WO2023051462A1 - Glp-1类似物的融合多肽 - Google Patents

Glp-1类似物的融合多肽 Download PDF

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WO2023051462A1
WO2023051462A1 PCT/CN2022/121356 CN2022121356W WO2023051462A1 WO 2023051462 A1 WO2023051462 A1 WO 2023051462A1 CN 2022121356 W CN2022121356 W CN 2022121356W WO 2023051462 A1 WO2023051462 A1 WO 2023051462A1
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glp
analog
fusion polypeptide
pharmaceutically acceptable
acceptable salt
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PCT/CN2022/121356
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English (en)
French (fr)
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孙磊
吕荟
张磊
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合肥天汇生物科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/26—Glucagons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/48—Drugs for disorders of the endocrine system of the pancreatic hormones
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity

Definitions

  • the invention belongs to the field of polypeptides, in particular, it relates to fusion polypeptides of GLP-1 analogues.
  • Glucagon-like peptide-1 is a peptide derived from proglucagon, which is secreted by endocrine cells when the human body takes in nutrients. It can stimulate the expression of insulin gene and insulin release; inhibit the release of insulin glucagon; inhibit the desire of food intake, have the effect of weight loss; stimulate the proliferation and differentiation of ⁇ cells; have a protective effect on the apoptosis of ⁇ cells. It has wide application in the field of treating diabetes.
  • GLP-1 N-terminally truncated products of GLP-1
  • GLP-1(7-36) N-terminally truncated products of GLP-1
  • GLP-1(7-37) N-terminally truncated products of GLP-1
  • Natural GLP-1 has many disadvantages in the treatment of diabetes, for example, it is easily degraded by dipeptidyl peptidase IV (DPP-IV) in vivo.
  • DPP-IV dipeptidyl peptidase IV
  • GLP-1 analogues are currently on the market. NovoNordisk has developed Liraglutide (Liraglutide) (US Patent No. 6,268,343) with 1.8mgs.c./day of once-daily administration, and it was approved in 2010.
  • Liraglutide Liraglutide
  • Semaglutide GLP-1 analogue semaglutide
  • USFDA International Publication No. WO 2006/097537 A2
  • the present invention relates to a fusion polypeptide of a GLP-1 analog or a pharmaceutically acceptable salt thereof, wherein the fusion polypeptide of the GLP-1 analog comprises a GLP-1(7-37) analog, a GLP-1(7-37 ) analogs of C-terminal extensions, and fusion protein fragments.
  • the fusion protein fragment is a ⁇ -sheet fragment of the fusion protein.
  • the fusion protein fragment has 30-80 amino acids.
  • the fusion protein fragment has the amino acid sequence shown below
  • the GLP-1(7-37) analog substantially retains the function of native GLP-1(7-37), preferably, the native GLP-1(7-37 ) comprises, or consists of, the amino acid sequence shown below: HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG (SEQ ID NO: 1).
  • said GLP-1(7-37) analogue is corresponding to the 7th to the 37th position of the native GLP-1(7-37). Any amino acid at the position is substituted by a first cysteine, the C-terminal extension of the GLP-1(7-37) analog contains a second cysteine, and the first cysteine acid and said second cysteine form a disulfide bond, preferably said first cysteine and said second cysteine form an intramolecular disulfide bond.
  • the interval between the first cysteine and the second cysteine is at most 40 amino acids in length, preferably, Between 35 and 3 amino acids, more preferably between 30 and 5 amino acids, more preferably between 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22 , 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 amino acids, or any range between the above two points.
  • the GLP-1 analogue compared with the natural GLP-1(7-37) shown in SEQ ID NO: 1, the GLP-1 analogue also has Comprising at most 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 conservative amino acid modifications, optionally, the conservative amino acid modifications include amino acid substitutions, deletions and and/or added without altering the activity or function of GLP-1(7-37).
  • the conservative amino acid modification can be any amino acid substitution, deletion and/or addition known to those skilled in the art that does not change the activity or function of GLP-1(7-37).
  • the C-terminal extension of the GLP-1(7-37) analogue is a peptide segment comprising 1-15 amino acids .
  • the C-terminal extension of said GLP-1(7-37) analogues is at most 15, 14, 13, 12, Peptides composed of 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid, one of which is cysteine, and the rest are any amino acids except cysteine.
  • one amino acid of the C-terminal extension of the GLP-1(7-37) analogue is cysteine, and the rest For glycine or arginine.
  • the C-terminal extension of the GLP-1(7-37) analogue is selected from C, GCGR, or GCGGGGGG.
  • a fusion polypeptide of a GLP-1 analog or a pharmaceutically acceptable salt thereof is provided, wherein the fusion polypeptide of the GLP-1 analog has the formula from N-terminus to C-terminus Structure shown in (I):
  • the X is a GLP-1(7-37) analogue
  • the E is a C-terminal extension of the GLP-1(7-37) analogue
  • the Y is a fusion protein fragment.
  • said GLP-1(7-37) analog substantially retains the natural GLP-1 ( 7-37), preferably, the natural GLP-1 (7-37) comprises the amino acid sequence shown below, or consists of it:
  • the GLP-1 (7-37) analog is corresponding to the natural GLP-1 (7 -37) is substituted by a first cysteine at any of amino acids 7 to 37), the C-terminal extension of the GLP-1(7-37) analogue comprising a second cysteine , and said first cysteine and said second cysteine form a disulfide bond, preferably said first cysteine and said second cysteine form an intramolecular disulfide bond .
  • the interval between the first cysteine and the second cysteine is at most is 40 amino acids in length, preferably at intervals of 35 to 3 amino acids, more preferably at intervals of 30 to 5 amino acids, more preferably at intervals of 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 amino acids, or between the above two Any range between points.
  • the C-terminal extension segment of the GLP-1(7-37) analog comprises Peptides of 1-15 amino acids.
  • the C-terminal extension of the GLP-1 (7-37) analog is at most Peptides composed of 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid, one of which is cysteine, and the rest are cysteine Any amino acid other than amino acid.
  • one of the C-terminal extensions of the GLP-1 (7-37) analog is cysteine, and the rest are glycine or arginine.
  • the C-terminal extension segment of the GLP-1 (7-37) analog is selected from C, GCGR, or GCGGGGGG.
  • the fusion protein fragment is selected from the ⁇ -sheet fragment of the fusion protein.
  • the fusion protein fragment is shown in SEQ ID NO: 2, or it has at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity.
  • the fusion protein fragment of the present invention is as follows:
  • a fusion polypeptide of a GLP-1 analog or a pharmaceutically acceptable salt thereof is provided, wherein the fusion polypeptide of the GLP-1 analog has the formula from N-terminus to C-terminus Structure shown in (II):
  • the Y is a fusion protein fragment
  • the L' is a linker or does not exist
  • the X is a GLP-1 (7-37) analog
  • the E is the GLP-1 (7-37) analog C-terminal extension.
  • L' is a linker
  • it is a length of at most 15, 14, 13, 12, 11 , 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acids
  • a peptide linker consisting of any amino acid, preferably, L' is a linker containing arginine and/or lysine.
  • said GLP-1(7-37) analog substantially retains the natural GLP-1 ( 7-37), preferably, the natural GLP-1 (7-37) comprises the amino acid sequence shown below, or consists of it:
  • the GLP-1 (7-37) analog is in the corresponding natural GLP-1 (7 -37) is substituted by a first cysteine at any of amino acids 7 to 37), the C-terminal extension of the GLP-1(7-37) analogue comprising a second cysteine , and said first cysteine and said second cysteine form a disulfide bond, preferably said first cysteine and said second cysteine form an intramolecular disulfide bond .
  • the interval between the first cysteine and the second cysteine is at most is 40 amino acids in length, preferably at intervals of 35 to 3 amino acids, more preferably at intervals of 30 to 5 amino acids, more preferably at intervals of 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 amino acids, or between the above two Any range between points.
  • the C-terminal extension segment of the GLP-1(7-37) analog comprises Peptides of 1-15 amino acids.
  • the C-terminal extension of the GLP-1 (7-37) analog is at most Peptides composed of 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid, one of which is cysteine, and the rest are cysteine Any amino acid other than amino acid.
  • one of the C-terminal extensions of the GLP-1 (7-37) analog is cysteine, and the rest are glycine or arginine.
  • the C-terminal extension segment of the GLP-1(7-37) analog is selected from C, GCGR, or GCGGGGGG.
  • the fusion protein fragment is selected from the ⁇ sheet fragment of the fusion protein.
  • the fusion protein fragment is shown in SEQ ID NO: 2, or it has at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity.
  • the fusion protein fragment of the present invention is as follows:
  • the present invention provides a fusion polypeptide of a GLP-1 analog, which has the amino acid sequence shown in any one of SEQ ID NO: 3-5.
  • the invention provides a nucleotide sequence encoding a fusion polypeptide of a GLP-1 analog of the invention.
  • the present invention provides a vector comprising a nucleotide sequence encoding a fusion polypeptide of a GLP-1 analog of the present invention.
  • the present invention relates to a pharmaceutical composition, which comprises the fusion polypeptide of the GLP-1 analog according to the present invention and a pharmaceutically acceptable carrier or excipient.
  • the present invention provides fusion polypeptides of GLP-1 analogues according to the present invention or pharmaceutically acceptable salts thereof, and compositions comprising them are used for the treatment of non-insulin-dependent diabetes mellitus, insulin-dependent diabetes mellitus or obesity use in medicines for disease.
  • GLP-1(7-37) analogue refers to a polypeptide that substantially retains GLP-1(7-37) activity or function, which is identical to naturally occurring GLP-1(7-37) Conservative modifications of up to 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid, including amino acid substitutions, compared to (such as SEQ ID NO: 1) , deletion and/or addition, the "conservative modification” refers to the replacement of amino acids in the protein with other amino acids having similar characteristics (such as charge, side chain size, hydrophobicity/hydrophilicity, main chain conformation and rigidity, etc.), Such as not to alter the activity or function of GLP-1(7-37).
  • the term "substantially retains the function of natural GLP-1 (7-37)” means that those skilled in the art can determine that the GLP-1 (7-37) described herein is similar to Compared with native GLP-1 (7-37), the function and/or reduction and/or reduction is no more than 50%, preferably no more than 45%, no more than 40%, no more than 35%, no more than 30% , not exceeding 25%, not exceeding 20%, not exceeding 19%, not exceeding 18%, not exceeding 17%, not exceeding 16%, not exceeding 15%, not exceeding 14%, not exceeding 13%, not exceeding 12% , not exceeding 11%, not exceeding 10%, not exceeding 9%, not exceeding 8%, not exceeding 7%, not exceeding 6%, not exceeding 5%, not exceeding 4%, not exceeding 3%, not exceeding 2% , not more than 1%.
  • salts of the present invention are synthesized from parent compounds containing basic or acidic moieties by methods described in .
  • these salts can be prepared by reacting the free acid or base forms of these compounds with the appropriate base or acid in water or in an organic solvent or a mixture of the two.
  • Acid addition salts include mono- or di-salts formed with acids selected from the group consisting of acetic acid, hydrochloric acid, hydroiodic acid, phosphoric acid, nitric acid, sulfuric acid, citric acid, lactic acid, succinic acid, maleic acid, malic acid, Isethionic acid, fumaric acid, benzenesulfonic acid, toluenesulfonic acid, sulfuric acid, methanesulfonic acid (mesylate), ethanesulfonic acid, naphthalenesulfonic acid, valeric acid, propionic acid, butyric acid, malonic acid , glucuronic acid and lactobionic acid.
  • a particular salt is the hydrochloride.
  • Another specific salt is acetate.
  • a salt can be formed with an organic or inorganic base to produce a suitable cation.
  • suitable inorganic cations include, but are not limited to, alkali metal ions such as Li + , Na + and K + ; alkaline earth metal cations such as Ca 2+ and Mg 2+ ; and other cations. Such as Al 3+ or Zn + .
  • Suitable organic cations include, but are not limited to, ammonium ions (ie, NH 4 + ) and substituted ammonium ions (eg, NH 3 R + , NH 2 R 2 + , NHR 3 + , NR 4 + ).
  • suitable substituted ammonium ions are those derived from: methylamine, ethylamine, diethylamine, propylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine , benzylamine, phenylbenzylamine, choline, meglumine and tromethamine, and amino acids such as lysine and arginine.
  • An example of a common quaternary ammonium ion is N(CH 3 ) 4 + .
  • beta-sheet fragment of a fusion protein refers to a fragment of a fusion protein having a beta-sheet structure.
  • polypeptide refers to any polymeric chain of amino acids.
  • peptide and protein are used interchangeably with the term “polypeptide” and also refer to a polymeric chain of amino acids.
  • polypeptide encompasses natural or artificial proteins, protein fragments and polypeptide analogs of the protein's amino acid sequence. Unless contradicted by context, the term “polypeptide” encompasses fragments and variants (including fragments of variants) thereof.
  • isolated protein or "isolated polypeptide” is a protein or polypeptide that, according to its origin or source of derivation, is separated from naturally related components that accompany it in its native state, substantially free of Other proteins, are expressed by cells from different species, or do not occur in nature.
  • a polypeptide that is chemically synthesized or synthesized in a cellular system different from the cell from which it is naturally derived is “isolated” from its naturally associated components.
  • a protein consisting of one or more polypeptide chains may also be rendered substantially free of naturally associated components by isolation using protein purification techniques well known in the art.
  • recovery refers to the process of rendering a chemical substance, such as a polypeptide, substantially free of naturally associated components by isolation, for example, using protein purification techniques well known in the art.
  • isolated nucleic acid refers to a polynucleotide (e.g., a polynucleotide of genomic, cDNA, or synthetic origin, or some combination thereof) that has been separated by human intervention from all or a portion of the polynucleotide with which it naturally occurs, is operably linked to a polynucleotide that is not naturally linked, or would not exist as part of a larger sequence in nature.
  • a polynucleotide e.g., a polynucleotide of genomic, cDNA, or synthetic origin, or some combination thereof
  • identity refers to the sequence similarity between two proteins or polypeptides. When a position in both compared sequences is occupied by the same amino acid residue, eg, if a position is occupied by the same amino acid residue in both polypeptides, then the molecules are identical at that position.
  • algorithms suitable for determining percent sequence identity and percent sequence similarity are the BLAST and BLAST2.0 algorithms.
  • vector is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • plasmid refers to a circular double-stranded DNA loop into which other DNA segments can be ligated.
  • viral vector in which additional DNA segments can be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in the host cell into which they are introduced (eg, bacterial vectors with a bacterial origin of replication and episomal mammalian vectors).
  • vectors eg, non-episomal mammalian vectors
  • certain vectors are capable of directing the expression of genes to which they are operably linked.
  • Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors").
  • expression vectors useful in recombinant DNA techniques are usually in the form of plasmids.
  • plasmid and vector are used interchangeably, since plasmids are the most commonly used form of vectors.
  • the invention is intended to include such other forms of expression vectors, such as viral vectors (eg, replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
  • the term "recombinant host cell” is intended to mean a cell into which foreign DNA has been introduced.
  • the host cell comprises two or more (eg, multiple) nucleic acids encoding antibodies or binding proteins, eg, the host cells described in US Pat. No. 7,262,028.
  • Such terms are intended to refer not only to the particular subject cell, but also to the progeny of such cells.
  • progeny may actually differ from the parental cell because certain modifications may occur in the progeny, due to mutations or environmental influences, but are still included within the scope of the term "host cell” as used herein.
  • host cells include prokaryotic and eukaryotic cells.
  • eukaryotic cells include protists, fungi, plant and animal cells.
  • a particularly useful prokaryotic host cell is E. coli and its derivatives.
  • Particularly useful mammalian cell lines include, but are not limited to, CHO, HEK293, COS, NSO, SP2, and PER.C6.
  • HEK 293E cells are particularly useful as mammalian host cells in transient expression systems for recombinant proteins.
  • CHO cells are particularly useful as stably transfected mammalian host cells for the production of recombinant proteins.
  • a particularly useful insect cell line is the Sf9 cell line and its derivatives.
  • a particularly useful fungal host cell is Saccharomyces cerevisiae and its derivatives.
  • Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, cell culture, tissue culture, and transformation (eg, electroporation, lipofection, transformation).
  • Enzymatic reactions and purification techniques can be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein.
  • the foregoing techniques and procedures can generally be performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).
  • Figure 1 shows that there is a linear relationship between protein concentration and A450 in the ELISA pre-experiment
  • Figure 2A to Figure 2D show the affinity fitting curve of the fusion polypeptide of the GLP-1 analog of the present invention detected by ELISA;
  • FIG. 3 shows the enzymatic degradation of DPP-4.
  • the present invention relates to the fusion polypeptides of the following GLP-1 analogues, which respectively have the following amino acid sequences:
  • each of the above-mentioned G1, G2 and G4 polypeptides contains an intrachain disulfide bond formed by two cysteines therein.
  • cysteines form an intrachain disulfide bond.
  • cysteines form an intrachain disulfide bond.
  • cysteines form an intrachain disulfide bond.
  • GLP-1 drugs on diabetes is based on the combination of GLP-1 and GLP-1 receptors, which activates the G protein coupling signaling pathway and promotes the secretion of insulin by pancreatic ⁇ cells. Therefore, the fusion polypeptide of GLP-1 analogues is prepared. The ability to bind to the GLP-1 receptor is equivalent to the natural GLP-1 protein.
  • PBS Phosphate Buffered Saline
  • PBS Phosphate Buffered Saline
  • PBS Phosphate Buffered Saline
  • the INS-1 rat insulinoma cells were cultured in a 37°C incubator to a confluence of more than 80%, and then subcultured.
  • the passaged INS-1 rat insulinoma cells were added to a 96-well plate, and the volume of cells added to each well was 100ul, and cultured in the 96-well plate until the cells adhered to the wall. Gently wash the 96-well plate with PBS buffer three times to remove unattached cells. Add 100ul of 4% paraformaldehyde to each well and let stand at room temperature for 10 minutes to solidify the cells.
  • INS-1 rat insulinoma cells were embedded in 96-well plates.
  • the fusion polypeptide of the GLP-1 analog of the present invention is combined with the GLP-1 receptor on the cell surface, the fusion polypeptide of the unbound GLP-1 analog is washed away, the GLP-1 receptor is blocked, and human GLP1 (7 -36)
  • the antibody in the ELISA kit reacts with the fusion polypeptide of the GLP-1 analogue bound to the GLP-1 receptor, and the reaction mechanism is similar to the double-antibody sandwich method.
  • the ELISA kit was taken out of the refrigerator in advance and equilibrated to room temperature.
  • the required wells in the 96-well plate were blocked with BSA to eliminate the non-specific reaction between the cells and the sample to be tested. 1% BSA was added to each reaction well and blocked at 37°C for 40 minutes.
  • the 96-well plate was placed on a microplate reader for absorbance detection at a wavelength of 450 nm.
  • the cell surface The GLP-1 receptor and the GLP-1 antibody in the kit have different binding abilities to GLP-1, so it is necessary to conduct a preliminary experiment and use human GLP-1 protein to measure GLP-1 on the surface of INS-1 rat insulinoma cells The binding ability of the receptor, determine the optimal reactant concentration range and linearity.
  • Pre-experimental protocol Dilute human GLP-1(7-36) protein with PBS to obtain different protein concentrations, gradually dilute downwards from the highest concentration of 0.1mg/ml, dilute each concentration tenfold, and finally dilute to 0.1ng/ml ml.
  • Human GLP-1(7-36) protein with different concentrations was detected by the ELISA kit according to the method in 4.1 to obtain the detection results of absorbance at 450nm. Two samples of each concentration were used in parallel, and PBS was used as a blank control. The detection scheme is shown in Table 1 below:
  • the fusion polypeptides G1, G2, and G4 of the three GLP-1 analogs prepared were combined with the GLP-1 receptor on the surface of INS-1 rat insulinoma cells.
  • the concentration gradient of the fusion polypeptide of the analog is set to 100ng/ml, 33ng/ml, 10ng/ml, 3.3ng/ml, 1ng/ml, and the fusion polypeptide of the three analogs of G1, G2, and G4 are diluted to
  • human GLP-1 (7-36) protein was used as a positive control
  • PBS was used as a blank control.
  • Three parallel samples at each concentration point above were tested according to the above ELISA experimental procedures. The detection scheme is shown in Table 2 below:
  • the fusion polypeptides G1, G2, and G4 of the three GLP-1 analogues prepared were combined with the GLP-1 receptor on the surface of INS-1 rat insulinoma cells, and the A 450 results obtained after the ELISA reaction were as follows:
  • a 0 is the average value of the blank control 0.865
  • a ⁇ is the 450nm absorbance of 3.550 when the human GLP-1(7-36) protein is saturated when the pre-experiment is obtained
  • [x] tot is the total receptor Concentration
  • the numerical value obtained through the calculation of the pre-experimental results is 303nM.
  • Kd The dissociation constant, detects the affinity between the antibody and the receptor.
  • Kd itself remains constant and is not affected by the concentration of ligands and receptors.
  • Kd is equal to the concentration of ligand at which half of the receptor is bound by the ligand. The smaller the Kd, the slower the dissociation and the stronger the affinity.
  • the coefficient of determination R 2 is used to judge the fitting degree of the fitting curve to the sample data. The higher the coefficient of determination, the better the fitting effect of the model, that is, the stronger the ability of the model to explain the dependent variable.
  • GLP-1 binds to the GLP-1 receptor and promotes the secretion of insulin by pancreatic ⁇ cells.
  • GLP-1 in the human body is easily degraded by DPP-4 enzyme and loses its function. Therefore, the prepared GLP-1 Whether the fusion polypeptide of the analog can resist the degradation of the DPP-4 enzyme, thereby prolonging the action time of the fusion polypeptide of the GLP-1 analog, is the object of this experiment.
  • the fusion polypeptide of the three GLP-1 analogues prepared was digested with excess DPP-4 enzyme, and the ELISA reaction was used to measure the different time of the DPP-4 enzyme acting on the fusion polypeptide of the three GLP-1 analogues under the same conditions
  • human GLP-1 protein (7-36) was used as a control, and the maintenance time of the fusion polypeptide of the GLP-1 analogue was simulated in vitro to characterize the hypoglycemic effect duration.
  • the amount of the fusion polypeptide of the GLP-1 analog remaining after being digested by DPP-4 enzyme at different time points is determined by ELISA reaction, and the generated Enzyme digestion curve, to determine the degradation resistance of the fusion polypeptide of the GLP-1 analogue.
  • the amount of fusion polypeptide of human GLP-1(7-36) and GLP-1 analog remaining in the reaction system is determined by ELISA reaction
  • the ELISA kit was taken out of the refrigerator in advance and equilibrated to room temperature.
  • the 96-well plate was placed on a microplate reader for absorbance detection at a wavelength of 450 nm.
  • Human GLP-1(7-36) was used as the substrate for the reaction, divided into two groups, and diluted with PBS at pH 7.4 to concentrations of 0.1ug/ml and 10ng/ml. Thaw the DPP-4 enzyme and dilute it with PBS to a concentration of 10ug/ml. Add 400ul of PBS to the reaction system first, then add 50ul of substrate and enzyme, the total reaction volume is 500ul. The reaction was carried out in a water bath with a temperature of 30°C.
  • AUC G1 G2 G4 Human GLP-1(7-36) The total area 5.137 1.342 2.469 1.025 standard error 0.1388 0.04715 0.09012 0.05201 95% confidence interval 4.865-5.409 1.249-1.434 2.292-2.646 0.9232-1.127
  • PBS Phosphate Buffered Saline
  • PBS Phosphate Buffered Saline
  • PBS Phosphate Buffered Saline
  • Gender and quantity 70 were purchased, and 62 were screened into the group, all of which were male.
  • Animal age the animals were 6-8 weeks old at the time of administration.
  • Drinking water sterile filtered purified water, purified water prepared by the Millipore Elix water purifier, and then filtered and sterilized by a 0.22 ⁇ m sterile filter.
  • Feed SPF grade KM mouse maintenance compound feed, the nutritional ingredients meet the general quality standards of GB14924.1-2001, GB14924.2-2001, GB14924.3-2010 experimental animal compound feed, production unit: Beijing Keao Xieli Feed Co., Ltd.
  • mice 52 KM mice were injected with streptozotocin according to their body weight, and each mouse was injected intraperitoneally with streptozotocin at a dose of 40 mg/kg every two days to make a mouse model of impaired glucose tolerance, with a total of 3 injections.
  • One week after the injection fast for 12 hours to measure fasting blood sugar (basal blood sugar), and then give glucose solution at 2.5g/kg orally, with a volume of 0.10-0.15ml/10g, and measure blood sugar after 30 minutes of gavage.
  • the model mice whose blood glucose level increased by more than 200% in 30 minutes were sorted from high to low according to the increase, and the highest and lowest increases were removed, and 40 model mice were selected according to the increase and included in the impaired glucose tolerance mice.
  • Model 52 KM mice were injected with streptozotocin according to their body weight, and each mouse was injected intraperitoneally with streptozotocin at a dose of 40 mg/kg every two days to make a mouse model of impaired glucose tolerance,
  • mice 40 impaired glucose tolerance model mice were divided into model control group, GLP-1 group, G1 group, G2 group, G4 group, a total of 5 groups, 8 mice in each group, and were divided into balanced groups according to the increase of blood glucose level and body weight. Another 8 unmodeled mice were selected as the NGT (Normal Glucose Tolerance, normal control) group.
  • NGT Normal Glucose Tolerance, normal control
  • the molecular weight of liraglutide is 3751.
  • the initial dose is 0.6 mg per day. After 1 week, the dose should be increased to 1.2 mg.
  • the recommended daily dose should not exceed 1.8 mg.
  • the molar amount of administration is converted, which is equivalent to the dosage of GLP-1 analogues for adults is 0.32 ⁇ mol/day.
  • the dosage conversion relationship between a mouse with a body weight of 20g and a human with a weight of 70Kg is 0.0026:1, that is, the converted dosage for a mouse with a body weight of about 20g is 0.83nmol/day.
  • Glucose was administered orally after the first administration, and measured at different times, and glucose was given again on the second day and measured at different times.
  • mice were administered at about 9:30 am on the first day after grouping.
  • the NGT group and the model control group were subcutaneously injected with normal saline, and the animals in GLP-1, G1, G2, and G4 were injected subcutaneously with the corresponding human GLP-1 (7-36) and the fusion polypeptide of the GLP-1 analogue of the present invention. All were administered by a single subcutaneous injection on the back of the neck. For nine consecutive days, the drug was administered every other day at around 9:30 in the morning, and the drug was administered five times in total.
  • the dosing regimen is as shown in Table 7:
  • Oral Glucose Tolerance Test also known as Oral Glucose Tolerance Test (OGTT) is a series of plasma glucose concentration measurements within 2 hours after oral administration of a certain amount of glucose. For a long time, OGTT has been adopted by countries all over the world as the gold standard for diagnosing diabetes. After a normal person takes a certain amount of glucose, the blood sugar concentration temporarily rises, but the blood sugar concentration can return to the normal level within 2 hours, while the patients with abnormal glucose metabolism will have different time periods and different degrees of blood sugar rise after taking glucose. All clinically found patients with suspected diabetes mellitus can be checked by oral glucose tolerance test except those who are contraindicated in the test. Can understand the body's ability to regulate glucose metabolism.
  • OGTT Oral Glucose Tolerance Test
  • the glucose dose of 2.5g/kg was given to the mice by intragastric administration, and the blood glucose concentration was measured by taking blood from the tip of the tail at 30min, 60min, 90min, and 120min within 2 hours after the administration of sugar.
  • blood was collected from the tail vein of the mice to detect the fasting blood sugar.
  • 2.5 g/kg glucose solution (20% glucose solution, 10 ml/Kg) was given by intragastric administration.
  • Blood glucose was measured at 30min, 60min, 90min and 120min after gavage.
  • another 2 g/kg glucose solution (20% glucose solution, 10 ml/Kg) was given by intragastric administration. Blood glucose was measured at 30min, 60min, 90min and 120min after gavage.
  • Blood collection method use a scalpel to take blood from the tail tip of the mouse, discard the first drop of blood, and collect the second drop of blood for testing blood sugar.
  • Detection index blood sugar level.
  • each cage contains 4 mice of the same group. Change food at about 8:30 every morning, weigh the food placed in each cage, as F0, and weigh the remaining food when changing food the next day, as F1, F1-F0 is the 4 small cages in the cage. The daily food intake of mice.
  • each cage contains 4 mice of the same group. Change the water at about 8:30 every morning, weigh the water placed in each cage, as W0, and weigh the remaining water when changing the water the next day, as W1, W1-W0 is the 4 small cages in the cage. Daily water intake of mice.
  • mice were weighed every two days in the morning, and the fasting body weight was obtained on the 9th day due to fasting before administration.
  • AUC blood glucose-time curve
  • the basal blood glucose of the mice in the NGT group and after the modeling was completed and the blood glucose values of the mice in each group before administration and 30 imn after the glucose administration are shown in Table 8:
  • the basal blood glucose of the model control group and each administration group increased slightly, but There was no statistically significant difference (P>0.05); there was no significant difference between each administration group and the model control group (P>0.05).
  • the 30-min blood glucose and the increase rate of blood glucose in the model control group and each administration group were higher than those in the NGT group, and the difference was statistically significant (P ⁇ 0.01). There was no significant difference in basal blood glucose, 30-min blood glucose and blood glucose increase between the model control group and each administration group (P>0.05).
  • the body weight changes during the administration of each group are shown in Table 9: Compared with the NGT group, the body weight of the mice in the model control group and each administration group decreased, and the difference was statistically significant on the 9th day (P ⁇ 0.05). Compared with the model control group, there was no significant difference in the body weight of the G1 and G2 groups during the administration period (P>0.05), and the body weight of the G4 group decreased on the 9th day, but the difference was not statistically significant (P>0.05). ⁇
  • the food intake and water intake of the mice in each group during the administration period are shown in Table 10 and Table 11: Compared with the mice in the NGT group, the food intake of the model control group increased, and the water intake only increased on the first to second days of administration, but The difference was not statistically significant (P>0.05); there was no significant difference in the food intake of the mice in the G1 and G2 groups, and the food intake and water intake of the G4 group decreased on the 1st to 2nd day and the 7th to 8th day, but the difference was not statistically significant Scientific significance (P>0.05).
  • the food intake and water intake of the G1 group decreased on the 4th to 5th and 7th to 8th days of administration; the food intake and water intake of the G2 group decreased for 3 times, and the reduction rate was lower than that of the G1 group. Slightly larger; the G4 group can reduce the food intake and water intake of the mice on the 1st to 2nd day and 7th to 8th day after administration, and the food intake and water intake of the mice on the 4th to 5th day increase significantly.
  • P>0.05 there was no statistical difference between the three administration groups and the model control group (P>0.05).
  • mice in each group 4.4 OGTT detection after the last administration of mice in each group
  • the OGTT detection situation after the last administration of mice in each group is shown in Table 12 and Table 13: after the first administration of glucose to the mice in each group, the blood sugar rose to the highest at 30 minutes, decreased at 60 minutes, and dropped to a lower level at 120 minutes; After the first dose of glucose, the blood sugar increased again, and the change of blood sugar was consistent with the first dose of glucose, but the change was slightly smaller.
  • the first OGTT test after the last administration showed that the blood glucose at each time point in the G1 group and the G4 group decreased significantly.
  • the blood sugar decreased at the time point, but it was not as obvious as that of the G1 group. Therefore, the hypoglycemic effect of G1 was significant and lasted for a long time.
  • the first OGTT and the second OGTT test after the last administration showed that the blood glucose in the G2 group decreased, indicating that G2 has a certain hypoglycemic effect.
  • Prediabetes includes impaired fasting glucose regulation and impaired glucose tolerance.
  • Patients with impaired glucose tolerance are a high-risk group of type 2 diabetes and cardiovascular disease, late detection or no intervention, it is easy to develop diabetes.
  • KM mice model of impaired glucose tolerance was established by intraperitoneal injection of 40 mg/kg streptozotocin 3 times (1 time/3d). This animal model does not cause impaired fasting glucose, only impaired glucose tolerance. There was no significant difference in the fasting blood glucose (basal blood glucose) of the mice in each group after the modeling was completed and before administration. However, 30 minutes after the mice were given glucose, the blood glucose and the increase rate of blood glucose were significantly higher than those of the normal group, indicating that the KM mouse model of abnormal diabetic urine volume was successfully established in this experiment.
  • the administration method of this test is subcutaneous injection of 0.02mL/monkey, the cycle is 9 days, and the administration frequency is q2d, a total of 5 times.
  • the drug was administered after fasting for 9 hours, the blood glucose was detected 1 hour after the drug administration, and then two consecutive OGTT tests were performed.
  • the blood glucose at 1 hour after administration, the blood glucose at the first and second OGTT 120 minutes were not significantly affected, and the blood glucose values at the first OGTT 30 minutes, 60 minutes and the second OGTT 30 minutes were significantly increased.
  • G1 has obvious hypoglycemic effect, and the hypoglycemic effect lasts for a long time.
  • G4 has a hypoglycemic effect, the hypoglycemic effect lasts for a long time, and the hypoglycemic effect is not as good as G1.
  • G2 has a certain hypoglycemic effect.

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Abstract

提供了GLP-1类似物的融合多肽。具体地，提供了GLP-1类似物的融合多肽或其可药用盐，其中所述GLP-1类似物的融合多肽包含GLP-1(7-37)类似物、GLP-1(7-37)类似物的C-末端延伸区段，和融合蛋白片段；进一步地提供了包含所述GLP-1类似物的融合多肽或其可药用盐的组合物、其制备方法以及用途。

Description

GLP-1类似物的融合多肽
本申请要求申请日为2021年9月29日的中国专利申请CN202111150471.7的优先权。本申请引用上述中国专利申请的全文。
技术领域
本发明属于多肽领域，具体地说，涉及GLP-1类似物的融合多肽。
背景技术
胰高血糖素样肽-1(Glucagon-like peptide-1，GLP-1)是由高血糖素原衍生出来的肽段，在人体摄入营养成分时候由内分泌细胞分泌出来。它具有刺激胰岛素基因的表达和胰岛素释放；抑制胰岛血糖素的释放；抑制食物摄取的欲望，有减肥功效；刺激β细胞的增值和分化；对β细胞的凋亡有保护作用。在治疗糖尿病领域具有广泛的应用。
存在两种GLP-1两种N末端截短的产物，GLP-1(7-36)和GLP-1(7-37)，其被发现识别胰腺受体，并且确定为体内的活性种类。天然GLP-1在糖尿病治疗上具有诸多缺点，例如，它在体内易被二肽基肽酶IV(DPP-IV)快速降解。
目前已有多种GLP-1类似物上市，NovoNordis k开发了具有1.8mgs.c./天的每天一次给药的利拉鲁肽(Liraglutide)(美国专利号6,268,343)，并且于2010年批准，另外，GLP-1类似物索马鲁肽(Semaglutide)(国际公布号WO 2006/097537 A2)由USFDA批准。上述两种已上市的GLP-1类似物都有脂肪酸侧链，不易生产。
因此，目前仍然需要对GLP-1进行改进，以获得便于生产，并具有改善功能的GLP-1类似物。
发明内容
本发明涉及一种GLP-1类似物的融合多肽或其可药用盐，其中所述GLP-1类似物的融合多肽包含GLP-1(7-37)类似物、GLP-1(7-37)类似物的C-末端延伸区段，和融合蛋白片段。
在本发明的一个实施方案中，所述融合蛋白片段为融合蛋白的β折叠片段。
在本发明的一个实施方案中，所述融合蛋白片段具有30～80个氨基酸。
在本发明的一个实施方案中，所述融合蛋白片段具有如下所示的氨基酸序列
Figure PCTCN2022121356-appb-000001
或与其具有至少70％、75％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％的序列同一性。
在本发明的一个实施方案中，所述GLP-1(7-37)类似物基本上保留了天然GLP-1(7-37)的功能，优选地，所述天然GLP-1(7-37)包含如下所示的氨基酸序列， 或由其组成：HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG(SEQ ID NO：1)。
在本发明所述的GLP-1类似物的融合多肽的一个实施方案中，所述GLP-1(7-37)类似物在对应于天然GLP-1(7-37)的第7至第37位的任一氨基酸被第一半胱氨酸取代，所述GLP-1(7-37)类似物的C-末端延伸区段中包含第二半胱氨酸，并且所述第一半胱氨酸和所述第二半胱氨酸形成二硫键，优选地，所述第一半胱氨酸和所述第二半胱氨酸形成分子内二硫键。
在本发明所述的GLP-1类似物的融合多肽的一个实施方案中，所述所述第一半胱氨酸与所述第二半胱氨酸间隔至多为40个氨基酸长度，优选地，间隔35至3个氨基酸，更优选地，间隔30至5个氨基酸，更优选地，间隔35、34、33、32、31、30、29、28、27、26、25、24、23、22、21、20、19、18、17、16、15、14、13、12、11、10、9、8、7、6、5个氨基酸，或间隔上述两点间的任一范围。
在根据本发明所述的GLP-1类似物的融合多肽的一个实施方案中，所述GLP-1类似物与SEQ ID NO：1所示的天然GLP-1(7-37)相比，还包含至多10个、9个、8个7个、6个、5个、4个、3个、2个或1个保守氨基酸修饰，任选地，所述保守氨基酸修饰包括氨基酸的取代、缺失和/或添加，而不改变GLP-1(7-37)的活性或功能。任选地，所述保守氨基酸修饰可以为本领域技术人员公知的任何不改变GLP-1(7-37)的活性或功能氨基酸的取代、缺失和/或添加。
在本发明所述的GLP-1类似物的融合多肽的进一步的实施方案中，所述GLP-1(7-37)类似物的C-末端延伸区段为包含1-15个氨基酸的肽段。
在本发明所述的GLP-1类似物的融合多肽的进一步的实施方案中，所述GLP-1(7-37)类似物的C-末端延伸区段为至多15、14、13、12、11、10、9、8、7、6、5、4、3、2或1个氨基酸组成的肽段，其中一个氨基酸为半胱氨酸，其余为除半胱氨酸以外的任意氨基酸。
在本发明所述的GLP-1类似物的融合多肽的进一步的实施方案中，所述GLP-1(7-37)类似物的C-末端延伸区段的一个氨基酸为半胱氨酸，其余为甘氨酸或精氨酸。
在本发明所述的GLP-1类似物的融合多肽的进一步的实施方案中，所述GLP-1(7-37)类似物的C-末端延伸区段选自C、GCGR、或GCGGGGGG。
在本发明的另一个实施方案中，提供了一种GLP-1类似物的融合多肽或其可药用盐，其中所述GLP-1类似物的融合多肽从N-末端至C-末端具有式(I)所示的结构:
X-E-Y   (I)
其中，所述X为GLP-1(7-37)类似物，所述E为GLP-1(7-37)类似物的C-末端延伸区段，所述Y为融合蛋白片段。
在如式(I)所示的GLP-1类似物的融合多肽或其可药用盐的一个实施方案中，所述GLP-1(7-37)类似物基本上保留了天然GLP-1(7-37)的功能，优选地，所述天 然GLP-1(7-37)包含如下所示的氨基酸序列，或由其组成：
Figure PCTCN2022121356-appb-000002
在如式(I)所示的GLP-1类似物的融合多肽或其可药用盐的一个实施方案中，所述GLP-1(7-37)类似物在对应于天然GLP-1(7-37)的第7至第37位的任一氨基酸被第一半胱氨酸取代，所述GLP-1(7-37)类似物的C-末端延伸区段中包含第二半胱氨酸，并且所述第一半胱氨酸和所述第二半胱氨酸形成二硫键，优选地，所述第一半胱氨酸和所述第二半胱氨酸形成分子内二硫键。
在如式(I)所示的GLP-1类似物的融合多肽或其可药用盐的一个实施方案中，所述所述第一半胱氨酸与所述第二半胱氨酸间隔至多为40个氨基酸长度，优选地，间隔35至3个氨基酸，更优选地，间隔30至5个氨基酸，更优选地，间隔35、34、33、32、31、30、29、28、27、26、25、24、23、22、21、20、19、18、17、16、15、14、13、12、11、10、9、8、7、6、5个氨基酸，或间隔上述两点间的任一范围。
在如式(I)所示的GLP-1类似物的融合多肽或其可药用盐的一个实施方案中，所述GLP-1类似物与SEQ ID NO：1所示的天然GLP-1(7-37)相比，还包含至多10个、9个、8个7个、6个、5个、4个、3个、2个或1个保守氨基酸修饰。
在如式(I)所示的GLP-1类似物的融合多肽或其可药用盐的一个实施方案中，所述GLP-1(7-37)类似物的C-末端延伸区段为包含1-15个氨基酸的肽段。
在如式(I)所示的GLP-1类似物的融合多肽或其可药用盐的一个实施方案中，所述GLP-1(7-37)类似物的C-末端延伸区段为至多15、14、13、12、11、10、9、8、7、6、5、4、3、2或1个氨基酸组成的肽段，其中一个氨基酸为半胱氨酸，其余为除半胱氨酸以外的任意氨基酸。
在如式(I)所示的GLP-1类似物的融合多肽或其可药用盐的一个实施方案中，所述GLP-1(7-37)类似物的C-末端延伸区段的一个氨基酸为半胱氨酸，其余为甘氨酸或精氨酸。
在如式(I)所示的GLP-1类似物的融合多肽或其可药用盐的一个实施方案中，所述GLP-1(7-37)类似物的C-末端延伸区段选自C、GCGR、或GCGGGGGG。
在如式(I)所示的GLP-1类似物的融合多肽或其可药用盐的一个实施方案中，所述融合蛋白片段选自融合蛋白的β折叠片段。
在如式(I)所示的GLP-1类似物的融合多肽或其可药用盐的一个实施方案中，所述融合蛋白片段如SEQ ID NO：2所示，或与其具有至少70％、75％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％的序列同一性。
在如式(I)所示的GLP-1类似物的融合多肽或其可药用盐的一个实施方案中，本发明的融合蛋白片段如下所示：
Figure PCTCN2022121356-appb-000003
或与其具有至少70％、75％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％的序列同一性。
在本发明的另一个实施方案中，提供了一种GLP-1类似物的融合多肽或其可药用盐，其中所述GLP-1类似物的融合多肽从N-末端至C-末端具有式(II)所示的结构:
Y-L’-X-E   (II)
其中，所述Y为融合蛋白片段，所述L’为接头或不存在，所述X为GLP-1(7-37)类似物，所述E为GLP-1(7-37)类似物的C-末端延伸区段。
在如式(II)所示的GLP-1类似物的融合多肽或其可药用盐的一个实施方案中，当L’为接头时，其为长度为至多15、14、13、12、11、10、9、8、7、6、5、4、3、2或1个氨基酸的任意氨基酸组成的肽接头，优选地，L’为包含精氨酸和/或赖氨酸的接头。
在如式(II)所示的GLP-1类似物的融合多肽或其可药用盐的一个实施方案中，所述GLP-1(7-37)类似物基本上保留了天然GLP-1(7-37)的功能，优选地，所述天然GLP-1(7-37)包含如下所示的氨基酸序列，或由其组成：
Figure PCTCN2022121356-appb-000004
在如式(II)所示的GLP-1类似物的融合多肽或其可药用盐的一个实施方案中，所述GLP-1(7-37)类似物在对应于天然GLP-1(7-37)的第7至第37位的任一氨基酸被第一半胱氨酸取代，所述GLP-1(7-37)类似物的C-末端延伸区段中包含第二半胱氨酸，并且所述第一半胱氨酸和所述第二半胱氨酸形成二硫键，优选地，所述第一半胱氨酸和所述第二半胱氨酸形成分子内二硫键。
在如式(II)所示的GLP-1类似物的融合多肽或其可药用盐的一个实施方案中，所述所述第一半胱氨酸与所述第二半胱氨酸间隔至多为40个氨基酸长度，优选地，间隔35至3个氨基酸，更优选地，间隔30至5个氨基酸，更优选地，间隔35、34、33、32、31、30、29、28、27、26、25、24、23、22、21、20、19、18、17、16、15、14、13、12、11、10、9、8、7、6、5个氨基酸，或间隔上述两点间的任一范围。
在如式(II)所示的GLP-1类似物的融合多肽或其可药用盐的一个实施方案中，所述GLP-1类似物与SEQ ID NO：1所示的天然GLP-1(7-37)相比，还包含至多10个、9个、8个7个、6个、5个、4个、3个、2个或1个保守氨基酸修饰。
在如式(II)所示的GLP-1类似物的融合多肽或其可药用盐的一个实施方案中，所述GLP-1(7-37)类似物的C-末端延伸区段为包含1-15个氨基酸的肽段。
在如式(II)所示的GLP-1类似物的融合多肽或其可药用盐的一个实施方案中，所述GLP-1(7-37)类似物的C-末端延伸区段为至多15、14、13、12、11、10、9、8、7、6、5、4、3、2或1个氨基酸组成的肽段，其中一个氨基酸为半胱氨酸，其余为除半胱氨酸以外的任意氨基酸。
在如式(II)所示的GLP-1类似物的融合多肽或其可药用盐的一个实施方案中，所述GLP-1(7-37)类似物的C-末端延伸区段的一个氨基酸为半胱氨酸，其余为甘氨酸或精氨酸。
在如式(II)所示的GLP-1类似物的融合多肽或其可药用盐的一个实施方案中，所述GLP-1(7-37)类似物的C-末端延伸区段选自C、GCGR、或GCGGGGGG。
在如式(II)所示的GLP-1类似物的融合多肽或其可药用盐的一个实施方案中，所述融合蛋白片段选自融合蛋白的β折叠片段。
在如式(II)所示的GLP-1类似物的融合多肽或其可药用盐的一个实施方案中，所述融合蛋白片段如SEQ ID NO：2所示，或与其具有至少70％、75％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％的序列同一性。
在如式(II)所示的GLP-1类似物的融合多肽或其可药用盐的一个实施方案中，本发明的融合蛋白片段如下所示：
Figure PCTCN2022121356-appb-000005
在本发明的一个实施方案中，本发明提供了一种GLP-1类似物的融合多肽，其具有SEQ ID NO：3-5中任一项所示的氨基酸序列。
G1：
Figure PCTCN2022121356-appb-000006
G2：
Figure PCTCN2022121356-appb-000007
G4：
Figure PCTCN2022121356-appb-000008
另一方面，本发明提供了编码本发明的GLP-1类似物的融合多肽的核苷酸序列。
又一方面，本发明提供了载体，其包含编码本发明的GLP-1类似物的融合多肽的核苷酸序列。
另一方面，本发明涉及一种药物组合物，其包含根据本发明所述的的GLP-1类似物的融合多肽和药学可接受的载体或赋形剂。
另一方面，本发明提供了根据本发明的GLP-1类似物的融合多肽或其可药用盐，以及包含其的组合物在制备用于治疗非胰岛素依赖性糖尿病、胰岛素依赖性糖尿病或肥胖症的药物中的用途。
另一方面，本发明提供了治疗非胰岛素依赖性糖尿病、胰岛素依赖性糖尿病或肥胖症的方法，其包括向有此需要的受试者施用治疗有效量的根据本发明的 GLP-1类似物的融合多肽或其可药用盐，以及包含其的组合物。
术语
除非有相反陈述，在说明书和权利要求书中使用的术语具有下述含义。
如本文所用的术语“GLP-1(7-37)类似物”指的是基本上保留GLP-1(7-37)活性或功能的多肽，其与天然存在的GLP-1(7-37)(如SEQ ID NO：1)相比，具有至多10个、9个、8个7个、6个、5个、4个、3个、2个或1个氨基酸的保守修饰，包括氨基酸的取代、缺失和/或添加，所述“保守修饰”是指用具有类似特征(例如电荷、侧链大小、疏水性/亲水性、主链构象和刚性等)的其它氨基酸置换蛋白中的氨基酸，使得不改变GLP-1(7-37)的活性或功能。
如本文所用，术语“基本上保留了天然GLP-1(7-37)的功能”，指的是本领域技术人员通过常规技术手段，能够确定本文所述的GLP-1(7-37)类似物与天然GLP-1(7-37)相比，功能和/或降低和/或减少不超过50％，优选地，不超过45％、不超过40％、不超过35％、不超过30％、不超过25％、不超过20％、不超过19％、不超过18％、不超过17％、不超过16％、不超过15％、不超过14％、不超过13％、不超过12％、不超过11％、不超过10％、不超过9％、不超过8％、不超过7％、不超过6％、不超过5％、不超过4％、不超过3％、不超过2％、不超过1％。
如本文所用的术语“可药用盐”，是指GLP-1类似物的融合多肽的盐形式。可以通过常规化学方法，例如在Pharmaceutical Salts：Properties,Selection,and Use，P.Heinrich Stahl(编)、Camille G.Wermuth(编)ISBN：3-90639-026-8精装388页，2002年8月中描述的方法，从含有碱性或酸性部分的母体化合物来合成本发明的盐。通常，可以通过水中、或在有机溶剂中、或在两者的混合物中，使这些化合物的游离酸或碱形式与适当的碱或酸进行反应，来制备这些盐。
能够与多种无机和有机酸形成酸加成盐(单盐或二盐)。酸加成盐的示例包括与选自以下的酸形成的单盐或二盐：乙酸、盐酸、氢碘酸、磷酸、硝酸、硫酸、柠檬酸、乳酸、琥珀酸、马来酸、苹果酸、羟乙基磺酸、富马酸、苯磺酸、甲苯磺酸、硫酸、甲磺酸(甲磺酸盐)、乙磺酸、萘磺酸、戊酸、丙酸、丁酸、丙二酸、葡萄糖醛酸和乳糖酸。一种具体的盐是盐酸盐。另一种具体的盐是乙酸盐。
如果化合物是阴离子化合物，或具有可以是阴离子的官能团(例如，-COOH可以是-COO -)，则可以与有机或无机碱形成盐，产生合适的阳离子。合适的无机阳离子的示例包括但不限于碱金属离子，如Li +、Na +和K +；碱土金属阳离子，如Ca 2+和Mg 2+；和其他阳离子。如Al 3+或Zn +。合适的有机阳离子的示例包括但不限于铵离子(即NH 4 +)和取代的铵离子(例如，NH 3R +、NH 2R 2 +、NHR 3 +、NR 4 +)。一些合适的取代铵离子的示例是衍生自以下的那些：甲胺、乙胺、二乙胺、丙胺、二环己胺、三乙胺、丁胺、乙二胺、乙醇胺、二乙醇胺、哌嗪、苄胺、苯基苄胺、胆碱、葡甲胺和氨丁三醇，以及氨基酸，如赖氨酸和精氨酸。常见的季铵离子的示例是N(CH 3) 4 +。
术语“融合蛋白的β折叠片段”是指融合蛋白的具有β折叠结构的片段。
术语“多肽”是指氨基酸的任何聚合链。术语“肽”和“蛋白”与术语”多肽”可互换使用，并且也指氨基酸的聚合链。术语“多肽”涵盖天然或人工蛋白、蛋白片段和蛋白氨基酸序列的多肽类似物。除非上下文矛盾，否则术语“多肽”涵盖其片段和变体(包括变体的片段)。
术语“分离的蛋白”或“分离的多肽”是如下的蛋白或多肽：根据其衍生的起源或来源，其与在其天然状态下伴随的天然相关组分分离、基本上不含来自同一物种的其他蛋白、由来自不同物种的细胞表达、或者在自然界中不存在。因此，化学合成的或在不同于其天然来源的细胞的细胞系统中合成的多肽是从其天然相关组分中“分离的”。还可以使用本领域众所周知的蛋白纯化技术，通过分离使由一条或多条多肽链组成的蛋白基本上不含天然相关组分。
术语“回收”是指通过分离，例如使用本领域众所周知的蛋白纯化技术，使化学物质例如多肽基本上不含天然相关组分的过程。
术语“分离的核酸”是指多核苷酸(例如，基因组、cDNA或合成来源的多核苷酸，或其某种组合)，其通过人为干预从与其一起天然存在的全部或部分多核苷酸分离、与不是天然连接的多核苷酸可操作地连接、或不会作为天然中较大序列的一部分存在。
如本文所用术语多肽的序列“同一性”是指两个蛋白或多肽之间的序列相似性。当两个比较序列中的位置均被相同氨基酸残基占据时，例如如果两个多肽的一个位置都被同一个氨基酸残基占据时，那么所述分子在该位置是一致的。适于确定序列同一性百分比和序列相似性百分比的算法的实例是BLAST和BLAST2.0算法。
如本文所用，术语“载体”旨在指能够转运已与其连接的另一核酸的核酸分子。载体的一种类型是“质粒”，其是指环状双链DNA环，其中可以连接其他DNA区段。载体的另一种类型是病毒载体，其中可以将其他DNA区段连接到病毒基因组中。某些载体能够在引入它们的宿主细胞中自主复制(例如，具有细菌复制起点的细菌载体和游离型哺乳动物载体)。在导入宿主细胞后，其他载体(例如，非游离型哺乳动物载体)可以整合到宿主细胞的基因组中，从而与宿主基因组一起复制。此外，某些载体能够指导与其可操作连接的基因的表达。此类载体在本文中称为“重组表达载体”(或简称为“表达载体”)。通常，在重组DNA技术中有用的表达载体通常是质粒的形式。在本说明书中，“质粒”和“载体”可以互换使用，因为质粒是最常用的载体形式。然而，本发明旨在包括具有等同功能的此类其他形式的表达载体，例如病毒载体(例如复制缺陷型逆转录病毒、腺病毒和腺相关病毒)。
术语“重组宿主细胞”(或简称为“宿主细胞”)旨在指已将外源DNA引入其中的细胞。在一个实施方案中，宿主细胞包含两个或更多个(例如，多个)编码抗体或结合蛋白的核酸，例如美国专利号7,262,028中描述的宿主细胞。此类术语不仅 旨在指特定的受试细胞，而且还指此类细胞的后代。因为由于突变或环境影响，某些修饰可能在后代中发生，所以此类后代实际上可能与亲本细胞不同，但仍包括在本文所用的术语“宿主细胞”的范围内。在一个实施方案中，宿主细胞包括原核和真核细胞。在另一个实施方案中，真核细胞包括原生生物、真菌、植物和动物细胞。一种特别有用的原核宿主细胞是大肠杆菌及其衍生物。特别有用的哺乳动物细胞系包括但不限于CHO、HEK293、COS、NS0、SP2和PER.C6。HEK 293E细胞在用于重组蛋白的瞬时表达系统中作为哺乳动物宿主细胞特别有用。CHO细胞特别用作稳定转染的哺乳动物宿主细胞，以产生重组蛋白。特别有用的昆虫细胞系是Sf9细胞系及其衍生物。特别有用的真菌宿主细胞是酿酒酵母(Saccharomyces cerevisiae)及其衍生物。
标准技术可用于重组DNA、寡核苷酸合成、细胞培养、组织培养和转化(例如，电穿孔、脂转染、转化)。酶促反应和纯化技术可以根据制造商的说明书或如本领域通常完成的或如本文所述的进行。前述技术和步骤通常可以根据本领域公知的常规方法来执行，并且如本说明书通篇所引用和讨论的各种一般性和更具体的参考文献中所述。参见，例如，Sambrook et al.,Molecular Cloning:A Laboratory Manual,第2版(Cold Spring Harbor Laboratory Press,Cold Spring Harbor,N.Y.,1989)。
附图说明
图1显示ELISA预实验中蛋白浓度和A450呈线性关系；
图2A至图2D显示ELISA检测的本发明GLP-1类似物的融合多肽的亲和力拟合曲线；
图3显示DPP-4的酶降解作用。
具体实施方式
为了更详细的说明本发明，本说明书提供了下列具体实施方案，但本发明的方案并非仅限于此。实施例中未注明具体条件的实验方法，通常按照常规条件，或按照原料或商品制造厂商所建议的条件。未注明具体来源的试剂，为市场购买的常规试剂。
本发明涉及了以下GLP-1类似物的融合多肽，其分别具有如下氨基酸序列：
G1：
Figure PCTCN2022121356-appb-000009
G2：
Figure PCTCN2022121356-appb-000010
G4：
Figure PCTCN2022121356-appb-000011
Figure PCTCN2022121356-appb-000012
并且，上述G1、G2和G4多肽各自包含由其中的两个半胱氨酸形成的链内二硫键。
实施例1 GLP-1类似物的融合多肽的制备
G1的制备：
G1的氨基酸序列：
Figure PCTCN2022121356-appb-000013
并且，其中的两个半胱氨酸形成链内二硫键。
采用固相合成法按照G1的氨基酸顺序连接氨基酸，脱除保护基和树脂，得到GLP-1类似物融合多肽的中间体粗品，用水或10～20％的乙腈溶解，采用反相C8-HPLC纯化，使用乙腈-水-三氟乙酸分离纯化，浓缩，冻干，得到GLP-1类似物融合多肽中间体的纯品，用水或10～20％的乙腈溶解，用碳酸氢铵或DMSO氧化形成二硫键，纯化，得到GLP-1类似物的融合多肽，MS测定分子量为8212.4Da，符合预期的G1蛋白的分子量。
G2的制备：
G2氨基酸序列：
Figure PCTCN2022121356-appb-000014
并且，其中的两个半胱氨酸形成链内二硫键。
采用固相合成法按照G2的氨基酸顺序连接氨基酸，脱除保护基和树脂，得到GLP-1类似物的融合多肽的中间体粗品，用水或10～20％的乙腈溶解，采用反相C8-HPLC纯化,使用乙腈-水-三氟乙酸分离纯化，浓缩，冻干，得到GLP-1类似物的融合多肽的中间体纯品，用水或10～20％的乙腈溶解，用碳酸氢铵或DMSO氧化形成二硫键，纯化，得GLP-1类似物的融合多肽，MS测定分子量为8210.4Da，符合预期的G2蛋白的分子量。
G4的制备：
G4氨基酸序列：
Figure PCTCN2022121356-appb-000015
并且，其中的两个半胱氨酸形成链内二硫键。
采用固相合成法按照G4的氨基酸顺序连接氨基酸，脱除保护基和树脂，得到GLP-1类似物的融合多肽的中间体粗品，用水或10～20％的乙腈溶解，采用反相C8-HPLC纯化,使用乙腈-水-三氟乙酸分离纯化，浓缩，冻干，得到GLP-1类似物的融合多肽的中间体纯品，用水或10～20％的乙腈溶解，用碳酸氢铵或DMSO氧 化形成二硫键，纯化，得到GLP-1类似物的融合多肽，MS测定分子量为7828.2Da，符合预期的G4蛋白的分子量。
实施例2 GLP-1类似物的融合多肽与GLP-1受体亲和力实验
1)实验目的
GLP-1类药物对糖尿病的治疗效果是基于GLP-1与GLP-1受体结合后，启动G蛋白偶联信号通路，促使胰岛β细胞分泌胰岛素，因此制备的GLP-1类似物的融合多肽与GLP-1受体的结合能力与天然GLP-1蛋白相当。通过酶联免疫吸附试验，测定本发明制备的GLP-1类似物的融合多肽与INS胰岛素细胞上的GLP-1受体结合的亲和力，并与人GLP-1蛋白与INS胰岛素细胞上的GLP-1受体结合的亲和力进行对比，确认本发明的GLP-1类似物的融合多肽在蛋白质序列及空间结构上的改变是否会影响其与细胞表面GLP-1受体的结合能力。
2)受试物
G1:
溶剂：磷酸缓冲盐溶液(PBS)
A280：4.68
折算蛋白质浓度：3.05mg/ml
折算摩尔浓度：0.387mmol/L
G2:
溶剂：磷酸缓冲盐溶液(PBS)
A280：6.18
折算蛋白质浓度：3.95mg/ml
折算摩尔浓度：0.502mmol/L
G4:
溶剂：磷酸缓冲盐溶液(PBS)
A280：0.78
折算蛋白质浓度：0.48mg/ml
折算摩尔浓度：0.061mmol/L
阳性对照
人GLP-1(7-36)
生产商：中肽生化有限公司，货号：GLUC-010A，规格：1mg/瓶
3)试剂
人GLP1(7-36)ELISA试剂盒：
生产商：Abcam公司，产品货号：ab184857。
大鼠胰岛素瘤细胞：
生产商：上海复祥生物科技有限公司，产品货号：INS-1。
4)实验方法
4.1 ELISA反应底物的制备
将INS-1大鼠胰岛素瘤细胞在37℃培养箱中培养至超过80％的汇合度后，进行传代培养。将传代后的INS-1大鼠胰岛素瘤细胞加入96孔板中，每孔加入的细胞体积为100ul，在96孔板培养至细胞贴壁。用PBS缓冲液轻轻冲洗96孔板，冲洗三次，去除未贴壁的细胞。在每孔中加入100ul的4％的多聚甲醛，在室温下静置10分钟以固化细胞。固化后将多聚甲醛倒出，再在每孔加入甘油至终浓度为含2％甘油的pH7.5的PBS缓冲液100ul处理5分钟。获得包埋INS-1大鼠胰岛素瘤细胞的96孔板用于ELISA反应。
在96孔板上包埋INS-1大鼠胰岛素瘤细胞。使本发明的GLP-1类似物的融合多肽与细胞表面的GLP-1受体相结合，洗掉未结合的GLP-1类似物的融合多肽，封闭GLP-1受体，再用人GLP1(7-36)ELISA试剂盒中抗体与结合在GLP-1受体上的GLP-1类似物的融合多肽相互反应，反应机理类似于双抗体夹心法。
4.2 ELISA试验操作步骤
ELISA试剂盒预先从冰箱中取出，平衡至室温。
使用前准备以下试剂：
取出试剂盒中的10×清洗液PT用9倍体积的去离子水稀释到所需量。
取出试剂盒中的捕获抗体和检测抗体按1:1等比例混合，再用四倍体积抗体稀释液稀释到所需量，得到抗体混合液。
将96孔板中所需要用到的孔用BSA进行封闭，以消除细胞与待测样品的非特异性反应。将1％的BSA加满各反应孔，在37℃下封闭40分钟。
在96孔板每孔中加入50ul的待测样品或对照品。
再在每孔中加入50ul的抗体混合液。
将板盖好后，室温下置于酶标板振荡器上以400rpm的速度振荡孵育1小时。
将板中的反应液倒出，再在每孔中加入350ul的清洗液PT清洗三次。每次应将液体倒空并吸净。
在每孔中加入100ul的TMB，置于酶标板振荡器上在黑暗处以400rpm的速度振荡孵育15分钟，进行显色反应。
在每孔中加入100ul的终止液，置于酶标板振荡器上以400rpm的速度振荡1min使其混匀以终止反应。
将96孔板置于酶标仪上用450nm波长进行吸光度检测。
预实验
因为用自行制备的包埋有INS-1大鼠胰岛素瘤细胞的96孔板取代商品化的人GLP1(7-36)ELISA试剂盒中包埋了GLP-1抗体的96孔板，细胞表面的GLP-1受体与试剂盒中GLP-1抗体对GLP-1的结合能力会有区别，所以需要对开展预实验，用人GLP-1蛋白来测定INS-1大鼠胰岛素瘤细胞表面GLP-1受体的结合能力，确 定最适反应物浓度范围及线性。同时摸索ELISA试剂盒反应中，当人GLP-1(7-36)蛋白饱和时的450nm吸光度，并通过饱和时的人GLP-1(7-36)蛋白浓度来估算INS-1大鼠胰岛素瘤细胞表面GLP-1受体的浓度。
预实验方案：用PBS稀释人GLP-1(7-36)蛋白获得不同的的蛋白浓度，从最高浓度0.1mg/ml开始向下逐步稀释，每个浓度稀释十倍，最终稀释至0.1ng/ml。将不同浓度的人GLP-1(7-36)蛋白用ELISA试剂盒按4.1的方法进行受体结合检测，获得450nm吸光度的检测结果，每个浓度平行两个样，使用PBS作为空白对照。检测方案见下表1：
表1
Figure PCTCN2022121356-appb-000016
GLP-1类似物的融合多肽与GLP-1受体结合实验
根据预实验的结果，对所制备的三个GLP-1类似物的融合多肽G1、G2、G4进行与INS-1大鼠胰岛素瘤细胞表面GLP-1受体的结合实验，将三个GLP-1类似物的融合多肽的浓度梯度设置为100ng/ml，33ng/ml，10ng/ml，3.3ng/ml，1ng/ml，用PBS将G1、G2、G4三个类似物的融合多肽分别稀释到上述蛋白浓度，以人GLP-1(7-36)蛋白作为阳性对照，以PBS作为空白对照。以上每个浓度点三个平行样，按照上述ELISA实验步骤进行检测，检测方案见下表2：
表2
Figure PCTCN2022121356-appb-000017
数据处理
人GLP-1(7-36)及GLP-1类似物的融合多肽的Kd值的计算使用GraphPad Prism 8.0软件。
计算方法，通过GraphPad Prism 8.0软件用单一位点亲和力方法进行拟合，获得Kd值。曲线中的横坐标为受测蛋白质摩尔浓度，纵坐标为
Figure PCTCN2022121356-appb-000018
公式中A 0为空白对照在450nm时的吸光度；Ai为每个检测浓度实测450nm吸光度的平均值；A ∞为饱和时的450nm吸光度，该数值通过预实验测得；[x] tot为受体总浓度，该数值通过预实验结果计算获得。
5)实验结果
预实验
通过预实验确认固化的INS-1大鼠胰岛素细胞表面GLP-1受体与野生型GLP-1的结合效率，确认最适反应浓度，并得到饱和时的人GLP-1(7-36)蛋白浓度。
表3
Figure PCTCN2022121356-appb-000019
可看出在人GLP-1(7-36)蛋白浓度为1ug/ml时，ELISA反应达到饱和，饱和时抗体摩尔浓度为1*1000*1000/3300(人GLP-1(7-36)蛋白分子量)＝303nM，饱和时450nm吸光度为3.550。
以双倒数作图，可见1ng/ml至1000ng/ml时蛋白浓度和A450呈线性关系(参见图1)。
正式实验
对所制备的三个GLP-1类似物的融合多肽G1、G2、G4进行与INS-1大鼠胰岛素瘤细胞表面GLP-1受体的结合实验，通过ELISA反应后检测所得A 450结果如下：
表4
Figure PCTCN2022121356-appb-000020
Figure PCTCN2022121356-appb-000021
按照公式
Figure PCTCN2022121356-appb-000022
对上述数据进行处理计算，A 0为空白对照的平均值0.865，A ∞为预实验时得到的人GLP-1(7-36)蛋白饱和时的450nm吸光度3.550，[x] tot为受体总浓度，该数值通过预实验结果计算获得，为303nM。
经计算后，表中的数据转换为下表5数值
表5
Figure PCTCN2022121356-appb-000023
将上表数据导入GraphPad Prism 8.0软件用单一位点亲和力方法进行拟合，得到图2A-2D，并获得解离常数Kd值和决定系数R 2。
解离常数Kd，检测抗体和受体间亲和力。在温度，PH值，盐浓度等影响亲和力的因素不变的情况下，Kd本身维持恒定，不受配体和受体浓度的影响。Kd等于一半的受体被配体结合时配体的浓度。Kd越小解离越慢，亲和力越强。
决定系数R 2，判断拟合曲线对样本数据的拟合程度，决定系数越高，模型的拟合效果越好，即模型解释因变量的能力越强。
从图2A至图2D中可以看出，各组实验所获得的R 2均在0.95以上，曲线对 样本数据的拟合程度较佳，其中G1的Kd值为0.5845，亲和力显著优于人GLP-1(7-36)的2.447；G4的Kd值为1.345，亲和力略好于人GLP-1(7-36)，但处于同一个量级；G2的Kd值为14.44，亲和力相对于人GLP-1(7-36)下降明显。
实施例3 GLP-1类似物的融合多肽的DPP-4酶酶解拮抗作用
1)实验目的
GLP-1起到与GLP-1受体相结合，促使胰岛β细胞分泌胰岛素的作用，但人体内的GLP-1极易被DPP-4酶所降解而失去功能，因此所制备的GLP-1类似物的融合多肽是否能够抵御DPP-4酶的降解，从而延长所述GLP-1类似物的融合多肽作用时间，是本实验的考察目标。用过量的DPP-4酶对制备的三个GLP-1类似物的融合多肽进行酶切，用ELISA反应测量在相同条件下DPP-4酶对三个GLP-1类似物的融合多肽作用不同时间后GLP-1类似物的融合多肽的残留量，以人GLP-1蛋白(7-36)作为对照，通过体外试验模拟GLP-1类似物的融合多肽在体内维持作用时间，从而表征降糖效果持续时间。
2)实验材料
DPP-4酶
生产商：Sigma-Aldrich，产品货号：D4943-1VL,规格：酶浓度119ug/ml。
ELISA试剂盒
生产商：Abcam公司，产品货号：ab184857。
3)实验方法
在同样的反应条件下，以人GLP-1(7-36)蛋白作为对照，通过ELISA反应测定不同时间点经DPP-4酶酶切后残留的GLP-1类似物的融合多肽的量，生成酶切曲线，确定GLP-1类似物的融合多肽的抗降解能力。
ELISA试验操作步骤
反应体系残留人GLP-1(7-36)及GLP-1类似物的融合多肽的量通过ELISA反应测定
ELISA试剂盒预先从冰箱中取出，平衡至室温。
使用前准备以下试剂：
取出试剂盒中的10×清洗液PT用9倍体积的去离子水稀释到所需量
取出试剂盒中的捕获抗体和检测抗体按1:1等比例混合，再用四倍体积抗体稀释液稀释到所需量，得到抗体混合液。
在96孔板每孔中加入50ul的待测样品或对照品。
再在每孔中加入50ul的抗体混合液
将板盖好后，室温下置于酶标板振荡器上以400rpm的速度振荡孵育1小时
将板中的反应液倒出，再在每孔中加入350ul的清洗液PT清洗三次。每次应将液体倒空并吸净。
在每孔中加入100ul的TMB，置于酶标板振荡器上在黑暗处以400rpm的速度振荡孵育15分钟，进行显色反应。
在每孔中加入100ul的终止液，置于酶标板振荡器上以400rpm的速度振荡1min使其混匀以终止反应。
将96孔板置于酶标仪上用450nm波长进行吸光度检测。
预实验
通过预实验确定合适的酶与底物的浓度。
以人GLP-1(7-36)作为反应的底物，分为两组，分别用pH为7.4的PBS稀释至浓度为0.1ug/ml，10ng/ml。将DPP-4酶解冻后用PBS稀释至浓度10ug/ml。在反应体系中先加入PBS 400ul，然后底物与酶各加50ul，总反应体积500ul。反应在水浴锅中进行，水浴温度30℃。
取样检测时间点：加酶前，0时，0.1h，0.5h，1h，2h，4h。在相应时间点分别取样按上述中的ELISA实验步骤检测吸光度。
正式实验
根据预实验的结果将G1、G2、G4和人GLP-1(7-36)分别用pH为7.4的PBS稀释到10ng/ml，DPP-4酶用PBS稀释到10ug/ml。按照人GLP-1(7-36)，G1，G2，G4分为四组，每组首先加入PBS 800ul，然后分别加入各组底物100ul，DPP-4酶100ul，总反应体积为1ml。
取样检测时间点：加酶前，0时，0.1h，0.5h，1h，2h，4h，8h，12h，24h。在相应时间点分别取样按上述ELISA实验步骤检测吸光度。
数据处理
以各组加酶前测得的ELISA反应结果作为基准，其他反应时间得到的ELISA反应结果与之相比，获得不同时间点残留的人GLP-1(7-36)及GLP-1类似物的融合多肽与未加酶的初始值的百分比。使用GraphPad Prism 8.0软件对人GLP-1(7-36)及GLP-1类似物的融合多肽随着时间的延长所剩余的百分比绘制曲线，计算线下峰面积AUC，通过人GLP-1(7-36)及GLP-1类似物的融合多肽在相同条件下与过量的DPP-4酶反应后的线下峰面积来表征人GLP-1(7-36)及GLP-1类似物的融合多肽在体内的降糖效果及持续时间。由于ELISA反应本身消耗一定的时间，0时的结果并不是真实的0时反应状态，而是完成ELISA反应后获得最快结果，由于ELISA反应的时间和试剂的干扰无法准确得出真正的0时结果。因此所有时间点均应视为扣除ELISA反应时间后的时长。
4)实验结果
表6
AUC G1 G2 G4 人GLP-1(7-36)
总面积 5.137 1.342 2.469 1.025
标准误差 0.1388 0.04715 0.09012 0.05201
95％置信区间 4.865-5.409 1.249-1.434 2.292-2.646 0.9232-1.127
从图3和上表可以看出，G2在DPP-4酶作用下的残留与人GLP-1(7-36)并无明显差异，不具备抑制DPP-4酶切的效果，而G4具有一定的抑制DPP-4酶降解的作用，其线下峰面积为2.469，超出人GLP-1(7-36)的1.025逾一倍。G1抑制DPP-4酶切的效果则非常显著，其线下峰面积为5.137，是人GLP-1(7-36)的5倍，并且在酶切24小时之后仍有20％左右的蛋白。这预示着G1在体内可以存在更长的时间，从而降糖效果维持时间更长。
实施例4 GLP-1类似物的融合多肽对链脲佐菌素致糖耐量受损小鼠模型的影响
1)实验目的及原理
构建糖耐量受损小鼠模型，通过模型鼠对制备的GLP-1类似物的融合多肽G1，G2，G4开展初步的药效研究，确认G1，G2，G4的降糖效果及持续时间，从而判断所制备的GLP-1类似物的融合多肽是否具备药用价值。进一步通过连续多次给药，观察连续给药对小鼠体重，摄食，摄水量的影响，并考察所制备的GLP-1类似物的融合多肽有无减轻体重的作用。
2)实验材料
受试物
G1
溶剂：磷酸缓冲盐溶液(PBS)
A280：4.68
折算蛋白质浓度：3.05mg/ml
折算摩尔浓度：0.387mmol/L
G2
溶剂：磷酸缓冲盐溶液(PBS)
A280：6.18
折算蛋白质浓度：3.95mg/ml
折算摩尔浓度：0.502mmol/L
G4
溶剂：磷酸缓冲盐溶液(PBS)
A280：0.78
折算蛋白质浓度：0.48mg/ml
折算摩尔浓度：0.061mmol/L
对照药
阴性对照品：
生理盐水
0.9％氯化钠，注射用，市售；
阳性对照品：
人GLP-1(7-36)
生产商：中肽生化有限公司，货号：GLUC-010A，规格：1mg/瓶
实验动物
实验动物动物种属：雄性，KM小鼠。
动物等级：SPF级。
性别和数量：购买70只，筛选62只入组，均为雄性。
动物年龄：动物给药时6～8周龄。
动物体重范围：动物给药时为18～25g。
动物来源：北京维通利华实验动物技术有限公司。
实验动物适应性观察
动物接收后进行适应性观察，每天1次，连续观察3天，内容包括一般精神状态、皮毛、活动情况等。
实验动物饲养观察条件
普通动物房：温度：20-26℃，相对湿度：40％-70％。
饮水：除菌过滤纯化水，通过美国密理博Elix纯水机制备的纯化水，再经0.22μm除菌滤器过滤除菌。
饲料：SPF级KM小鼠维持配合饲料，营养成分符合GB14924.1-2001，GB14924.2-2001，GB14924.3-2010试验动物配合饲料通用质量标准，生产单位：北京科澳协力饲料有限公司。
3)实验方法
3.1糖耐量受损(IGT,Impaired Glucose Tolerance)小鼠模型建立
将52只KM小鼠根据体重注射链脲佐菌素，每只小鼠按40mg/kg的剂量，隔两天腹腔注射链脲佐菌素制作糖耐量受损小鼠模型，共注射3次。注射完一周后，禁食12小时测空腹血糖(基础血糖)，然后按照2.5g/kg灌胃给予葡萄糖溶液，灌胃体积为0.10-0.15ml/10g，灌胃30min后再测定血糖值。取30min血糖值增加幅度大于200％的造模小鼠按增加幅度大小从高往低排序，去掉增加幅度最高和最低值，按增加幅度大小取40只造模小鼠纳入糖耐量受损小鼠模型。
分组
将40只糖耐量受损模型小鼠分为模型对照组、GLP-1组、G1组、G2组、G4组，共5组，每组8只，按血糖值增加幅度及体重大小均衡分组。另取8只未造模小鼠作为NGT(Normal Glucose Tolerance，正常对照)组。
给药剂量
以市售利拉鲁肽作为参考，利拉鲁肽的分子量为3751，起始剂量为每天0.6mg，1周后，剂量应增加至1.2mg，推荐每日剂量不超过1.8mg。按照标准剂量1.2mg/ 天折算给药摩尔量，相当于GLP-1类似物成人给药剂量为0.32μmol/天。
20g体重的小鼠与70Kg的人的给药剂量换算关系为0.0026:1，即体重20g左右的小鼠折算给药剂量为0.83nmol/天。参考文献药理实验方法学(第三版)徐叔云，2002
将G1，G2，G4分别用生理盐水稀释到20μmol/L，每次给药1nmol，相当于每只小鼠每次给药25μl/10g体重。
给药方案
多次给药，每次给药间隔一天。首次给药后口服葡萄糖，测量不同时间，第2天后再给葡萄糖再测不同时间。
小鼠分组完毕后第1天上午9：30左右开始给药。NGT组和模型对照组皮下注射生理盐水，GLP-1，G1，G2，G4各组动物皮下注射相应人GLP-1(7-36)及本发明的GLP-1类似物的融合多肽，所有动物均为单次颈背部皮下注射给药。连续九天，每隔一天给药一次，给药时间均为上午9：30左右，共给药五次。
给药方案如下表7：
表7给药方案
Figure PCTCN2022121356-appb-000024
3.2口服葡萄糖耐量试验(OGTT)
口服葡萄糖耐量试验又称糖耐量试验诊断检查(Oral Glucose Tolerance Test，OGTT)，是在口服一定量葡萄糖后2小时内做系列血浆内葡萄糖浓度的测定。长期以来，OGTT作为公认的诊断糖尿病的金标准为世界各国所采用。正常人服用一定量葡萄糖后，血糖浓度暂时性升高，但2h内血糖浓度又可恢复至正常水平，而 糖代谢异常的患者在服用葡萄糖后会出现不同时段、不同程度的血糖升高。凡是临床上发现患者有可疑糖尿病者除试验禁忌外的病人都可进行口服葡萄糖耐量试验检查。可了解机体对葡萄糖代谢的调节能力。
本实验中，小鼠的灌胃给予葡萄糖剂量为2.5g/kg，给糖后2小时内于30min、60min、90min、120min进行尾尖取血测量血糖浓度。
试验方案：
小鼠分组完毕后，第1天上午9：30左右开始给药，给药前9小时禁食不禁水，给药后1h，小鼠尾静脉取血检测空腹血糖。测定血糖后立即灌胃给予2.5g/kg葡萄糖溶液(20％葡萄糖溶液，10ml/Kg)。测定灌胃后30min、60min、90min、120min的血糖。第2天9：30再次进行灌胃给予2g/kg的葡萄糖溶液(20％葡萄糖溶液，10ml/Kg)。测定灌胃后30min、60min、90min、120min的血糖。
检测指标
血糖水平
取血方法：用手术刀片从小鼠尾尖处取血，第一滴血液弃去不用，收集第二滴血液用于检测血糖。
使用仪器：强生ONETOUCH UltraEasy TM稳豪倍易型血糖仪。
检测指标：血糖值。
摄食量
按笼具统计，每笼具为同组小鼠4只。每天早晨8:30左右换食，对每笼放置的食物进行称重，作为F0，第二天换食时，对剩余的食物进行称重，作为F1，F1-F0即为该笼4只小鼠当日摄食量。
摄水量
按笼具统计，每笼具为同组小鼠4只。每天早晨8:30左右换水，对每笼放置的水进行称重，作为W0，第二天换水时，对剩余的水进行称重，作为W1，W1-W0即为该笼4只小鼠当日摄水量。
体重
每隔两天清晨对小鼠进行称重，其中第9天由于给药前禁食，获得的是空腹体重。
数据统计
梯形公式计算血糖-时间曲线下面积(AUC)：AUC＝1/2(0min血糖值+15min血糖值)×0.25h+1/2(15min血糖值+30min血糖值)×0.25h+1/2(30min血糖值+60min血糖值)×0.5h……
使用统计学软件GraphPad Prism 8对数据进行处理。各时间点的血糖水平采用双因素方差分析进行各时间点的组间比较，如有统计学差异(P<0.05)，则以Bonferroni test同正常组和模型对照组进行比较，以P<0.05为具有统计学差异。
血糖-时间曲线下面积(AUC)采用单因素方差分析进行比较，如有统计学差 异(P<0.05)，则以Dunnett’s test同NGT组和模型对照组进行比较，以P<0.05为具有统计学差异。
由于摄水量和摄食量测量时，以每笼4只小鼠为统计，故每组由两个均值构成，亦采用单因素方差分析。
4)实验结果
4.1糖耐量受损小鼠造模结果
NGT组及造模完成后给药前各组小鼠基础血糖及给葡萄糖后30imn血糖值如表8所示：与NGT组比较，模型对照组和各给药组的基础血糖略所上升，但差异均无统计学意义(P＞0.05)；各给药组与模型对照组比较无明显差异(P＞0.05)。模型对照组和各给药组的30min血糖和血糖增幅较NGT组上升，差异有统计学意义(P＜0.01)。模型对照组和各给药组间的基础血糖、30min血糖及血糖增幅均无明显差异(P＞0.05)。以上数据表明各组之间基础血糖无明显差异，有较好的一致性。在给葡萄糖之后，模型对照组和各给药组与NGT组相比，血糖显著上升，造模是成功的，造模小鼠的糖耐量明显低于正常小鼠。在给葡萄糖之后，模型对照组和各给药组之间血糖无明显差异，表明各组间一致性较好，组间差异较小，不会影响实验的代表性。
表8 NGT组及造模完成后给药前各组小鼠基础血糖及给葡萄糖后30imn血糖值(
Figure PCTCN2022121356-appb-000025
n＝8)
Figure PCTCN2022121356-appb-000026
注：与NGT组比较， ▲P<0.05；与模型对照组组比，*P<0.05。
4.2给药期间体重变化
各组给药期间体重变化情况如表9所示：与NGT组比较，模型对照组和各给药组小鼠的体重有所下降，第9天时差异有统计学意义(P＜0.05)。与模型对照组比较，G1和G 2组给药期间体重均无明显差异(P＞0.05)，G4组第9天体重有所降低，但差异无统计学意义(P＞0.05)。·
表9给药期间各组小鼠体重(
Figure PCTCN2022121356-appb-000027
n＝8)
Figure PCTCN2022121356-appb-000028
注：与NGT组比较，*P<0.05，**P<0.01；与模型对照组比，▲P<0.05，▲▲P<0.01。
4.3给药期间摄食摄水量变化
各组小鼠给药期间摄食摄水量如表10和表11所示：与NGT组小鼠比较，模型对照组的摄食量均有所增加，摄水量仅给药第1～2天增加，但差异均无统计学意义(P＞0.05)；G1和G2组小鼠的摄食量无明显差异，G4组的1～2天及7～8天的摄食量和摄水量均降低，但差异无统计学意义(P＞0.05)。与模型对照组比较，G1组给药第4～5、7～8天的的摄食、摄水量有所降低；G2组的3次摄食、摄水量均有所降低，且降低的幅度较G1组稍大；G4组在给药后第1～2天及7～8天可降低小鼠摄食、摄水量，第4～5天小鼠摄食、摄水量明显增加。但给药的三组和模型对照组比较均无统计学差异(P＞0.05)。
表10 NGT组及造模给药各组小鼠摄食量统计(
Figure PCTCN2022121356-appb-000029
n＝2)
Figure PCTCN2022121356-appb-000030
注：与NGT组比较，▲P<0.05，▲▲P<0.01；与模型对照组比，*P<0.05，**P<0.01。
表11 NGT组及造模给药各组小鼠摄水量统计(
Figure PCTCN2022121356-appb-000031
n＝2)
Figure PCTCN2022121356-appb-000032
注：与NGT组比较，▲P<0.05，▲▲P<0.01；与模型对照组比，*P<0.05，**P<0.01。
4.4各组小鼠末次给药后OGTT检测
各组小鼠末次给药后OGTT检测情况如表12和表13所示：各组小鼠第1次给葡萄糖后在30min血糖升到最高，60min时降低，120min时降到更低；第2次给葡萄糖后血糖再一次升高，血糖变化情况与第1次给葡萄糖一致但变化稍小。与NGT组比较，模型对照组小鼠的给药后1h血糖、第1和第2次OGTT 120min血糖均无明显差异，说明基础血糖不受影响；第1次OGTT 30min、60min和第2次OGTT 30min血糖值显著升高(P＜0.01)，说明糖负荷后模型组小鼠血糖异常升高，存在明显的糖耐量受损。
与模型对照组相比，末次给药后的第一次OGTT检测显示，G1组和G4组各时间点血糖均下降明显，第二次OGTT时，G1组各时间点血糖下降明显，G4组各时间点血糖下降，但没有G1组下降明显，因此，G1降糖效果显著，持续时间长，G4降糖效果显著，持续时间长，降糖效果不如G1。
末次给药后的第一次OGTT和第二次OGTT检测显示，G2组血糖下降，说明G2具有一定的降糖效果。
表12各组小鼠末次给药后糖耐量检测血糖值(
Figure PCTCN2022121356-appb-000033
n＝8)
Figure PCTCN2022121356-appb-000034
注：与NGT组比较，▲表示P<0.05；与模型对照组比，*表示P<0.05。
表13第2次OGTT糖耐量检测血糖值(
Figure PCTCN2022121356-appb-000035
n＝8)
Figure PCTCN2022121356-appb-000036
Figure PCTCN2022121356-appb-000037
注：与NGT组比较，▲表示P<0.05；与模型对照组比，*表示P<0.05。
5)结论与讨论
糖尿病前期包括空腹血糖调节受损和糖耐量受损。糖耐量受损(im-paired glucose tolerance，IGT)患者是2型糖尿病和心血管疾病的高危人群，晚发现或未干预的，很容易发展成为糖尿病。
本试验通过3次(1次/3d)腹腔注射40mg/kg的链脲佐菌素，建立KM小鼠糖耐量受损模型。此动物模型不造成空腹血糖受损，仅存在糖耐量受损。本次试验造模结束后给药前各组小鼠的空腹血糖(基础血糖)无明显差异。而小鼠给予葡萄糖后30min，血糖和血糖增幅均较正常组明显升高，说明本实验成功建立了糖尿量异常KM小鼠模型。
本试验给药方式为皮下注射0.02mL/只，周期为9天，给药频率为q2d，共5次。试验第9天禁食9h后给药，检测给药后1h血糖，随即进行连续2次OGTT试验。模型组小鼠给药后1h血糖、第1和第2次OGTT 120min血糖均不受明显影响，第1次OGTT 30min、60min和第2次OGTT 30min血糖值显著升高。
综上，G1有明显的降糖作用，降糖效果持续时间长。G4有降糖作用，降糖效果持续时间长，降糖效果不如G1。G2具有一定的降糖作用。

Claims (25)

  1. 一种GLP-1类似物的融合多肽或其可药用盐，其中所述GLP-1类似物的融合多肽包含GLP-1(7-37)类似物、GLP-1(7-37)类似物的C-末端延伸区段，和融合蛋白片段。
  2. 根据权利要求1所述的GLP-1类似物的融合多肽或其可药用盐，其中所述GLP-1类似物的融合多肽从N-末端至C-末端具有式(I)所示的结构：
    X-E-Y   (I)
    其中，所述X为GLP-1(7-37)类似物，所述E为GLP-1(7-37)类似物的C-末端延伸区段，所述Y为融合蛋白片段。
  3. 根据权利要求1所述的GLP-1类似物的融合多肽或其可药用盐，其中所述GLP-1类似物的融合多肽从N-末端至C-末端具有式(II)所示的结构:
    Y-L’-X-E   (II)
    其中，所述Y为融合蛋白片段，所述L’为接头或不存在，所述X为GLP-1(7-37)类似物，所述E为GLP-1(7-37)类似物的C-末端延伸区段。
  4. 根据权利要求3所述的GLP-1类似物的融合多肽或其可药用盐，其中当L’为接头时，其为长度为至多15、14、13、12、11、10、9、8、7、6、5、4、3、2或1个氨基酸的任意氨基酸组成的肽接头。
  5. 根据权利要求4所述的GLP-1类似物的融合多肽或其可药用盐，其中L’为包含精氨酸和/或赖氨酸的接头。
  6. 根据权利要求1-5中任一项所述的GLP-1类似物的融合多肽或其可药用盐，其中所述GLP-1(7-37)类似物在对应于天然GLP-1(7-37)的第7至第37位的任一氨基酸被第一半胱氨酸取代，所述GLP-1(7-37)类似物的C-末端延伸区段中包含第二半胱氨酸，并且所述第一半胱氨酸和所述第二半胱氨酸形成二硫键。
  7. 根据权利要求6所述的GLP-1类似物的融合多肽或其可药用盐，其中所述第一半胱氨酸和所述第二半胱氨酸形成分子内二硫键。
  8. 根据权利要求1至7中任一项所述的GLP-1类似物的融合多肽或其可药用盐，其中所述GLP-1(7-37)类似物基本上保留了天然GLP-1(7-37)的活性和/或功能。
  9. 根据权利要求1至8中任一项所述的GLP-1类似物的融合多肽或其可药用 盐，其中所述GLP-1(7-37)类似物与SEQ ID NO：1所示的天然GLP-1(7-37)相比，还包含至多10个、9个、8个、7个、6个、5个、4个、3个、2个或1个保守氨基酸修饰。
  10. 根据权利要求1至9中任一项所述的GLP-1类似物的融合多肽或其可药用盐，其中所述GLP-1(7-37)类似物的C-末端延伸区段为包含1-15个氨基酸的肽段。
  11. 根据权利要求10所述的GLP-1类似物的融合多肽或其可药用盐，其中所述GLP-1(7-37)类似物的C-末端延伸区段为至多15、14、13、12、11、10、9、8、7、6、5、4、3、2或1个氨基酸组成的肽段，其中一个氨基酸为半胱氨酸，其余为除半胱氨酸以外的任意氨基酸。
  12. 根据权利要求11所述的GLP-1类似物的融合多肽或其可药用盐，其中所述GLP-1(7-37)类似物的C-末端延伸区段的一个氨基酸为半胱氨酸，其余为甘氨酸或精氨酸。
  13. 根据权利要求12所述的GLP-1类似物的融合多肽或其可药用盐，其中所述GLP-1(7-37)类似物的C-末端延伸区段选自C、GCGR、或GCGGGGGG。
  14. 根据权利要求1至13中任一项所述的GLP-1类似物的融合多肽或其可药用盐，其中所述融合蛋白片段选自融合蛋白的β折叠片段。
  15. 根据权利要求1-14中任一项所述的GLP-1类似物的融合多肽或其可药用盐，其中所述融合蛋白片段如SEQ ID NO：2所示，或与其具有至少70％、75％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％的序列同一性。
  16. 根据权利要求6-15中任一项所述的GLP-1类似物的融合多肽或其可药用盐，其中所述第一半胱氨酸与所述第二半胱氨酸间隔至多为40个氨基酸长度，优选地，间隔35至3个氨基酸，更优选地，间隔30至5个氨基酸。
  17. 根据权利要求1-16中任一项所述的GLP-1类似物的融合多肽或其可药用盐，其中所述GLP-1类似物的融合多肽具有SEQ ID NO：3-5中任一项所示的氨基酸序列，所述氨基酸序列中的两个半胱氨酸形成分子内二硫键。
  18. 分离的核酸，其编码根据权利要求1-17中任一项所述的GLP-1类似物的 融合多肽。
  19. 一种载体，其包含权利要求18所述的分离的核酸。
  20. 一种宿主细胞，其包含权利要求19所述的载体。
  21. 一种制备GLP-1类似物的融合多肽的方法，其包括在适当的条件下培养权利要求20所述的宿主细胞，表达和回收权利要求1-17中任一项所述的GLP-1类似物的融合多肽的步骤。
  22. 一种药物组合物，其包含根据权利要求1-17中任一项所述的GLP-1类似物的融合多肽或其可药用盐，以及药学可接受的载体或赋形剂。
  23. 根据权利要求1-17中任一项所述的GLP-1类似物的融合多肽或其可药用盐，或根据权利要求22所述的药物组合物，在制备用于治疗非胰岛素依赖性糖尿病、胰岛素依赖性糖尿病或肥胖症的药物中的用途。
  24. 根据权利要求1-17中任一项所述的GLP-1类似物的融合多肽或其可药用盐，或根据权利要求22所述的药物组合物，用作药物。
  25. 一种治疗非胰岛素依赖性糖尿病、胰岛素依赖性糖尿病或肥胖症的方法，其包括向有此需要的受试者施用治疗有效量的根据权利要求1-17中任一项所述的GLP-1类似物的融合多肽或其可药用盐，或根据权利要求22所述的药物组合物。
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