WO2023124847A1 - 一种长效glp-1衍生物 - Google Patents

一种长效glp-1衍生物 Download PDF

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WO2023124847A1
WO2023124847A1 PCT/CN2022/137104 CN2022137104W WO2023124847A1 WO 2023124847 A1 WO2023124847 A1 WO 2023124847A1 CN 2022137104 W CN2022137104 W CN 2022137104W WO 2023124847 A1 WO2023124847 A1 WO 2023124847A1
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glp
derivative
long
ethoxy
pharmaceutically acceptable
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French (fr)
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曹海燕
林兆生
朱志伟
王建宇
辛瑞
曹丙洲
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北京惠之衡生物科技有限公司
吉林惠升生物制药有限公司
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    • 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
    • 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
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the disclosure relates to the field of polypeptide technology and its derivatives, in particular to a long-acting GLP-1(7-37) derivative and its preparation method, pharmaceutical composition and medical application.
  • Diabetes mellitus is a group of carbohydrate, protein, fat and other metabolic disorders caused by absolute or relative insulin secretion deficiency and/or insulin utilization disorder, with hyperglycemia as the main symbol, which can be caused by various factors such as genetics and environment. Diabetes is one of the three leading causes of death in humans, and its mortality rate is second only to cardiovascular and cerebrovascular diseases and cancer.
  • Type 2 diabetes diabetes is mainly divided into type 1 diabetes and type 2 diabetes, and most of the patients are type 2 diabetes (according to statistics, accounting for about 90%).
  • Type 2 diabetes diabetes (diabetes mellitus type 2, T2DM), formerly known as non-insulin-dependent diabetes mellitus (NIDDM) or adult-onset diabetes (adult-onset diabetes), patients are characterized by hyperglycemia, relative lack of insulin, insulin resistance etc.
  • NIDDM non-insulin-dependent diabetes mellitus
  • adult-onset diabetes adult-onset diabetes
  • clinically used drugs for treating type 2 diabetes mainly include biguanides, sulfonylureas, thiazolidinediones, DPP-4 receptor inhibitors, SGLT-2 receptor inhibitors and GLP-1 derivatives.
  • GLP-1 derivatives are gradually becoming the main therapeutic drugs and research hotspots for type 2 diabetes due to their hypoglycemic effect similar to insulin, but at the same time there is almost no risk of hypoglycemia, weight loss effect and cardiovascular protection function. .
  • Glucagon-like peptide 1 (GLP-1) is a secreted hormone secreted by intestinal L cells, which can promote insulin secretion, inhibit the release of glucagon, stimulate the proliferation of pancreatic ⁇ -cells, and induce the regeneration of pancreatic ⁇ -cells. , prevent islet ⁇ cell apoptosis, improve insulin sensitivity and increase glucose utilization. Therefore, GLP-1 and its analogs and derivatives play an important role in the treatment of the occurrence and development of type 1 and type 2 diabetes.
  • GLP-1 analogs and glucagon are the same, and they have multiple functions such as glucose-dependent insulin secretion and biosynthesis, inhibition of glucagon secretion and gastric emptying (Fu Gang, Gong et al. Min, Xu Weiren. Research progress of glucagon-like peptide 1 and its receptor agonists[J]. Tianjin Medicine, 2012, 40(2):181-184.).
  • the glucagon level decreased significantly, and the fasting blood sugar level became normal after 4 hours; after the normal blood sugar level, although the GLP-1 infusion was continued, the patient's insulin level did not rise again, and the blood sugar level remained stable , no further decline, which shows that GLP-1, as a gut-derived hormone, is released into the blood under the stimulation of nutrients (especially carbohydrates), and its insulin secretion-stimulating effect is glucose concentration-dependent.
  • GLP-1 as a gut-derived hormone
  • GLP-1 reduces body weight through multiple pathways, including inhibiting gastrointestinal motility and gastric juice secretion, inhibiting appetite and food intake, and delaying the emptying of gastric contents.
  • GLP-1 can also act on the central nervous system (especially the hypothalamus) to suppress appetite and reduce food intake, thereby causing the body to feel full and reduce appetite, reducing calorie intake, and then achieving the goal of weight loss.
  • GLP-1 derivatives mainly include exenatide, liraglutide, dulaglutide, lixisenatide, exenatide microsphere preparations, albiglutide, polyethylene glycol Senatide and semaglutide (also known as semaglutide).
  • semaglutide is a representative of GLP-1 derivative drugs.
  • Semaglutide is a long-acting GLP-1 derivative developed by Novo Nordisk. The drug only needs to be administered by subcutaneous injection once a week, and it has been approved for marketing in many countries. Moreover, Novo Nordisk has developed oral formulations of semaglutide through formulation technology. From the structural point of view, semaglutide is the 26th Lys on the GLP-1 (7-37) chain connected to the side chain of AEEA, glutamic acid and octadecane fatty acid, and the 8th amino acid is used The non-natural amino acid aminoisobutyric acid (Aib) is obtained by replacing the original Ala.
  • semaglutide Compared with liraglutide, semaglutide has a longer fatty chain and increased hydrophobicity, but semaglutide is modified with short-chain AEEA, and its hydrophilicity is greatly enhanced. After modification, AEEA can not only bind tightly with albumin, cover the hydrolysis site of DPP-4 enzyme, but also reduce renal excretion, prolong the biological half-life, and achieve the effect of long circulation. Semaglutide has been proven in multiple clinical trials that combining different oral hypoglycemic drugs can effectively control blood sugar, and can make patients lose weight, reduce systolic blood pressure and improve islet ⁇ -cell function.
  • the object of the present invention is to provide a GPL-1 derivative having excellent weight loss and hypoglycemic ability and a preparation thereof.
  • the technical problem to be solved by the present disclosure is the current problem of providing a GPL-1 derivative with excellent weight loss and hypoglycemic ability and its preparation.
  • the embodiments of the present invention provide a GLP-1(7-37) polypeptide analog and long-acting derivatives thereof.
  • the long-acting GPL-1(7-37) derivative provided by the present invention has excellent weight loss and hypoglycemic ability, and its weight loss effect can even reach nearly twice that of semaglutide, and its hypoglycemic effect is also significantly better than that of semaglutide. Maglutide.
  • the present invention provides a GLP-1(7-37) analogue
  • the GLP-1(7-37) analogue is composed of a polypeptide having an amino acid sequence represented by the following formula:
  • X 8 is selected from V, I, T, L, G or S
  • X 26 is R or K
  • X 34 is R or K
  • X 35 is G or R
  • X 36 is G or R, wherein X 26 and Only one of X 34 is K.
  • the X 8 is selected from V, I or T, X 26 is R, and X 34 is K.
  • the X 8 is selected from I or T, X 26 is R, and X 34 is K.
  • the X 8 is selected from V, I, T, L, G or S, X 26 is R, X 34 is K, X 35 is R, and X 36 is G; further, the X 8 is selected from from V, I or T; more closely, the X is selected from I or T.
  • the present invention provides a long-acting GLP-1(7-37) derivative, the derivative comprising fatty acid side chains respectively connected to the K residues of the GLP-1(3-37) analogue,
  • the attachment to the side chain is preferably via the epsilon amino group on the K residue.
  • the amino acid sequence of the GLP-1 (7-37) analog is as SEQ ID NO.1 is shown.
  • the amino acid sequence of the GLP-1 (7-37) analog is as SEQ Shown in ID NO.2.
  • the GLP-1 (7-37) analog is Ile 8 Glu 22 Arg 26 Lys 34 Arg 35 Gly 36 -GLP-1 (7-37), the GLP-1 (7-37) analog
  • the amino acid sequence is shown in SEQ ID NO.3.
  • the GLP-1 (7-37) analog is Thr 8 Glu 22 Arg 26 Lys 34 Arg 35 Gly 36 -GLP-1 (7-37), the GLP-1 (7-37) analog
  • the amino acid sequence is shown in SEQ ID NO.4.
  • the fatty acid side chain structure used in the long-acting GLP-1(7-37) derivatives of the present invention is HOOC(CH 2 ) n CO-, wherein n is selected from 10- An integer of 24, more preferably an integer of 16-20.
  • the fatty acid side chain can be selected from HOOC(CH 2 ) 14 CO-, HOOC(CH 2 ) 15 CO-, HOOC(CH 2 ) 16 CO-, HOOC(CH 2 ) 17 CO-, HOOC(CH 2 ) 18 CO-, HOOC(CH 2 ) 19 CO-, HOOC(CH 2 ) 20 CO-, HOOC(CH 2 ) 21 CO- or HOOC(CH 2 ) 22 CO-, preferably, the fatty acid side chain
  • the structure is HOOC(CH 2 ) 16 CO-.
  • the fatty acid side chain is connected to the residue through a linker.
  • the fatty acid side chain is connected to the epsilon amino group of Lys at position X 26 or X 34 on the GLP-1(3-37) analog through a linker.
  • the linker is selected from:
  • m is an integer of 0-6, such as 0, 1, 2, 3, 4, 5, 6, etc.
  • n is an integer of 1-3, such as 1, 2, 3, etc.
  • s is an integer of 0-3,
  • t is an integer of 0-4, such as 0, 1, 2, 3, 4, etc.
  • p is an integer of 1-23, such as 1, 2, 3, 4, 5, 6 , 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, etc.
  • the joint is:
  • the derivative of the present invention comprises a fatty acid side chain connected to the epsilon amino group of lysine at position 34 of the GLP-1 (7-37) analogue, and the fatty acid
  • the side chain is HOOC(CH 2 ) 16 CO-, and the fatty acid side chain is connected to the ⁇ amino group of the above-mentioned 34th lysine via - ⁇ -Glu-OEG-OEG-.
  • the long-acting GLP-1 derivatives of the present invention are selected from the following compounds:
  • the inventors found that by changing some positions, especially after modifying the 35th and 36th amino acids of the GLP-1 (7-37) analogue polypeptide to amino acids Arg and Gly respectively, and then at specific The GLP-1 derivative obtained after the site is acylated has more excellent hypoglycemic and weight-reducing activities.
  • the present invention provides a recombinant engineered bacterium that highly expresses the GLP-1 analog
  • the engineered bacterium is preferably a recombinant Escherichia coli engineered bacterium, more preferably a recombinant Escherichia coli BL21 engineered bacterium.
  • the present invention provides a method for constructing a recombinant engineering bacterium of the GLP-1 analogue, the method comprising the steps of: (1) encoding the inclusion body sequence, the EK restriction sequence and the GLP-1 analogue The gene sequence is sequentially fused in series to prepare a gene expression fragment of the GLP-1 analog; (2) inserting the gene expression fragment into a prokaryotic expression plasmid to obtain an expression plasmid of the GLP-1 analog; (3) inserting the expression plasmid Transform into Escherichia coli to prepare recombinant engineering bacteria expressing the GLP-1 analogue.
  • the prokaryotic expression plasmid is pET-30a(+), and the gene expression fragment is inserted into the above plasmid through NdeI and XhoI sites, or the prokaryotic expression plasmid is pET-28a(+), and the gene expression fragment is inserted into the plasmid through NcoI and XhoI Insert the gene expression fragment into the above plasmid.
  • amino acid sequence of the inclusion body promoting sequence of the present invention is FKFEFKFE
  • EK enzyme cutting sequence is DDDDK
  • nucleic acid sequence of the gene expression fragment of the present invention is selected from one of SEQ ID NO.5-8.
  • the in vitro binding activity shows that compared with semaglutide, the long-acting GLP-1 derivative provided by the present invention maintains a comparable binding affinity to GLP-1R.
  • hypoglycemic experiments in animal models of diabetes show that the long-acting GLP-1 derivatives of the present invention have significantly better hypoglycemic effects than semaglutide, especially the hypoglycemic effects of HS-G3 and HS-G4 are particularly prominent .
  • Another important function of GLP-1 derivatives is their weight loss effect, which can be developed as a weight loss indication drug, and semaglutide has been approved by the US FDA as a weight loss indication drug.
  • the research results in the obese animal model of the present invention show that, compared with semaglutide, the GLP-1 derivatives of the present invention (especially HS-G3 and HS-G4) have an average
  • the weight loss effect is nearly twice or more than twice, and there is no risk of hypoglycemia. Therefore, the long-acting GLP-1 derivative of the present invention has broader commercial development value.
  • the present invention provides a pharmaceutical composition, comprising the long-acting GLP-1 derivative or a pharmaceutically acceptable salt thereof, and pharmaceutically acceptable excipients.
  • the present invention provides the use of the long-acting GLP-1 derivative or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof in the preparation of a drug for treating diabetes.
  • the present invention provides the application of the long-acting GLP-1 derivative or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof in the preparation of a weight loss preparation.
  • the GLP-1 derivatives provided by the present invention have more excellent hypoglycemic ability in diabetic patients;
  • the long-acting GLP-1 derivative provided by the present invention has more excellent and significant weight loss ability for normal people and diabetic patients, and has better application potential for weight loss.
  • Figure 1 is a histogram of the effect of long-acting GLP-1 derivatives on blood sugar changes in db/db mice:
  • model control group semaglutide group
  • HS-G1 group HS-G2 group
  • HS-G3 group HS-G4 group
  • Fig. 2 is a histogram of the effect of long-acting GLP-1 derivatives on the rate of change in body weight of db/db mice;
  • model control group semaglutide group, HS-G1 group, HS-G2 group, HS-G3 group and HS-G4 group;
  • Fig. 3 is a histogram of the effect of long-acting GLP-1 derivatives on the rate of change in body weight of normal mice;
  • model control group semaglutide group
  • HS-G3 group HS-G4 group
  • This embodiment provides a variety of long-acting GLP-1 (7-37) derivatives and preparation methods thereof, especially to construct a recombinant engineering bacterium capable of efficiently expressing the derivatives of the present invention.
  • the preparation method is as follows (with HS-G1 as example):
  • the present invention finally selects FKFEFKFE as the inclusion body-promoting sequence, DDDDK as the EK restriction sequence, and sequentially fuses the inclusion body-promoting sequence, the EK restriction sequence and the GLP-1 analog coding gene sequence in series to obtain the following:
  • the gene fragment shown in SEQ ID NO.5; through the NcoI and XhoI sites, the above fragment was inserted into the prokaryotic expression plasmid pET-28a(+) and verified by sequencing to obtain an expression plasmid called pET-28a(+)-Ile 8 Glu22Arg26Lys34 - Glp-1 ( 7-37).
  • the expressed bacterial liquid was centrifuged at 8000g for 30 minutes to obtain bacterial cell slurry.
  • the bacterial cell yield was about 300g bacteria/L fermentation broth.
  • the target protein expression level was measured for the bacterial cells harvested by centrifugation, and the expression level was not less than 10g/L.
  • the cell slurry that takes 100g step (3) is resuspended in the solution (50mM Tris-HCl, 50mM NaCl, pH8.0) of 500mL, ultrasonic 30min with ultrasonic cell pulverizer, to make cell breakage, gained homogenate is in 4 Centrifuge at 13000g for 30min at °C, collect the precipitate after centrifugation, and dissolve it in 8M urea, which is the sample before enzyme digestion;
  • the sample before enzyme digestion was concentrated by UniPS30-300 (purchased from Suzhou Nawei Technology Co., Ltd.) equilibrated with equilibrium solution 3 (10mM ammonium acetate, 20% acetonitrile) in advance, after rinsing with equilibrium solution 3, press 0- 100% eluent (10mM ammonium acetate, 80% acetonitrile) gradient elution, analyzed by SDS-PAGE, the purity of the GLP-1 intermediate product generated by the above purification process is higher than 70%;
  • Fatty acid modification add water to the Ile 8 Glu 22 Arg 26 Lys 34 -GLP-1(7-37) obtained in step (4) to prepare a 4-6mg/mL solution, add 1M sodium hydroxide to adjust the pH to 11.0-11.5 , shake well to dissolve the protein completely, and quantify the concentration of the peptide by HPLC; weigh it according to the molar ratio of peptide to octadecanedioic acid mono-tert-butyl-glutamic acid (1-tert-butyl ester)-AEEA-AEEA-OSU- 1:4
  • the fatty acid powder was dissolved in acetonitrile, the polypeptide sample was mixed with the fatty acid solution, and the mixture was allowed to stand at 4°C for one hour, then the sample was diluted 5 times with water, and the pH was adjusted to 4.8 with 1M citric acid (or 10% acetic acid) to terminate the reaction. Put it at 4°C for acid precipitation for 10 minutes, after acid
  • Fatty acid deprotection and purification add TFA to the obtained precipitate to a final peptide concentration of about 10 mg/mL, shake to dissolve the precipitate, leave it at room temperature for 30 minutes for deprotection, then drop in 4M NaOH to adjust the pH to 7.5-8.5 to terminate the reaction;
  • the eluted peak was diluted 3 times with water, adjusted to pH 4.80 by acid precipitation, acid precipitation at 4°C for 30 min, centrifuged, and the precipitate was redissolved in PBST buffer with pH 7.0 and then frozen at -80°C to obtain N- ⁇ 34 -[2 -(2-[2-(2-[2-(2-[4-(17-Carboxyheptadecanylamino)-4(S)-carboxybutyrylamino]ethoxy)ethoxy]acetamido )ethoxy]ethoxy)acetyl][Ile 8 Glu 22 Arg 26 Lys 34 ]GLP-1(7-37) (HS-G1 for short).
  • the inclusion body promoting sequence, the EK restriction sequence and the GLP-1 analog coding gene sequence were sequentially fused in series to obtain the gene containing the coding Thr 8 Glu 22 Arg 26 Lys 34 -GLP-1( 7-37), Ile 8 Glu 22 Arg 26 Lys 34 Arg 35 Gly 36 -GLP-1 (7-37) and Thr 8 Glu 22 Arg 26 Lys 34 Arg 35 Gly 36 -GLP-1 (7-37) Fragments, the nucleotide sequences of which are shown in SEQ ID NO.6-8 respectively; wherein,
  • Thr 8 Glu 22 Arg 26 Lys 34 -GLP-1(7-37) was prepared in the same way as in steps (1)-(5) to obtain N- ⁇ 34 -[2-(2-[2-(2-[4-(17-Carboxyheptadecanoylamino)-4(S)-carboxybutyrylamino]ethoxy)ethoxy]acetamido)ethoxy]ethoxy)acetyl Base][Thr 8 Glu 22 Arg 26 Lys 34 ]-GLP-1(7-37) (referred to as HS-G2);
  • step (1)- (5) Basically the same method, but select the NdeI and XhoI sites, insert the above fragment into the prokaryotic expression plasmid pET-30a(+) and verify it by sequencing to obtain the expression plasmid (other steps are the same), and finally get: N- ⁇ 34 -[2-(2-[2-(2-[2-[2-[4-(17-carboxyheptadecylamino)-4(S)-carboxybutyrylamino]ethoxy)ethoxy Oxygen] acetamido) ethoxy] ethoxy) acetyl] [Ile 8 Glu 22 Arg 26 Lys 34 Arg 35 Gly 36 ] GLP-1 (7-37) (referred to as HS-G3)
  • the specific formula is: 1.133mg/mL Na 2 HPO 4 , 5.5mg/mL phenol, 14.0mg/mL propylene glycol, GLP-1 derivatives: 1200ng/mL, 300ng/mL, 75ng/mL, 18.75ng/mL, 4.6875ng /mL, 1.172ng/mL, 0.293ng/mL, 0.073ng/mL.
  • cAMP detection kit R&D
  • prepare the assay culture medium dilute the sample to 1200ng/mL step by step with the assay culture medium, and the single dilution factor is not more than 10 times;
  • a 4-fold serial dilution was performed, with a total of 8 gradients, and 2 replicate wells were made for each dilution.
  • Washing Discard the liquid, shake dry, add 300 ⁇ L of washing buffer to each well, put it into a microplate shaker for 1 min and then discard, repeat this 4 times, and pat dry on the filter paper.
  • Add sample and secondary antibody first add 50 ⁇ L secondary antibody to each well, transfer 100 ⁇ L cell lysate from each well of the cell plate to the well of the microtiter plate, seal the plate with a sealing film, mix well and put it into a microwell plate Shaker, incubate at room temperature for 2 hours.
  • Washing Discard the liquid, shake dry, add 300 ⁇ L of washing buffer to each well, put it into a microplate shaker for 1 min and then discard, repeat this 4 times, and pat dry on the filter paper.
  • Color development mix equal amounts of color development solution A and color development solution B, add 200 ⁇ L of the mixed solution to each well, put it into a microplate shaker, and develop color in the dark for 30 minutes.
  • Termination Add 100 ⁇ L of stop solution to each well to terminate the reaction.
  • the experimental animals are BKS-LeprREM/Gpt mice, 30 in number, 6 weeks old, male;
  • Experimental drug formulation 1.133 mg/mL Na 2 HPO 4 , 5.5 mg/mL phenol, 14.0 mg/mL propylene glycol, 0.045 mg/mL GLP-1 derivative.
  • the dosing frequency was once every 48 hours, four times, intraperitoneal subcutaneous injection.
  • Blood glucose level measure the blood glucose level before administration and 2h after administration at the first administration, and then measure the blood glucose value at 0h before administration and 2h after administration again in the following three administrations;
  • the 0h before re-administration is 48h described in Table 4 and DAY3, DAY5, DAY7 and DAY9 in Figure 1, which refers to 48h after the last administration and also 0h before re-administration.
  • each group of data from left to right is the model control group, semaglutide, HS-G1, HS-G2, HS-G3 and HS-G4;
  • the strain is BKS-LeprREM/Gpt mice, the number is 30, 6 weeks old, male;
  • Administration was performed in the same manner as shown in Table 3 in Embodiment 3, and the administration frequency was once every 48 hours, four times, and administered by intraperitoneal subcutaneous injection.
  • mice were weighed before each administration and 48 hours after the last administration;
  • Fig. 2 is a figure of the body weight change rate of the mice administered for 9 days;
  • GLP-1 derivative injection (HS-G3 and HS-G4), the specific formula is 1.133mg/mLNa 2 HPO 4 , 5.5mg/mL phenol, 14.0mg/mL propylene glycol, and GLP-1 derivative 0.045mg/ mL.
  • ND normal diet
  • HFD high-fat diet
  • mice The body weight of the HFD group was detected, the unmodeled mice were screened out, and the remaining mice were randomly grouped according to body weight, and divided into blank control group (used to judge whether the DIO model was successful, not participating in the drug test), positive control group (semaglutide ), experimental group 1, experimental group 2, model control group (blank solvent);
  • Body weight body weight is detected each time the drug is administered
  • test results of this embodiment are shown in Table 8, and the body weight change rate for 9 days after administration is shown in Figure 3;
  • Fig. 3 is the figure of the body weight change rate of normal mice administered for 9 days, as can be seen from Fig. 3 and table 8 results, for normal mice, the long-acting GLP-1 derivatives HS-G3 and HS-G4 provided by the present invention have peso Marutide's more excellent weight loss ability has achieved nearly 2 times or more than 2 times the weight loss effect of the former; at the same time, the long-acting GLP-1 derivatives of the present invention will not cause excessively low blood sugar and no hypoglycemia risk. Therefore, the long-acting GLP-1 derivatives of the present invention are more suitable for the development of indications for weight loss.
  • Embodiment 6 pharmacokinetic research
  • mice SD rats, 50 male mice with a body weight between 180-200g, 6-8 weeks old.
  • the long-acting GLP-1 derivatives HS-G3 and HS-G4 provided by the present invention have a better half-life than semaglutide in rats, and both of them have a higher half-life than semaglutide at different doses.
  • Marutide has a longer half-life about 1.5 times longer.
  • the GLP-1 derivatives disclosed in the present disclosure have good binding affinity to the GLP-1 receptor (GLP-1R), and compared with other marketed GLP-1 derivatives, the long-acting GLP-1 derivatives of the present invention
  • GLP-1R GLP-1 receptor
  • the compound has more obvious and excellent hypoglycemic ability and weight reducing ability, and has strong industrial applicability.

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Abstract

一种GLP‑1多肽类似物、多肽类似物的经脂肪酸酰化修饰的长效GLP‑1衍生物。所述长效GLP‑1衍生物具有与GLP‑1受体(GLP‑1R)的良好的结合亲和力，且与已上市的其他GLP‑1衍生物相比，具有更为明显和优异的降糖能力和减重能力。

Description

一种长效GLP-1衍生物
本公开要求于2021年12月28日提交中国专利局、申请号为202111629476.8、发明名称为“一种长效GLP-1衍生物”的中国专利申请的优先权，其全部内容通过引用结合在本公开中。
技术领域
本公开涉及多肽技术及其衍生物领域，尤其涉及一种长效GLP-1(7-37)衍生物及其制备方法、药物组合物和医药用途。
背景技术
糖尿病是一组因胰岛素绝对或相对分泌不足和/或胰岛素利用障碍引起的碳水化合物、蛋白质、脂肪等代谢紊乱性疾病，以高血糖为主要标志，可由遗传和环境等多种因素引起。糖尿病是人类三大致死疾病之一，它的死亡率仅次于心脑血管疾病和癌症。
糖尿病主要分为1型糖尿病和2型糖尿病，其中大多数患者为2型糖尿病患者(据统计，约占90％)。2型糖尿病(diabetes mellitus type 2，T2DM)，旧称非胰岛素依赖型糖尿病(noninsulin-dependent diabetes mellitus，NIDDM)或成人发病型糖尿病(adult-onset diabetes)，患者特征为高血糖、相对缺乏胰岛素、胰岛素抵抗等。目前，临床上使用的治疗2型糖尿病的药物主要有双胍类、磺酰脲类、噻唑烷二酮类、DPP-4受体抑制剂、SGLT-2受体抑制剂和GLP-1衍生物。而其中，GLP-1衍生物由于具有与胰岛素类似的降糖效果，但同时几乎无低血糖风险、兼具减重效果和心血管保护功能，正逐渐成为2型糖尿病的主要治疗药物和研究热点。
胰高血糖素样肽1(GLP-1)是一种由肠道L细胞分泌的促胰素，具有促进胰岛素分泌、抑制胰高血糖素的释放、刺激胰岛β细胞增殖、诱导胰岛β细胞再生、阻止胰岛β细胞凋亡、改善胰岛素敏感性和增加葡萄糖的利用等作用。因此，GLP-1及其类似物和衍生物在治疗1和2型糖尿病的发生、发展中起着重要作用。
GLP-1类似物和胰高血糖素的氨基酸序列有近一半相同，具有葡萄糖依赖性的促胰岛素分泌和生物合成、抑制胰高血糖素分泌及抑制胃排空等多种功能(付刚，龚珉，徐为人.胰高血糖素样肽1及其受体激动剂研究进展[J].天津医药，2012，40(2):181-184.)。
在文献Normalization of fasting hyperglycaemia by exogenous glucagon-like peptide 1(7-36amide)in type 2(non-insulin-dependent)diabetic patients([J].Diabetologia,Nauck M.，1993,36)中，作者对10例血糖控制不佳的2型糖尿病患者进行了研究，并在空腹状态下分别给予患者GLP-1或安慰剂，结果显示，患者在输注GLP-1后，其胰岛素和C肽水平显著增加，胰高血糖素水平显著降低，空腹血糖水平在4小时后变为正常；在血糖水平正常后，虽然仍持续输注GLP-1，患者的胰岛素水平却不会再升高，血糖水平也维持稳定，不再进一步下降，这说明GLP-1作为一种肠源性激素，其是在营养物质(特别是碳水化合物)的刺激下才释放入血的，其促胰岛素分泌作用呈葡萄糖浓度依赖性，能够在血糖升高时发挥降糖作用，抑制胰高糖素分泌，增加饱腹感，减少饥饿感，从而达到降低血糖作用。而在文献Effect of 6-week course of glucagon-like peptide 1 on glycaemic control,insulin sensitivity,and β-cell function in type 2 diabetes:a parallel-group study.([J].Lancet,Zander,Mette,Madsbad,et al.，2002)中，研究者的研究表明GLP-1通过多种途径产生降低体重的作用，包括抑制胃肠道蠕动和胃液分泌，抑制食欲及摄食，延缓胃内容物排空。此外，GLP-1还可作用于中枢神经***(特别是下丘脑)抑制食欲，减少进食量，从而使人体产生饱胀感和食欲下降，减少卡路里的摄入量，进而达到减轻体重的目的。
目前，已上市的GLP-1衍生物主要有艾塞那肽、利拉鲁肽、度拉糖肽、利司那肽、艾塞那肽微球制剂、阿必鲁肽、聚乙二醇洛塞那肽和索马鲁肽(又名司美鲁肽)。其中，索马鲁肽为GLP-1衍生物药物中的代表。
索马鲁肽(Semaglutide)是由诺和诺德公司研发的长效GLP-1衍生 物，该药物只需要进行每周一次的皮下注射给药，目前已在多国获批上市。而且，诺和诺德通过制剂技术，开发了索马鲁肽的口服制剂。从结构上看，索马鲁肽是将GLP-1(7-37)链上第26位Lys接上AEEA、谷氨酸和十八烷脂肪二酸侧链，并将其中第8位氨基酸采用非天然氨基酸氨基异丁酸(Aib)取代原Ala所得。与利拉鲁肽相比，索马鲁肽的脂肪链更长，疏水性增加，但是索马鲁肽经过短链的AEEA修饰，亲水性大大增强。AEEA修饰后不但可以与白蛋白紧密结合，掩盖DPP-4酶水解位点，还能降低肾***，延长生物半衰期，达到长循环的效果。索马鲁肽在多个临床试验研究已经证明联合不同的口服降糖药可以有效控制血糖，并能够使患者减轻体重、减少收缩压及改善胰岛β细胞功能。
因此，本发明的目的是提供一种具有优异的减重和降糖能力的GPL-1衍生物及其制剂。
发明内容
本公开要解决的技术问题是目前需要提供一种具有优异的减重和降糖能力的GPL-1衍生物及其制剂的问题。
为了解决上述技术问题，本发明实施例提供了一种GLP-1(7-37)多肽类似物及其长效衍生物。本发明提供的长效GPL-1(7-37)衍生物具有优异的减重和降糖能力，其减重效果甚至可以达到索马鲁肽的近两倍，降糖效果也明显优于索马鲁肽。
第一方面，本发明提供了一种GLP-1(7-37)类似物，所述GLP-1(7-37)类似物由下式所示的氨基酸序列的多肽组成：
HX 8EGTFTSDVSSYLEEQAAX 26EFIAWLVX 34X 35X 36G；
其中，X 8选自V、I、T、L、G或S，X 26为R或K，X 34为R或K，X 35为G或R，X 36为G或R，其中X 26和X 34中仅有一个为K。
优选地，所述X 8选自V、I或T，X 26为R，X 34为K。
优选地，所述X 8选自I或T，X 26为R，X 34为K。
更优选地，所述X 8选自V、I、T、L、G或S，X 26为R，X 34为K，X 35为R，X 36为G；进一步的，所述X 8选自V、I或T；更近一步地，所述 X 8选自I或T。
第二方面，本发明提供一种长效GLP-1(7-37)衍生物，所述衍生物包含分别与所述GLP-1(3-37)类似物K残基连接的脂肪酸侧链，优选通过K残基上的ε氨基与侧链连接。
当所述GLP-1(7-37)类似物为Ile 8Glu 22Arg 26Lys 34-GLP-1(7-37)时，所述GLP-1(7-37)类似物的氨基酸序列如SEQ ID NO.1所示。
当所述GLP-1(7-37)类似物为Thr 8Glu 22Arg 26Lys 34-GLP-1(7-37)时，所述GLP-1(7-37)类似物的氨基酸序列如SEQ ID NO.2所示。
当所述GLP-1(7-37)类似物为Ile 8Glu 22Arg 26Lys 34Arg 35Gly 36-GLP-1(7-37)时，所述GLP-1(7-37)类似物的氨基酸序列如SEQ ID NO.3所示。
当所述GLP-1(7-37)类似物为Thr 8Glu 22Arg 26Lys 34Arg 35Gly 36-GLP-1(7-37)时，所述GLP-1(7-37)类似物的氨基酸序列如SEQ ID NO.4所示。
作为本发明的一种优选技术方案，本发明所述长效GLP-1(7-37)衍生物中使用的脂肪酸侧链结构为HOOC(CH 2) nCO-，其中n为选自10-24的整数，更优选为16-20的整数。具体地，所述脂肪酸侧链可以选自HOOC(CH 2) 14CO-、HOOC(CH 2) 15CO-、HOOC(CH 2) 16CO-、HOOC(CH 2) 17CO-、HOOC(CH 2) 18CO-、HOOC(CH 2) 19CO-、HOOC(CH 2) 20CO-、HOOC(CH 2) 21CO-或HOOC(CH 2) 22CO-，优选地，所述脂肪酸侧链结构为HOOC(CH 2) 16CO-。
作为本发明的一种优选技术方案，所述脂肪酸侧链通过接头与残基连接。
作为本发明的一种优选技术方案，所述脂肪酸侧链通过接头与所述GLP-1(3-37)类似物上的位置X 26或X 34的Lys的ε氨基连接。
作为本发明的一种优选技术方案，所述接头选自：
Figure PCTCN2022137104-appb-000001
Figure PCTCN2022137104-appb-000002
其中，m为0-6的整数，例如0、1、2、3、4、5、6等，n为1-3的整数，例如1、2、3等，s为0-3的整数，例如0、1、2、3等，t为0-4的整数，例如0、1、2、3、4等，p为1-23的整数，例如1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23等。
作为本发明的一种具体实施方式，所述接头为：
Figure PCTCN2022137104-appb-000003
对于上述优选的接头部分(当n为1时)，按照IUPAC命名法，可表示为γ-Glu-OEG-OEG，即“2-[2-(2-氨基乙氧基)乙氧基]乙酰基”的简写。当选择HOOC(CH 2) 16CO-作为侧链时，上述侧链和接头的组合(酰基基团)，按照IUPAC命名法，可以被称为“[2-(2-[2-(2-[2-(2-)4-(17-羧基十七烷酰胺基)-4(S)-羧基丁酰基氨基]乙氧基)乙氧基]乙酰氨基)乙氧基]乙氧基)乙酰基]”。
作为本发明的一种具体实施方式，本发明所述衍生物包含与所述GLP-1(7-37)类似物的第34位的赖氨酸的ε氨基连接的脂肪酸侧链，所述脂肪酸侧链为HOOC(CH 2) 16CO-，所述脂肪酸侧链通过-γ-Glu-OEG-OEG-与上述第34位的赖氨酸的ε氨基连接。
因此，优选地，本发明所述长效GLP-1衍生物选自如下化合物：
N-ε 34-[2-(2-[2-(2-[2-(2-[4-(17-羧基十七烷酰胺基)-4(S)-羧基丁酰基氨基]乙氧基)乙氧基]乙酰氨基)乙氧基]乙氧基)乙酰基][Ile 8Glu 22Arg 26Lys 34]GLP-1(7-37)(简称为HS-G1)；或
N-ε 34-[2-(2-[2-(2-[2-(2-[4-(17-羧基十七烷酰胺基)-4(S)-羧基丁酰基氨基]乙氧基)乙氧基]乙酰氨基)乙氧基]乙氧基)乙酰基][Thr 8Glu 22Arg 26Lys 34]-GLP-1(7-37)(简称为HS-G2)；或
N-ε 34-[2-(2-[2-(2-[2-(2-[4-(17-羧基十七烷酰胺基)-4(S)-羧基丁酰基氨基]乙氧基)乙氧基]乙酰氨基)乙氧基]乙氧基)乙酰基][Ile 8Glu 22Arg 26Lys 34Arg 35Gly 36]GLP-1(7-37)(简称为HS-G3)；或
N-ε 34-[2-(2-[2-(2-[2-(2-[4-(17-羧基十七烷酰胺基)-4(S)-羧基丁酰基氨基]乙氧基)乙氧基]乙酰氨基)乙氧基]乙氧基)乙酰基][Thr 8Glu 22Arg 26Lys 34Arg 35Gly 36]GLP-1(7-37)(简称为HS-G4)。
在本发明中，发明人发现通过部分位点的变化，尤其是将GLP-1(7-37)类似物多肽第35位和第36位的氨基酸分别修饰为氨基酸Arg和Gly后，再在特定位点经过酰化后得到的GLP-1衍生物，具有更为优异的降糖和减重活性。
第三方面，本发明提供一种高表达所述GLP-1类似物的重组工程菌，所述工程菌优选为重组大肠杆菌工程菌，更优选为重组大肠杆菌BL21工程菌。
第四方面，本发明提供一种所述GLP-1类似物的重组工程菌的构建方法，所述方法包括步骤：(1)将促包涵体序列、EK酶切序列和GLP-1类似物编码基因序列依次串联融合，制备得到GLP-1类似物的基因表达片段；(2)将所述基因表达片段***原核表达质粒，得GLP-1类似物的表达质粒；(3)将所述表达质粒转入大肠杆菌，制备得到表达所述GLP-1类似物的重组工程菌。
优选地，所述原核表达质粒为pET-30a(+)，并通过NdeI和XhoI位点将基因表达片段***上述质粒，或所述原核表达质粒为pET-28a(+)，并通过NcoI和XhoI位点将基因表达片段***上述质粒。
优选地，本发明的促包涵体序列的氨基酸序列为FKFEFKFE，所述EK酶切序列为DDDDK。
更优选地，本发明所述基因表达片段的核酸序列选自SEQ ID NO.5-8之一。
体外结合活性表明，本发明提供的长效GLP-1衍生物相比于索马鲁肽，保持了与之相当的GLP-1R结合亲和力。但在糖尿病动物模型中的降糖实验表明，本发明的长效GLP-1衍生物具有显著优于索马鲁肽的降糖效果，尤其是HS-G3和HS-G4的降糖效果尤为突出。GLP-1衍生物的另外一个重要作用即是其减重效果，可开发为减重适应症药物，而且索马鲁肽作为减重适应症药物已经获得美国FDA批准。本发明肥胖动物模型中的研究结果表明，相比于索马鲁肽，本发明的GLP-1衍生物(尤其是HS-G3和HS-G4)在正常动物模型和糖尿病动物模型中，平均具有近两倍或两倍以上的减重效果，且不存在低血糖风险，因此，本发明的长效GLP-1衍生物具有更为广阔的商业开发价值。
第五方面，本发明提供了一种药物组合物，包括所述的长效GLP-1衍生物或其药学上可接受的盐，以及药学上可接受的辅料。
第六方面，本发明提供了所述的长效GLP-1衍生物或其药学上可接受的盐，或其药物组合物在制备治疗糖尿病的药物中的应用。
第七方面，本发明提供了所述的长效GLP-1衍生物或其药学上可接受的盐，或其药物组合物在制备减重制剂中的应用。
本公开实施例提供的上述技术方案与现有技术相比具有如下优点：
(1)本发明提供的GLP-1衍生物，在糖尿病患者中具有更优异的降糖能力；
(2)相比于索马鲁肽，本发明提供的长效GLP-1衍生物，对正常人群和糖尿病患者均具有更加优异且显著的减重能力，具备更优的减重应用潜力。
应当理解的是，以上的一般描述和后文的细节描述仅是示例性和解释性的，并不能限制本公开。
附图说明
此处的附图被并入说明书中并构成本说明书的一部分，示出了符合本公开的实施例，并与说明书一起用于解释本公开的原理。
为了更清楚地说明本公开实施例或现有技术中的技术方案，下面 将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，对于本领域普通技术人员而言，在不付出创造性劳动性的前提下，还可以根据这些附图获得其他的附图。
图1为长效GLP-1衍生物体对db/db小鼠血糖变化影响柱状图：
在图中，对于每组数据，从左至右依次为模型对照组、索马鲁肽组、HS-G1组、HS-G2组、HS-G3组和HS-G4组；
图2为长效GLP-1衍生物对db/db小鼠体重变化率影响的柱状图；
在图中，从左至右依次为模型对照组、索马鲁肽组、HS-G1组、HS-G2组、HS-G3组和HS-G4组；
图3为长效GLP-1衍生物对正常小鼠体重变化率影响的柱状图；
在图中，从左至右依次为模型对照组、索马鲁肽组、HS-G3组和HS-G4组。
具体实施方式
为使本公开实施例的目的、技术方案和优点更加清楚，下面将对本公开实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例是本公开的一部分实施例，而不是全部的实施例。基于本公开中的实施例，本领域普通技术人员在没有做出创造性劳动的前提下所获得的所有其他实施例，都属于本公开保护的范围。
实施例1长效GLP-1衍生物的制备
本实施例提供了多种长效GLP-1(7-37)衍生物及其制备方法，尤其是构建一种能高效表达本发明衍生物的重组工程菌，制备方法如下(以HS-G1为例)：
(1)构建编码Ile 8Glu 22Arg 26Lys 34-GLP-1(7-37)的表达质粒
通过大量研究和实验，本发明最终选择FKFEFKFE作为促包涵体序列，DDDDK作为EK酶切序列，并将促包涵体序列、EK酶切序列和GLP-1类似物编码基因序列依次串联融合，得到如SEQ ID NO.5所示基因片段；通过NcoI和XhoI位点，将上述片段***原核表达质粒pET-28a(+)中并测序验证，得到表达质粒，称作pET-28a(+)-Ile 8Glu 22Arg 26Lys 34-Glp-1(7-37)。
(2)构建表达Ile 8Glu 22Arg 26Lys 34-GLP-1(7-37)的重组工程菌
将BL21感受态细胞(TransGenBiotech)置于冰浴上融化(50μL)，加入步骤(1)构建的表达质粒，轻轻摇匀，并在冰浴中放置30min。继而42℃水浴热激30s，然后快速将离心管转移到冰浴中放置2min，该过程不要摇动离心管；
向离心管中加入500μL无菌的LB培养基(不含抗生素)，混匀后置于37℃，180rpm培养1h，使细菌复苏，然后吸取200μL已转化的感受态细胞加到含有卡那霉素抗性的LB琼脂培养基平板上，将细胞均匀涂开，将平板置于37℃至液体被吸收，倒置平板，37℃过夜培养，次日，使用接种环挑取转化平皿中的单克隆菌落，并接种于15mL的无菌LB培养基(含抗生素)，30℃过夜培养。将500μL过夜培养菌液，加入到无菌1.5ml离心管中，再加入500μL的50％无菌甘油混匀，得到甘油冻存菌，-80℃保存。
(3)重组工程菌的发酵表达
向50mL的2YT培养基中加入50μL甘油冻存菌液，同时加入50μL卡那霉素，混匀后放恒温振荡器中，37℃、200rpm过夜培养，得到一级种子培养液，测OD600＞5.0；
取过夜培养一级种子培养液40mL按照1:5的比例接入到200mL 2YT培养基中，同时加入200μL卡那霉素，混匀后放恒温振荡器中，37℃200rpm培养3h，OD600＞3.0，得二级种子培养液；
取二级种子液60mL，按1:10的比例接种于FDM培养基中(600mL)，在2L发酵罐内进行培养，检测培养菌液OD600值达到160左右的时候，开始接入IPTG(异丙基-β-D-硫代半乳糖苷)，终浓度为1mmol/L，30℃诱导培养24h结束培养，放罐离心；
表达的菌液以8000g离心30min获得菌体细胞浆，菌体收率约为300g菌/L发酵液，离心收获的菌体进行目的蛋白表达量测定，表达量均不低于10g/L。
(4)重组Ile 8Glu 22Arg 26Lys 34-GLP-1(7-37)的纯化
称取100g步骤(3)得到的细胞浆重悬于500mL的溶液(50mM Tris-HCl、50mM NaCl、pH8.0)中，用超声波细胞粉碎机超声30min，以使细胞破碎，所得匀浆在4℃下以13000g离心30min，离心完成后 收集沉淀，使用8M尿素溶解后即为酶切前样品；
将酶切前样品经事先用平衡液3(10mM乙酸铵，20％乙腈)平衡过的UniPS30-300(购自苏州纳微科技有限公司)进行浓缩，平衡液3淋洗后，再按0-100％洗脱液(10mM乙酸铵，80％乙腈)梯度洗脱，经SDS-PAGE分析，由上述纯化过程生成GLP-1中间产物纯度高于70％；
使用EK酶将标签序列切除：中间产物中加入pH＝7.4的20mM PB缓冲液使其稀释三倍，20℃条件下按照EK酶:中间产物＝1:15加入EK酶混匀后酶切过夜，经SDS-PAGE分析酶切率近80％；
Ile 8Glu 22Arg 26Lys 34-GLP-1(7-37)的精纯：利用平衡液3(10mM乙酸铵，20％乙腈)平衡过的UniPS30-300(购自苏州纳微科技有限公司)进行浓缩，平衡液3淋洗后，再按0-100％洗脱液(10mM乙酸铵，80％乙腈)梯度洗脱，经SDS-PAGE纯度约为90％；
洗脱样品中加入0.2M Na 2HPO 4使其终浓度为20mM，用1M柠檬酸调节pH至4.8-5.0，4℃酸沉过夜，SDS-PAGE检测收率90％以上，4℃下以13000g离心30min，收取沉淀放于-20℃保存，得到Ile 8Glu 22Arg 26Lys5 34-GLP-1(7-37)。
(5)制备长效GLP-1衍生物
脂肪酸修饰：步骤(4)得到的Ile 8Glu 22Arg 26Lys 34-GLP-1(7-37)中加水，配制成4～6mg/mL溶解液，加入1M氢氧化钠调整pH至11.0-11.5，摇匀使蛋白完全溶解，HPLC定量多肽浓度；按多肽与十八烷二酸单叔丁酯-谷氨酸(1-叔丁酯)-AEEA-AEEA-OSU-摩尔比1:4称取脂肪酸粉末溶于乙腈中，将多肽样品与脂肪酸溶液混合，将混合液于4℃静置一小时，然后样品加水稀释5倍，用1M柠檬酸(或10％乙酸)调pH至4.8终止反应，放于4℃静置酸沉10min，酸沉后以13000g，4℃离心30min，然后将沉淀放于-80℃保存；
脂肪酸脱保护与纯化：向得到的沉淀中加入TFA至多肽终浓度约10mg/mL，震荡使沉淀溶解，置于室温静置脱保护30min，然后滴入4M NaOH调节pH至7.5-8.5终止反应；
用蛋白纯化层析***(赛谱SDL100)将反应终止后的反应液按4mL/min流速，泵入事先用平衡液3(10mM乙酸铵，20％乙腈)平衡过的 UniPS10-300(购自苏州纳微科技有限公司)进行浓缩，平衡液3淋洗后，再按0-100％洗脱液(10mM乙酸铵，80％乙腈)梯度洗脱，收集洗脱峰经RP-HPLC检测纯度约为90％；
洗脱峰用水稀释3倍，酸沉调整pH至4.80，4℃酸沉30min，离心后沉淀中加入pH7.0的PBST缓冲液复溶后-80℃冻存，得到N-ε 34-[2-(2-[2-(2-[2-(2-[4-(17-羧基十七烷酰胺基)-4(S)-羧基丁酰基氨基]乙氧基)乙氧基]乙酰氨基)乙氧基]乙氧基)乙酰基][Ile 8Glu 22Arg 26Lys 34]GLP-1(7-37)(简称HS-G1)。
按照上述实施例中的步骤(1)，将促包涵体序列、EK酶切序列和GLP-1类似物编码基因序列依次串联融合，获得包含编码Thr 8Glu 22Arg 26Lys 34-GLP-1(7-37)、Ile 8Glu 22Arg 26Lys 34Arg 35Gly 36-GLP-1(7-37)和Thr 8Glu 22Arg 26Lys 34Arg 35Gly 36-GLP-1(7-37)的基因片段，其核苷酸序列分别如SEQ ID NO.6-8所示；其中，
Thr 8Glu 22Arg 26Lys 34-GLP-1(7-37)按照步骤(1)-(5)完全相同的方法，制备得到N-ε 34-[2-(2-[2-(2-[2-(2-[4-(17-羧基十七烷酰胺基)-4(S)-羧基丁酰基氨基]乙氧基)乙氧基]乙酰氨基)乙氧基]乙氧基)乙酰基][Thr 8Glu 22Arg 26Lys 34]-GLP-1(7-37)(简称HS-G2)；
Ile 8Glu 22Arg 26Lys 34Arg 35Gly 36-GLP-1(7-37)和Thr 8Glu 22Arg 26Lys 34Arg 35Gly 36-GLP-1(7-37)，按照步骤(1)-(5)基本相同的方法，但是选择通过NdeI和XhoI位点，将上述片段***原核表达质粒pET-30a(+)中并测序验证，得到表达质粒(其他步骤相同)，最后分别得到：N-ε 34-[2-(2-[2-(2-[2-(2-[4-(17-羧基十七烷酰胺基)-4(S)-羧基丁酰基氨基]乙氧基)乙氧基]乙酰氨基)乙氧基]乙氧基)乙酰基][Ile 8Glu 22Arg 26Lys 34Arg 35Gly 36]GLP-1(7-37)(简称HS-G3)，和，N-ε 34-[2-(2-[2-(2-[2-(2-[4-(17-羧基十七烷酰胺基)-4(S)-羧基丁酰基氨基]乙氧基)乙氧基]乙酰氨基)乙氧基]乙氧基)乙酰基][Thr 8Glu 22Arg 26Lys 34Arg 35Gly 36]GLP-1(7-37)(简称HS-G4)。
在实施例中，步骤(1)-(4)中构建的重组工程菌的表达情况如表1所示：
表1
Figure PCTCN2022137104-appb-000004
实施例2：体外细胞亲和力活性检测
(1)配制不同长效GLP-1衍生物注射液
具体配方为：1.133mg/mL Na 2HPO 4，5.5mg/mL苯酚，14.0mg/mL丙二醇，GLP-1衍生物：1200ng/mL、300ng/mL、75ng/mL、18.75ng/mL、4.6875ng/mL、1.172ng/mL、0.293ng/mL、0.073ng/mL。
选取培养状态良好的RIN-m5F细胞，弃去瓶中培养液，用Versene液(乙二胺四乙酸溶液)摇洗1次，加入0.05％TRYPSIN消化液消化，然后加入RPMI1640基础培养液终止消化，离心收集细胞，用RPMI1640基础培养液调整细胞密度为6.7×10 5个/mL，100μL/孔接种于96孔细胞培养板中，于37℃、5％CO 2条件下培养过夜。
用cAMP检测试剂盒(R&D)检测GLP-1类似物的衍生物的体外活性：配制测定培养液，用测定培养液分步稀释样品至1200ng/mL，单次稀释倍数不超过10倍；之后在96孔板中按照进行4倍系列稀释，共8个梯度，每个稀释度做2个复孔。
从培养箱中取出培养好的细胞培养板，弃掉旧培养液，在滤纸上吸干；将稀释好的溶液加入细胞板中，每孔100μL，置于37℃、5％CO 2条件下药物作用3.5min，倒掉细胞培养板上样品溶液，滤纸上吸干，将稀释好的细胞裂解液(1×Lysis Buffer)加到细胞培养板中，每孔加入120μL，-80℃冷冻-室温震荡融化。
(2)cAMP含量检测：
在96孔板架上排好所需板条，孵育一抗：每孔加入50μL一抗溶液，用封板膜封板后，放入微孔板振荡器室温孵育1小时。
洗涤：弃去液体，甩干，每孔加300μL洗涤缓冲液，放入微孔板振荡器震荡1min后弃去，如此重复4次，滤纸上拍干。
加样和二抗：在每孔先加入50μL二抗，从细胞板中每孔取100μL细胞裂解液转移到酶标板的孔中，用封板膜封板，混匀后放入微孔板振荡器，室温孵育2小时。
洗涤：弃去液体，甩干，每孔加300μL洗涤缓冲液，放入微孔板振荡器震荡1min后弃去，如此重复4次，滤纸上拍干。
显色：将显色液A和显色液B等量混匀，各孔加入200μL混合液，放入微孔板振荡器，避光显色30分钟。
终止：每孔加100μL终止液，终止反应。
测定：放入酶标仪，以570nm作为参比波长，450nm作为检测波长，检测各孔的OD值；30min之内完成检测，并绘制cAMP分析曲线图计算EC50值；试验数据采用四参数回归计算法进行处理，并计算出待测样品的EC50值，结果如表2所示：
表2
样品 HS-G1 HS-G2 HS-G3 HS-G4 索马鲁肽
EC50(nM) 4.814 9.269 3.842 2.463 1.892
由表中结果可知，本发明HS-G3和HS-G4相比于HS-G1和HS-G2具有更低的EC50值，其数值更接近索马鲁肽的EC50值，表明其具备与索马鲁肽相当的与受体的结合亲和力。
实施例3：db/db小鼠中降糖效果的研究
(1)实验材料：
实验动物为BKS-LeprREM/Gpt小鼠，数量30只，周龄为6周龄，雄性；
实验药物制剂：1.133mg/mL Na 2HPO 4，5.5mg/mL苯酚，14.0mg/mL丙二醇，0.045mg/mL GLP-1衍生物。
(2)实验方法：
a.造模与分组：
选取SPF级雄性6周BKS-LeprRem/Gpt小鼠30只，依据体重和血糖随机分组，分为6组，每组设置5只试验动物，分别为模型对照组(溶剂)，阳性对照组(索马鲁肽)，实验组1(HS-G1)、实验组2(HS-G2)、实验组3(HS-G3)、实验组4(HS-G4)；其中模型对照组腹腔皮下注射空白溶剂。
b.给药方式：
按照表3所示方式给药，给药频次为每48h一次，给药四次，腹腔皮下注射给药。
表3
Figure PCTCN2022137104-appb-000005
c.检测指标
血糖值：第一次给药时测定给药前血糖值及给药后2h血糖值，随后的三次给药测定再次给药前0h及给药后2h血糖值；
再次给药前0h为表4及图1中DAY3、DAY5、DAY7和DAY9中所述的48h，指上次给药后48h，也为再次给药前0h。
(3)实验结果
实验数据统计如表4所示，所述数据对应图1所示柱状图，在图中，每组数据从左到右依次为模型对照组、索马鲁肽、HS-G1、HS-G2、HS-G3和HS-G4；
表4：平均血糖情况
Figure PCTCN2022137104-appb-000006
本实验所有数据的统计分析均使用SPSS软件进行。所有数值以均数±标准差(mean±SD)表示。对于正态分布数据，采用单因素方差分析(one-way ANOVA)比较组间差异。对于所有分析，P<0.05认为有统计学意义。
从本实验的数据结果(表4及图1)中可以看出，本发明的衍生物在几乎每次给药2h和48h时，均有显著优于索马鲁肽的降糖效果；其中，HS-G3效果最为明显，降糖效果最佳，同样优于与其序列类似的HS-G1；而HS-G4降糖效果在优于索马鲁肽的同时，同样优于HS-G2。可见，本发明的GLP-1衍生物具备非常优异的降糖效果。
实施例4：db/db小鼠中减重效果的研究
(1)实验材料：
品系为BKS-LeprREM/Gpt小鼠，数量30只，6周龄，雄性；
实验药物制剂：1.133mg/ml Na 2HPO 4，5.5mg/ml苯酚，14.0mg/ml丙二醇，0.045mg/ml GLP-1衍生物
(2)实验方法
a.造模与分组：
选取SPF级雄性6周BKS-LeprRem/Gpt小鼠30只，依据体重和血糖随机分组，分为6组，每组设置5只试验动物，分别为模型对照组 (溶剂)，阳性对照组(索马鲁肽)，实验组1(HS-G1)、实验组2(HS-G2)、实验组3(HS-G3)、实验组4(HS-G4)。其中模型对照组腹腔皮下注射空白溶剂。
b.给药方式：
按照实施3中表3所示相同方式给药，给药频次为每48h一次，给药四次，腹腔皮下注射给药。
c.检测指标：
体重：每次给药前及最后一次给药后48h对小鼠进行称重；
摄食量：第一次给药前每笼小鼠设置固定食量(200g)，随后在每次(包括第一次)给药后的48h对小鼠摄食量进行测定。
(3)实验结果：
记录小鼠9天的体重变化数据，结果表5所示，图2为小鼠给药9天的体重变化率图；
表5
Figure PCTCN2022137104-appb-000007
本实验所有数据的统计分析均使用SPSS软件进行。所有数值以均数±标准差(mean±SD)表示。对于正态分布数据，采用单因素方差分析(one-way ANOVA)比较组间差异。对于所有分析，P<0.05认为有统计学意义。
同时，记录小鼠9天的摄食量情况，其中，摄食量抑制率＝(模型 对照组摄食量的平均值-给药组摄食量平均值)/模型对照组摄食量的平均值，结果如表6所示：
表6
Figure PCTCN2022137104-appb-000008
从记录小鼠减重和摄食量数据的表5-6和图2可以看出，本发明提供的长效GLP-1衍生物相比索马鲁肽具有更优的减重效果和更好的摄食量抑制，且差异明显。尤其HS-G3和HS-G4，体重变化率达到了索马鲁肽的2倍以上，减重效果尤为显著，即使对比本发明的HS-G1和HS-G2，其同样有接近1.5-2倍的减重效果。
实施例5：正常小鼠中减重效果研究
(1)实验材料
a.实验制剂：
配制GLP-1衍生物注射液(HS-G3和HS-G4)，具体配方为1.133mg/mLNa 2HPO 4，5.5mg/mL苯酚，14.0mg/mL丙二醇，以及GLP-1衍生物0.045mg/mL。
b.动物实验：
选取健康SPF级雄性6-8周龄的KM小鼠50只，体重18-20g，分为正常饮食(ND)组、HFD(高脂饲料)组，其中正常饮食组包含10只小鼠，饲喂普通维持饲料，HFD组包含40只小鼠，饲喂60％高脂饲料，饲喂周期为5-8周；
对HFD组检测体重，筛除未成模小鼠，剩下小鼠依据体重随机分 组，分为空白对照组(用于判断DIO模型是否成功，不参与药物试验)、阳性对照组(索玛鲁肽)、实验组1、实验组2、模型对照组(空白溶剂)；
(2)实验方法
a.给药方式：
每两天给药一次，给药途径为腹腔注射，给药四次，具体给药剂量等内容见表7：
表7
Figure PCTCN2022137104-appb-000009
b.检测指标：
体重：每次给药时对体重进行检测；
(3)实验结果
本实施例测试结果如表8所示，给药9天的体重变化率如图3所示；
表8
Figure PCTCN2022137104-appb-000010
本实验所有数据的统计分析均使用SPSS软件进行，所有数值以均 数±标准差(mean±SD)表示；对于正态分布数据，采用单因素方差分析(one-wayANOVA)比较组间差异；对于非正态分布数据，采用Kruskal-WallisH检验或Mann-WhitneyU检验分析组间差异；相关性分析采用Spearman检验；对于所有分析，P<0.05认为有统计学意义。
图3为正常小鼠给药9天的体重变化率图，由图3和表8结果可知，对于正常小鼠，本发明提供的长效GLP-1衍生物HS-G3和HS-G4具有比索马鲁肽更优异的减重能力，达到了前者的近2倍及2倍以上的减重效果；同时，本发明的长效GLP-1衍生物不会导致过度的血糖较低，无低血糖风险。因此，本发明的长效GLP-1衍生物更适于降重适应症的开发。
另外，在一组针对HS-G1和HS-G2在正常鼠模型中的实验结果表明，两者具备与索马鲁肽相当的减重效果，对应索马鲁肽8.19％的体重变化率，分别具有8.94％和6.26％的体重变化率。
实施例6：药代动力学研究
(1)实验材料
实验小鼠：SD大鼠，50只，体重在180-200g之间，6-8周龄的雄性小鼠。
实验制剂：索马鲁肽、HS-G3、HS-G4注射液，配方为1.133mg/mLNa 2HPO 4，5.5mg/mL苯酚，14.0mg/mL丙二醇，以及GLP-1衍生物分别为0.015mg/mL、0.045mg/mL和0.075mg/mL。
(2)实验方法
a.分组情况：按照表9对实验大鼠进行分组：
b.给药方式：单次给药，给药途径为腹腔皮下注射，具体给药剂量等内容见表9：
表9
Figure PCTCN2022137104-appb-000011
Figure PCTCN2022137104-appb-000012
(3)实验结果
检测不同实验的不同剂量的半衰期，其结果见表10：
表10
Figure PCTCN2022137104-appb-000013
本实验所有数据的统计分析均使用SPSS软件进行。所有数值以均数±标准差(mean±SD)表示。对于正态分布数据，采用单因素方差分析(one-way ANOVA)比较组间差异。对于非正态分布数据，采用Kruskal-Wallis H检验或Mann-Whitney U检验分析组间差异。相关性分析采用Spearman检验。对于所有分析，P<0.05认为有统计学意义。
由上述实验中结果可知，本发明提供的长效GLP-1衍生物HS-G3和HS-G4在大鼠体内具有比索马鲁肽更优异的半衰期，两者在不同剂量下均具有相对于索马鲁肽约1.5倍以上的长半衰期。
需要说明的是，在本文中，诸如“第一”和“第二”等之类的关系术语仅仅用来将一个实体或者操作与另一个实体或操作区分开来，而不一定要求或者暗示这些实体或操作之间存在任何这种实际的关系或者顺序。而且，术语“包括”、“包含”或者其任何其他变体意在涵盖非排他性的包含，从而使得包括一系列要素的过程、方法、物品或者设备不仅包括那些要素，而且还包括没有明确列出的其他要素，或者是还包括为这种过程、方法、物品或者设备所固有的要素。在没有更多限制的情况下，由语句“包括一个……”限定的要素，并不排除在包括所述要素的过程、方法、物品或者设备中还存在另外的相同要素。
以上所述仅是本公开的具体实施方式，使本领域技术人员能够理解或实现本公开。对这些实施例的多种修改对本领域的技术人员来说将是显而易见的，本文中所定义的一般原理可以在不脱离本公开的精神或范围的情况下，在其它实施例中实现。因此，本公开将不会被限制于本文所示的这些实施例，而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。
工业实用性
本公开公开的GLP-1衍生物具有与GLP-1受体(GLP-1R)的良好的结合亲和力，且与已上市的其他GLP-1衍生物相比，本发明的长效GLP-1衍生物具有更为明显和优异的降糖能力和减重能力，具有很强的工业实用性。

Claims (22)

  1. 一种长效GLP-1衍生物或其在药学上可接受的盐，其特征在于，所述长效GLP-1衍生物中的GLP-1(7-37)类似物的氨基酸序列为：
    HX 8EGTFTSDVSSYLEEQAAX 26EFIAWLVX 34X 35X 36G；
    其中，X 8选自V、I、T、L、G或S，X 26为R或K，X 34为R或K，X 35为G或R，X 36为G或R，且X 26和X 34中仅有一个为K；
    所述衍生物包含与所述GLP-1(7-37)类似物的第26位或第34位中的氨基酸K连接的脂肪酸侧链。
  2. 根据权利要求1所述的长效GLP-1衍生物或其药学上可接受的盐，其特征在于，X 8选自V、I或T，优选选自I或T。
  3. 根据权利要求2所述的长效GLP-1衍生物或其药学上可接受的盐，其特征在于，X 35为R，X 36为G。
  4. 根据权利要求3所述的长效GLP-1衍生物或其药学上可接受的盐，其特征在于，X 26为R，X 34为K。
  5. 根据权利要求1-4任一项所述的长效GLP-1衍生物或其药学上可接受的盐，其特征在于，所述衍生物通过氨基酸K残基上的ε氨基与脂肪酸侧链连接。
  6. 根据权利要求5所述的长效GLP-1衍生物或其药学上可接受的盐，其特征在于，所述脂肪酸侧链选自HOOC(CH 2) 14CO-、HOOC(CH 2) 15CO-、HOOC(CH 2) 16CO-、HOOC(CH 2) 17CO-、HOOC(CH 2) 18CO-、HOOC(CH 2) 19CO-、HOOC(CH 2) 20CO-、HOOC(CH 2) 21CO-或HOOC(CH 2) 22CO-。
  7. 根据权利要求6所述的长效GLP-1衍生物或其药学上可接受的盐，其特征在于，所述脂肪酸侧链选自HOOC(CH 2) 16CO-。
  8. 根据权利要求1-7中的任一项所述的长效GLP-1衍生物或其药学上可接受的盐，其特征在于，所述脂肪酸侧链通过接头与氨基酸K连接。
  9. 根据权利要求8所述的长效GLP-1衍生物或其药学上可接受的盐，其特征在于，所述接头选自：
    Figure PCTCN2022137104-appb-100001
    其中，m为0-6的整数，n为1-3的整数，s为0-3的整数，t为0-4的整数，p为1-23的整数。
  10. 根据权利要求9所述的长效GLP-1衍生物或其药学上可接受的盐，其特征在于，所述接头为：
    Figure PCTCN2022137104-appb-100002
  11. 根据权利要求9或10所述的长效GLP-1衍生物或其药学上可接受的盐，其特征在于，所述长效GLP-1衍生物为：
    N-ε 34-[2-(2-[2-(2-[2-(2-[4-(17-羧基十七烷酰胺基)-4(S)-羧基丁酰基氨基]乙氧基)乙氧基]乙酰氨基)乙氧基]乙氧基)乙酰基][Ile 8Glu 22Arg 26Lys 34]GLP-1(7-37)；或
    N-ε 34-[2-(2-[2-(2-[2-(2-[4-(17-羧基十七烷酰胺基)-4(S)-羧基丁酰基氨基]乙氧基)乙氧基]乙酰氨基)乙氧基]乙氧基)乙酰基][Thr 8Glu 22Arg 26Lys 34]-GLP-1(7-37)；或
    N-ε 34-[2-(2-[2-(2-[2-(2-[4-(17-羧基十七烷酰胺基)-4(S)-羧基丁酰基氨基]乙氧基)乙氧基]乙酰氨基)乙氧基]乙氧基)乙酰基][Ile 8Glu 22Arg 26Lys 34Arg 35Gly 36]GLP-1(7-37)；或
    N-ε 34-[2-(2-[2-(2-[2-(2-[4-(17-羧基十七烷酰胺基)-4(S)-羧基丁酰基氨基]乙氧基)乙氧基]乙酰氨基)乙氧基]乙氧基)乙酰基][Thr 8Glu 22Arg 26Lys 34Arg 35Gly 36]GLP-1(7-37)。
  12. 一种表达GLP-1(7-37)类似物的重组工程菌，其特征在于，所述GLP-1(7-37)类似物序列为：
    HX 8EGTFTSDVSSYLEEQAAX 26EFIAWLVX 34X 35X 36G，
    其中，X 8选自V、I、T、L、G或S，X 26为R或K，X 34为R或K，X 35为G或R，X 36为G或R，其中X 26和X 34中仅有一个为K；
    所述重组工程菌为重组大肠杆菌工程菌，所述重组工程菌由如下制备方法得到：
    (1)将促包涵体序列、EK酶切序列和GLP-1类似物编码基因序列依次串联融合，得到GLP-1类似物的基因表达片段；
    (2)将所述基因表达片段***原核表达质粒，得到GLP-1类似物的表达质粒；
    (3)将所述表达质粒转入大肠杆菌，得到表达所述GLP-1类似物的重组工程菌。
  13. 根据权利要求12所述的重组工程菌，其特征在于，X 35为R，X 36为G。
  14. 根据权利要求13所述的重组工程菌，其特征在于，X 8选自I或T。
  15. 根据权利要求14所述的重组工程菌，其特征在于，X 26为R，X 34为K。
  16. 根据权利要求12-15中的任一项所述的重组工程菌，其特征在于，步骤(2)中所述原核表达质粒为pET-28a(+)或pET-30a(+)。
  17. 根据权利要求16所述的重组工程菌，其特征在于，所述大肠杆菌为重组BL21大肠杆菌。
  18. 根据权利要求12-15中的任一项所述的重组工程菌，其特征 在于，所述促包涵体序列的氨基酸序列为FKFEFKFE，所述EK酶切序列为DDDDK。
  19. 根据权利要求18所述的重组工程菌，其特征在于：所述基因表达片段的核酸序列选自SEQ ID NO.5-8。
  20. 一种药物组合物，其特征在于，包括权利要求1-11中的任一项所述的长效GLP-1衍生物或其药学上可接受的盐，以及药学上可接受的辅料。
  21. 权利要求1-11中的任一项所述的长效GLP-1衍生物或其药学上可接受的盐、权利要求20所述的药物组合物在制备治疗糖尿病的药物中的应用。
  22. 权利要求1-11中的任一项所述的长效GLP-1衍生物或其药学上可接受的盐、权利要求20所述的药物组合物在制备减重药物中的应用。
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