WO2023124847A1 - Dérivé de glp-1 à action prolongée - Google Patents

Dérivé de glp-1 à action prolongée 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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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

L'invention concerne un analogue de polypeptide GLP-1 et un dérivé de GLP-1 à action prolongée de ce dernier. Le dérivé de GLP-1 à action prolongée est modifié par acylation d'acide gras. Le dérivé de GLP-1 à action prolongée a une bonne affinité de liaison avec un récepteur de GLP-1 (GLP-1R), et par comparaison avec d'autres dérivés de GLP-1 sur le marché, le dérivé de GLP-1 à action prolongée a des capacités d'hypoglycémie et de perte de poids plus évidentes et excellentes.
PCT/CN2022/137104 2021-12-28 2022-12-07 Dérivé de glp-1 à action prolongée WO2023124847A1 (fr)

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CN114774496B (zh) * 2022-06-21 2022-10-04 北京惠之衡生物科技有限公司 一种高密度发酵制备glp-1类似物的方法
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CN115536739B (zh) * 2022-07-04 2023-04-14 北京惠之衡生物科技有限公司 一种glp-1受体和gcg受体共激动多肽衍生物的制备方法
CN116284433B (zh) * 2022-07-04 2023-09-12 北京惠之衡生物科技有限公司 一种胰岛素和glp-1的缀合物及其应用
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