xaa indicates Aib, α-aminoacid
<220>
<221> misc_feature
<222> (31)..(31)
<223>C-terminal of Gly connects NH2
<400> 36
His Ala Pro Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Lys Xaa Arg Gly
20 25 30
<210> 37
<211> 30
<212> PRT
<213>artificial sequence
<220>
<223>Precursor Peptide
<220>
<221> misc_feature
<222> (29)..(29)
<223>Xaa indicates Aib, α-aminoacid
<220>
<221> misc_feature
<222> (30)..(30)
<223>C-terminal of Arg connects NH2
<400> 37
His Ala Pro Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Lys Xaa Arg
20 25 30
<210> 38
<211> 31
<212> PRT
<213>artificial sequence
<220>
<223>Precursor Peptide
<220>
<221> misc_feature
<222> (29)..(29)
<223>Xaa indicates Aib, α-aminoacid
<220>
<221> misc_feature
<222> (31)..(31)
<223>C-terminal of Gly connects NH2
<400> 38
His Ala Pro Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Arg Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Lys Xaa Arg Gly
20 25 30
<210> 39
<211> 30
<212> PRT
<213>artificial sequence
<220>
<223>Precursor Peptide
<220>
<221> misc_feature
<222> (29)..(29)
<223>Xaa indicates Aib, α-aminoacid
<220>
<221> misc_feature
<222> (30)..(30)
<223>C-terminal of Arg connects NH2
<400> 39
His Ala Pro Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Arg Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Lys Xaa Arg
20 25 30
<210> 40
<211> 29
<212> PRT
<213>artificial sequence
<220>
<223>Precursor Peptide
<220>
<221> misc_feature
<222> (29)..(29)
<223>C-terminal of Gly connects NH2
<400> 40
His Ala Pro Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Arg Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Lys Gly
20 25
<210> 41
<211> 31
<212> PRT
<213>artificial sequence
<220>
<223>Precursor Peptide
<220>
<221> misc_feature
<222> (29)..(29)
<223>Xaa indicates Aib, α-aminoacid
<220>
<221> misc_feature
<222> (31)..(31)
<223>C-terminal of Gly connects NH2
<400> 41
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Lys Xaa Arg Gly
20 25 30
<210> 42
<211> 30
<212> PRT
<213>artificial sequence
<220>
<223>Precursor Peptide
<220>
<221> misc_feature
<222> (29)..(29)
<223>Xaa indicates Aib, α-aminoacid
<220>
<221> misc_feature
<222> (30)..(30)
<223>C-terminal of Arg connects NH2
<400> 42
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Lys Xaa Arg
20 25 30
<210> 43
<211> 31
<212> PRT
<213>artificial sequence
<220>
<223>Precursor Peptide
<220>
<221> misc_feature
<222> (2)..(2)
<223>D type amino acid
<220>
<221> misc_feature
<222> (29)..(29)
<223>Xaa indicates Aib, α-aminoacid
<220>
<221> misc_feature
<222> (31)..(31)
<223>C-terminal of Gly connects NH2
<400> 43
His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Lys Xaa Arg Gly
20 25 30
<210> 44
<211> 30
<212> PRT
<213>artificial sequence
<220>
<223>Precursor Peptide
<220>
<221> misc_feature
<222> (2)..(2)
<223>D type amino acid
<220>
<221> misc_feature
<222> (29)..(29)
<223>Xaa indicates Aib, α-aminoacid
<220>
<221> misc_feature
<222> (30)..(30)
<223>C-terminal of Arg connects NH2
<400> 44
His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Lys Xaa Arg
20 25 30
<210> 45
<211> 31
<212> PRT
<213>artificial sequence
<220>
<223>Precursor Peptide
<220>
<221> misc_feature
<222> (29)..(29)
<223>Xaa indicates Aib, α-aminoacid
<220>
<221> misc_feature
<222> (31)..(31)
<223>C-terminal of Gly connects NH2
<400> 45
His Ala Pro Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Xaa Lys Gly
20 25 30
<210> 46
<211> 30
<212> PRT
<213>artificial sequence
<220>
<223>Precursor Peptide
<220>
<221> misc_feature
<222> (29)..(29)
<223>Xaa indicates Aib, α-aminoacid
<220>
<221> misc_feature
<222> (30)..(30)
<223>C-terminal of Lys connects NH2
<400> 46
His Ala Pro Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Xaa Lys
20 25 30
<210> 47
<211> 31
<212> PRT
<213>artificial sequence
<220>
<223>Precursor Peptide
<220>
<221> misc_feature
<222> (29)..(29)
<223>Xaa indicates Aib, α-aminoacid
<220>
<221> misc_feature
<222> (31)..(31)
<223>C-terminal of Gly connects NH2
<400> 47
His Ala Pro Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Arg Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Xaa Lys Gly
20 25 30
<210> 48
<211> 30
<212> PRT
<213>artificial sequence
<220>
<223>Precursor Peptide
<220>
<221> misc_feature
<222> (29)..(29)
<223>Xaa indicates Aib, α-aminoacid
<220>
<221> misc_feature
<222> (30)..(30)
<223>C-terminal of Lys connects NH2
<400> 48
His Ala Pro Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Arg Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Xaa Lys
20 25 30
<210> 49
<211> 31
<212> PRT
<213>artificial sequence
<220>
<223>Precursor Peptide
<220>
<221> misc_feature
<222> (29)..(29)
<223>Xaa indicates Aib, α-aminoacid
<220>
<221> misc_feature
<222> (31)..(31)
<223>C-terminal of Gly connects NH2
<400> 49
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Xaa Lys Gly
20 25 30
<210> 50
<211> 30
<212> PRT
<213>artificial sequence
<220>
<223>Precursor Peptide
<220>
<221> misc_feature
<222> (29)..(29)
<223>Xaa indicates Aib, α-aminoacid
<220>
<221> misc_feature
<222> (30)..(30)
<223>C-terminal of Lys connects NH2
<400> 50
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Xaa Lys
20 25 30
<210> 51
<211> 31
<212> PRT
<213>artificial sequence
<220>
<223>Precursor Peptide
<220>
<221> misc_feature
<222> (29)..(29)
<223>Xaa indicates Aib, α-aminoacid
<220>
<221> misc_feature
<222> (31)..(31)
<223>C-terminal of Gly connects NH2
<400> 51
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Arg Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Xaa Lys Gly
20 25 30
<210> 52
<211> 30
<212> PRT
<213>artificial sequence
<220>
<223>Precursor Peptide
<220>
<221> misc_feature
<222> (29)..(29)
<223>Xaa indicates Aib, α-aminoacid
<220>
<221> misc_feature
<222> (30)..(30)
<223>C-terminal of Lys connects NH2
<400> 52
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Arg Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Xaa Lys
20 25 30
<210> 53
<211> 31
<212> PRT
<213>artificial sequence
<220>
<223>Precursor Peptide
<220>
<221> misc_feature
<222> (2)..(2)
<223>D type amino acid
<220>
<221> misc_feature
<222> (29)..(29)
<223>Xaa indicates Aib, α-aminoacid
<220>
<221> misc_feature
<222> (31)..(31)
<223>C-terminal of Gly connects NH2
<400> 53
His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Xaa Lys Gly
20 25 30
<210> 54
<211> 30
<212> PRT
<213>artificial sequence
<220>
<223>Precursor Peptide
<220>
<221> misc_feature
<222> (2)..(2)
<223>D type amino acid
<220>
<221> misc_feature
<222> (29)..(29)
<223>Xaa indicates Aib, α-aminoacid
<220>
<221> misc_feature
<222> (30)..(30)
<223>C-terminal of Lys connects NH2
<400> 54
His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Xaa Lys
20 25 30
<210> 55
<211> 31
<212> PRT
<213>artificial sequence
<220>
<223>Precursor Peptide
<220>
<221> misc_feature
<222> (2)..(2)
<223>D type amino acid
<220>
<221> misc_feature
<222> (29)..(29)
<223>Xaa indicates Aib, α-aminoacid
<220>
<221> misc_feature
<222> (31)..(31)
<223>C-terminal of Gly connects NH2
<400> 55
His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Arg Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Xaa Lys Gly
20 25 30
<210> 56
<211> 30
<212> PRT
<213>artificial sequence
<220>
<223>Precursor Peptide
<220>
<221> misc_feature
<222> (2)..(2)
<223>D type amino acid
<220>
<221> misc_feature
<222> (29)..(29)
<223>Xaa indicates Aib, α-aminoacid
<220>
<221> misc_feature
<222> (30)..(30)
<223>C-terminal of Lys connects NH2
<400> 56
His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Arg Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Xaa Lys
20 25 30
<210> 57
<211> 31
<212> PRT
<213>artificial sequence
<220>
<223>polypeptide
<400> 57
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Cys
20 25 30
<210> 58
<211> 33
<212> PRT
<213>artificial sequence
<220>
<223>polypeptide
<220>
<221> misc_feature
<222> (29)..(29)
<223> Xaa can be any naturally occurring amino acid
<400> 58
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Xaa Arg Gly Ala
20 25 30
Cys
Claims (10)
1. a kind of Precursor Peptide of glucagon-like peptide-1 analogs or its pharmaceutically acceptable salt, which is characterized in that institute
Precursor Peptide is stated with amino acid sequence shown in following general formula I；
General formula I
HX1X2GTFTSDVSSYLEEX3AAX4EFIX5WLVKX6X7X8X9；
Wherein, the X1、X2、X3、X4、X5、X6、X7、X8And X9Indicate arbitrary amino acid.
2. Precursor Peptide according to claim 1 or its pharmaceutically acceptable salt, which is characterized in that the Precursor Peptide
Sequence as shown in any bar in SEQ ID NO:1-56.
3. a kind of glucagon-like peptide-1 analogs or its pharmaceutically acceptable salt, which is characterized in that the pancreas hyperglycemia
Plain -1 analog of sample peptide or its pharmaceutically acceptable salt are Precursor Peptide of any of claims 1 or 2 or it can pharmaceutically connect
The polyethylene glycol conjugate for the salt received.
4. glucagon-like peptide-1 analogs according to claim 3 or its pharmaceutically acceptable salt, feature exist
In the average molecular weight range of the polyethylene glycol is 5-50KDa, more preferably 20-50KDa；It is further preferably 40-
50Kda。
5. glucagon-like peptide-1 analogs according to claim 3 or 4 or its pharmaceutically acceptable salt, feature
It is, the polyethylene glycol is straight chain type, branching type polyethylene glycol；
Preferably, the polyethylene glycol is selected from the branching type polyethylene glycol of average molecular weight range 40-50KDa, further preferably 40-
The two branching type polyethylene glycol of 50KDa.
6. glucagon-like peptide-1 analogs according to claim 5 or its pharmaceutically acceptable salt, feature exist
In, the polyethylene glycol by the activated group on exposed terminated groups be conjugated in glucagon-like peptide-1 analogs or its pharmaceutically
Acceptable salt；
Preferably, the activated group is selected from dimaleoyl imino, sulfydryl, succinimido, aldehyde radical, halogen；More preferably
For dimaleoyl imino.
7. glucagon-like peptide-1 analogs according to claim 6 or its pharmaceutically acceptable salt, feature exist
In the polyethylene glycol is conjugated in lysine side-chain or the cysteine side of SEQ ID NO:1-56 any bar by activated group
Chain；It is highly preferred that the polyethylene glycol is conjugated in any bar in SEQ ID NO:9-12,17-20 by activated group.
8. a kind of pharmaceutical composition, described pharmaceutical composition include Precursor Peptide as claimed in claim 1 or 2 or its pharmaceutically
Acceptable salt or described pharmaceutical composition include the glucagon-like-peptide-1 class as described in any one of claim 3-7
Like object or its pharmaceutically acceptable salt；
Preferably, described pharmaceutical composition also includes pharmaceutically acceptable carrier and/or auxiliary material；It is highly preferred that the carrier
And/or auxiliary material is selected from one of water-soluble filler, pH adjusting agent, stabilizer, water for injection or osmotic pressure regulator or more
Kind；
Preferably, the water-soluble filler be selected from mannitol, D-40, sorbierite, polyethylene glycol, glucose,
One of lactose or galactolipin etc. are a variety of；The pH adjusting agent, which is selected from citric acid, phosphoric acid, lactic acid, tartaric acid, hydrochloric acid etc., to be had
Machine or inorganic acid and potassium hydroxide, sodium hydroxide, ammonium hydroxide, sodium carbonate, potassium carbonate, ammonium carbonate, saleratus, carbonic acid
One of the physiologically acceptable inorganic base such as hydrogen sodium or bicarbonate ammonium salt or salt are a variety of；The stabilizer is selected from EDTA-
2Na, sodium thiosulfate, sodium pyrosulfite, sodium sulfite, dipotassium hydrogen phosphate, sodium bicarbonate, sodium carbonate, arginine, lysine,
Glutamic acid, aspartic acid, polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, carboxyl/hydroxylated cellulose or derivatives thereof as
One of HPC, HPC-SL, HPC-L or HPMC, cyclodextrin, lauryl sodium sulfate or trishydroxymethylaminomethane etc. or more
Kind；The osmotic pressure regulator is sodium chloride and/or potassium chloride.
9. Precursor Peptide according to claim 1 or 2 or its pharmaceutically acceptable salt, or as appointed in claim 3-7
Glucagon-like peptide-1 analogs described in one or its pharmaceutically acceptable salt are being prepared for treating diabetes, fertilizer
Purposes in fat, metabolic syndrome pharmaceutical composition.
10. pharmaceutical composition according to claim 8 is preparing the medicine for treating diabetes, obesity, metabolic syndrome
Purposes in object.
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| CN201710660343.4A CN109384839B (en) | 2017-08-04 | 2017-08-04 | Glucagon-like peptide-1 analogs and uses thereof |
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|---|---|---|---|
| CN201710660343.4A CN109384839B (en) | 2017-08-04 | 2017-08-04 | Glucagon-like peptide-1 analogs and uses thereof |
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Cited By (6)
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| CN111574597A (en) * | 2020-05-07 | 2020-08-25 | 中国科学院微生物研究所 | anti-HIV polypeptide modified by high molecular weight PEG (polyethylene glycol), 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 |
| CN113336840A (en) * | 2020-03-02 | 2021-09-03 | 武汉帕肽生物医药有限责任公司 | Stapled peptides, methods of making and uses thereof |
| CN114874314A (en) * | 2021-12-28 | 2022-08-09 | 北京惠之衡生物科技有限公司 | Long-acting GLP-1 derivative |
| WO2023001186A1 (en) * | 2021-07-20 | 2023-01-26 | 派格生物医药(苏州)股份有限公司 | Polypeptide conjugate and application thereof in preparation of drug for treating diseases related to glucose metabolism |
| CN115947821A (en) * | 2021-10-09 | 2023-04-11 | 合肥天汇生物科技有限公司 | A GLP-1 analogue |
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| WO2007124461A2 (en) * | 2006-04-20 | 2007-11-01 | Amgen Inc. | Glp-1 compounds |
| CN106554408A (en) * | 2015-09-30 | 2017-04-05 | 天津药物研究院有限公司 | - 1 analog dimer of long-acting glucagon peptide and its application |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| 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 |
| CN112898404B (en) * | 2019-12-03 | 2024-11-12 | 天津药物研究院有限公司 | A long-acting modified glucagon peptide analog or its salt and its use |
| CN113336840A (en) * | 2020-03-02 | 2021-09-03 | 武汉帕肽生物医药有限责任公司 | Stapled peptides, methods of making and uses thereof |
| CN111574597A (en) * | 2020-05-07 | 2020-08-25 | 中国科学院微生物研究所 | anti-HIV polypeptide modified by high molecular weight PEG (polyethylene glycol), preparation method and application thereof |
| WO2023001186A1 (en) * | 2021-07-20 | 2023-01-26 | 派格生物医药(苏州)股份有限公司 | Polypeptide conjugate and application thereof in preparation of drug for treating diseases related to glucose metabolism |
| CN115947821A (en) * | 2021-10-09 | 2023-04-11 | 合肥天汇生物科技有限公司 | A GLP-1 analogue |
| CN115947821B (en) * | 2021-10-09 | 2023-06-30 | 合肥天汇生物科技有限公司 | GLP-1 analogues |
| CN114874314A (en) * | 2021-12-28 | 2022-08-09 | 北京惠之衡生物科技有限公司 | Long-acting GLP-1 derivative |
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|---|---|
| CN109384839B (en) | 2021-03-09 |
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