WO2024008058A1 - 一种glp-1受体和gcg受体共激动多肽衍生物及其应用 - Google Patents

一种glp-1受体和gcg受体共激动多肽衍生物及其应用 Download PDF

Info

Publication number
WO2024008058A1
WO2024008058A1 PCT/CN2023/105659 CN2023105659W WO2024008058A1 WO 2024008058 A1 WO2024008058 A1 WO 2024008058A1 CN 2023105659 W CN2023105659 W CN 2023105659W WO 2024008058 A1 WO2024008058 A1 WO 2024008058A1
Authority
WO
WIPO (PCT)
Prior art keywords
pharmaceutically acceptable
receptor
acceptable salt
agonist polypeptide
glp
Prior art date
Application number
PCT/CN2023/105659
Other languages
English (en)
French (fr)
Inventor
曹海燕
林兆生
辛瑞
曹丙洲
Original Assignee
北京惠之衡生物科技有限公司
惠升生物制药股份有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 北京惠之衡生物科技有限公司, 惠升生物制药股份有限公司 filed Critical 北京惠之衡生物科技有限公司
Publication of WO2024008058A1 publication Critical patent/WO2024008058A1/zh

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/605—Glucagons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the present invention relates to the field of polypeptide technology and its derivatives, in particular to a GLP-1 receptor and GCG receptor co-agonist polypeptide derivative and its application.
  • Type 2 diabetes diabetes (diabetes mellitus type 2, T2DM), formerly known as noninsulin-dependent diabetes mellitus (NIDDM) or adult-onset diabetes (adult-onset diabetes), is characterized by hyperglycemia, relative lack of insulin, and insulin deficiency. Resistance etc.
  • the drugs used clinically to treat 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 treatment drugs and research hotspots for type 2 diabetes because they have similar hypoglycemic effects as insulin, almost no risk of hypoglycemia, and have both weight loss effects and cardiovascular protective functions.
  • GLP-1 (glucagon-like peptide-1) is a glucose-dependent hypoglycemic peptide hormone secreted by L cells in the terminal jejunum, ileum and colon. It specifically binds to the GLP-1 receptor and exerts hypoglycemic effects. effect.
  • the main advantage of GLP-1 is that it has a blood glucose-dependent incretin secretion effect and avoids the risk of hypoglycemia that is often present in the treatment of diabetes.
  • GLP-1 can also prevent the degeneration of pancreatic beta cells, stimulate the proliferation and differentiation of beta cells, and improve the progression of diabetes from the source.
  • GLP-1 also has the effects of inhibiting gastric acid secretion, delaying gastric emptying, suppressing appetite, etc., and has a partial weight loss effect. However, if you need to achieve better weight loss, you generally need to increase the dosage. High-dose administration of GLP-1 derivatives is prone to gastrointestinal side effects, and often results in a narrow therapeutic window due to poor tolerability. narrow.
  • GCG is a hormone produced in the ⁇ -cells of the pancreas. It acts on the liver under stress conditions such as cold and hunger, decomposing glycogen in the liver and raising blood sugar. In fact, GCG also has the effects of promoting fat degradation, fat oxidation, and heating in the body (Diabetologia, 2017, 60, 1851-1861). Long-term administration can show weight loss effects by increasing energy metabolism, but these effects of GCG Beneficial effects on energy metabolism are prevented by its inherent glycemic effects. Therefore, dual-target co-agonists with GLP-1 receptor and GCG receptor activation activities can use GCG to promote fat degradation, fat oxidation and other functions in the body, achieving better weight loss than GLP-1 single-target agonists. Significant enhancement in efficacy.
  • Oxyntomodulin is a short peptide hormone secreted by intestinal epithelial L-cells. It is a peptide hormone composed of 37 amino acids. It is the product of post-transcriptional processing of the glucagon gene.
  • the 69th amino acid region includes the entire sequence of glucagon, and extends an 8-peptide at the C terminus, so it is also called “glucagon-37", which acts as an activating glucagon-like peptide-1
  • glucagon-37 acts as an activating glucagon-like peptide-1
  • polypeptide GLP-1/GCG receptor dual agonists include: CN201911103118.6, CN201780013643.1, CN201680021972.6, CN201580030150.X, CN201380048137.8, WO2008/071972, WO2008/ 101017 , WO2009/155258, WO2010/096052, WO2010/096142, WO2011/075393, WO2008/152403, etc., but currently, there is no relevant dual agonist on the market.
  • the present invention provides a GLP-1 receptor and GCG receptor co-agonist polypeptide derivative and its application.
  • the term "semaglutide” refers to a GLP-1 derivative having the peptide skeleton and overall compound structure in CAS registration number 910463-68-2.
  • GLP-1 receptor agonist may be defined as a compound capable of binding to and activating the GLP-1 receptor.
  • GCG receptor agonist may be defined as a compound capable of binding to and activating GCG receptors.
  • the "GLP-1 receptor and GCG receptor co-agonist polypeptide” can exhibit at least about 10% to about 500% or more activity against the glucagon receptor relative to natural glucagon, and also exhibit about at least 10% to about 200% or greater activity at the GLP-1 receptor relative to native GLP-1.
  • peptide encompasses a sequence of 3 or more amino acids and usually less than 50 amino acids, where the amino acids are naturally occurring or non-naturally occurring amino acids.
  • Non-naturally occurring amino acids refer to amino acids that do not occur naturally in the body, but which may be incorporated into the peptide structures described herein.
  • the term "derivative" related to a peptide means a peptide or its analogue that has been chemically modified (such as covalent modification, etc.). Typical modifications include amides, sugars, alkyl groups, acyl groups, esters, etc.
  • amino acid "modification” refers to the substitution, addition or deletion of amino acids, including substitution or addition with any of the 20 common amino acids in human proteins as well as atypical or non-naturally occurring amino acids.
  • the term "pharmaceutically acceptable salt” refers to a salt of a polypeptide or protein that retains the biological activity of the parent.
  • pharmaceutically acceptable excipient broadly refers to any component other than the active therapeutic ingredient. Excipients may be inert substances, inactive substances and/or non-pharmaceutically active substances.
  • aliphatic diacid includes linear or branched aliphatic dicarboxylic acids, It has at least two carbon atoms and is saturated or unsaturated.
  • Non-limiting examples of aliphatic diacids are succinic acid, adipic acid, suberic acid, sebacic acid, dodecanedioic acid, tetradecanedioic acid, hexadecanedioic acid, heptadecanedioic acid, stearic acid Alkanedioic acid and eicosanedioic acid.
  • the present invention provides a GLP-1 receptor and GCG receptor co-agonist polypeptide derivative or a pharmaceutically acceptable salt thereof, and its polypeptide has the binding activity of GLP-1 and GCG dual receptors.
  • the amino acid sequence of the polypeptide derivative or its pharmaceutically acceptable salt is as follows: X 1 X 2 X 3 GTFTSDYSX 12 YLX 15 X 16 X 17 X 18 AX 20
  • X 1 is selected from H or Y;
  • X 2 is selected from V or Aib;
  • X 3 is selected from Q or H;
  • X 12 is selected from K, R or S;
  • X 15 is selected from E or D;
  • X 16 is selected from E, S or A;
  • X 17 is selected from K, R or Q;
  • X 18 is selected from K, R or A;
  • X 20 is selected from K, Q, R or H;
  • X 21 is selected from E or D;
  • X 23 is selected from V or I;
  • X 24 is selected from E or K;
  • X 27 is selected from L or E;
  • X 28 is selected from E, K or R;
  • X 29 is selected from G, A-NH 2 or A;
  • X 30 is none or selected from GPSSG, PSG or GPSSG-NH 2 ;
  • X 30 is not GPSSG-NH 2 ;
  • amino acid sequence of the co-agonist polypeptide is selected from:
  • HVQGTFTSDYSKYLDEKAAKDFIEWLEKA-NH 2 (SEQ ID NO. 12);
  • the derivative is connected to the fatty acid side chain through the ⁇ amino group on the amino acid K residue at position 17, 20 or 24 of the polypeptide, that is, the polypeptide
  • the epsilon amino group of K17, K20 or K24 is connected to the fatty acid side chain.
  • the "K number” represents the lysine (K) at the position represented by the "number” in the co-agonist polypeptide sequence, and its ⁇ -amino group is connected to the side chain; for example, "K20" represents the 20th position in the corresponding co-agonist polypeptide sequence. position of lysine, and indicates that the ⁇ -amino group of the lysine is connected to the corresponding fatty acid side chain.
  • the "-NH 2 " at the tail end of the co-agonist polypeptide sequence is represented by replacing the hydroxyl group in the carboxyl group of the tail amino acid with "-NH 2 ", that is, modifying the COOH of the tail amino acid to CONH 2 .
  • the specific structure is as follows:
  • the fatty acid side chain is selected from HOOC(CH 2 ) n CO- ⁇ -Glu-(AEEA) 2 -, n is any integer from 14 to 22.
  • the fatty acid side chain is selected from HOOC(CH 2 ) 16 CO- ⁇ -Glu-(AEEA) 2 - or HOOC(CH 2 ) 18 CO- ⁇ -Glu-(AEEA) 2 -.
  • HOOC(CH 2 ) 16 CO- ⁇ -Glu-(AEEA) 2 - can be called “[2-(2-[2-(2-[2-(2-)4-(17 -Carboxyheptadecanoyl)-4(S)-carboxybutyrylamino]ethoxy)ethoxy]acetamido)ethoxy]ethoxy)acetyl]”.
  • HOOC(CH 2 ) 18 CO- ⁇ -Glu-(AEEA) 2 - can be called “[2-(2-[2-(2-[2-(2-)4-(19 -Carboxynonadecanamido)-4(S)-carboxybutyrylamino]ethoxy)ethoxy]acetamido)ethoxy]ethoxy)acetyl]”.
  • the derivative of the present invention contains a fatty acid side chain connected to the epsilon amino group of lysine at position 17, 20 or 24 of the polypeptide, preferably the fatty acid
  • the side chain is HOOC(CH 2 ) 16 CO- ⁇ -Glu-(AEEA) 2 - or HOOC(CH 2 ) 18 CO- ⁇ -Glu-(AEEA) 2 -.
  • co-agonistic polypeptide derivatives of the present invention are selected from any one of the co-agonistic polypeptide derivatives shown in Table 2 below:
  • co-agonist polypeptide derivatives described in the above table are all composed of GLP-1 receptor and GCG receptor co-agonist polypeptides (co-agonist polypeptide sequences in the table) connected to fatty acid side chains, where the side chains are connected to the specified positions in the polypeptide sequence.
  • the ⁇ -amino group of amino acid K is also composed of GLP-1 receptor and GCG receptor co-agonist polypeptides (co-agonist polypeptide sequences in the table) connected to fatty acid side chains, where the side chains are connected to the specified positions in the polypeptide sequence.
  • polypeptide derivatives or pharmaceutically acceptable salts thereof according to the present invention can be prepared by chemical synthesis, or by fermentation, expression, purification and modification of recombinant engineering bacteria.
  • the present invention provides a pharmaceutical composition, including the polypeptide derivative described in the first aspect or a pharmaceutically acceptable salt thereof, and pharmaceutically acceptable excipients.
  • the pharmaceutical composition is an injection preparation, tablet, capsule, syrup, granule, block, emulsion or compound preparation.
  • the pharmaceutical composition of the present invention can be administered parenterally, such as subcutaneous injection, intradermal injection, intravenous injection, intramuscular injection, intraperitoneal injection, etc.
  • the pharmaceutical composition for parenteral administration is an injection preparation, which is composed of the polypeptide derivative described in the first aspect or a pharmaceutically acceptable salt thereof as an effective raw material, plus pharmaceutically acceptable excipients.
  • the pharmaceutically acceptable excipients are selected from one or more of buffers, osmotic pressure regulators, preservatives, stabilizers, co-solvents, antioxidants, pH regulators and bacteriostatic agents.
  • the auxiliary materials of the composition are 0.1-10 mg/mL buffer, 5-40 mg/mL osmotic pressure regulator and 1-10 mg/mL preservative.
  • the amount of the buffer is 0.1-10 mg/mL, 0.5-5 mg/mL or 0.8-2 mg/mL, such as 1 mg/mL or 1.5 mg/mL; the buffer is preferably disodium hydrogen phosphate.
  • the amount of the osmotic pressure regulator is 5-40 mg/mL, 8-30 mg/mL or 10-20 mg/mL, such as 14 mg/mL or 17 mg/mL; the osmotic pressure regulator is preferably propylene glycol or glycerol. , more preferably propylene glycol.
  • the amount of the preservative is 1-10 mg/mL, 1-8 mg/mL or 1-6 mg/mL, such as 2 mg/mL or 5 mg/mL; the preservative is preferably phenol or m-cresol, more Phenol is preferred.
  • the pharmaceutical composition of the present invention can be administered orally, such as directly orally, mixed into drinking water or food, or intragastrically administered.
  • the pharmaceutical composition delivered orally is a solid composition, which is composed of the polypeptide derivative or its pharmaceutically acceptable salt described in the first aspect as an effective raw material, plus a penetration enhancer and other ingredients. This is because oral drugs are The body's absorption depends on the absorption-promoting effect of penetration enhancers.
  • the pharmaceutical composition includes any co-agonist polypeptide derivative or a pharmaceutically acceptable salt thereof and N-(8-(2-hydroxybenzoyl)amino)octanoate described in Table 1, wherein The amount of the co-agonist polypeptide derivative or a pharmaceutically acceptable salt thereof is 5-20 mg, and the N-(8-(2-hydroxybenzoyl)amino)octanoate is preferably N-(8-(2 Sodium hydroxybenzoyl)amino)octanoate (SNAC), and the amount of SNAC is 200-400 mg, and the oral preparation is preferably an oral tablet.
  • SNAC N-(8-(2 Sodium hydroxybenzoyl)amino)octanoate
  • the amount of the co-agonist polypeptide derivative or a pharmaceutically acceptable salt thereof is 5 mg, 10 mg, 15 mg or 20 mg; the amount of the SNAC is 250-350 mg, more preferably 250mg, 280mg, 300mg, 320mg or 350mg.
  • the pharmaceutical composition further comprises pharmaceutically acceptable excipients selected from one or more of binders, fillers, disintegrants, lubricants and glidants.
  • the excipients of the composition are 0.1-10% w/w binder, 5-40% w/w filler and 0.1-10% w/w lubricant.
  • the amount of the adhesive is 0.1-10% w/w, 0.2-4% w/w, 0.5-3% w/w, such as 1% w/w or 2% w/w;
  • the binder is preferably povidone.
  • the amount of the filler is 5-40% w/w, 10-30% w/w or 5-25% w/w, such as 10.9% w/w, 18% w/w, 19.5% w/w, Or 20.5% w/w; the filler is preferably microcrystalline cellulose.
  • the amount of the lubricant is 0.1-10% w/w, 0.5-5% w/w or 1-3.5% w/w, such as 1% w/w; the lubricant is preferably magnesium stearate.
  • the present invention provides the use of the polypeptide derivative or a pharmaceutically acceptable salt thereof as described in the first aspect, or the pharmaceutical composition as described in the second aspect in the preparation of a drug for treating diabetes.
  • the present invention provides the use of the polypeptide derivative or a pharmaceutically acceptable salt thereof as described in the first aspect, or the pharmaceutical composition as described in the second aspect in the preparation of weight loss medicines.
  • the GLP-1 receptor and GCG receptor co-agonist polypeptide derivatives provided by the present invention have dual GLP-1 and GCG receptor agonistic activities, and have prolonged action time. While effectively lowering blood sugar, they have significantly excellent weight loss effects. effect.
  • This example provides a variety of GLP-1 receptor and GCG receptor co-agonist polypeptide derivatives and their preparation methods.
  • This example is a chemical synthesis method, and the preparation method is as follows (taking C-004 as an example):
  • the protective amino acid used to connect the side chain is Fmoc-Lys(dde)-OH, and the last protected amino acid is Boc-His(Trt)-OH.
  • the crude peptide is eluted, and the chromatographic conditions are: Column (30mm*250mm), mobile phase 1: 0.05M ammonium acetate (aqueous ammonia adjusts pH to about 8.0), mobile phase 2: acetonitrile.
  • the elution gradient was acetonitrile varying from 31% to 38% in 25 minutes, resulting in a sample with greater than 95% purity.
  • the acetonitrile was removed from the obtained C-004 solution using a rotary evaporator, and then freeze-dried using a freeze dryer to obtain a white solid powder.
  • HEK293/Luc/GLP1R cells in good culture status, discard the culture medium in the bottle, wash once with PBS buffer, add 0.05% Trypsin digestion solution for digestion for 3 minutes, then add DMEM basal medium to terminate digestion, and centrifuge to collect the cells.
  • Fire-Lumi Luciferase Assay Kit to detect the in vitro GLP-1 receptor binding activity of co-agonistic peptide derivatives: Prepare the assay culture medium, and use the assay culture medium to dilute the sample step by step to 320nM, with a single dilution factor not exceeding 10 times. ; Then perform a 5-fold serial dilution in a 96-well plate, a total of 8 gradients, and make 2 duplicate wells for each dilution.
  • test data is processed using the four-parameter regression calculation method to obtain the EC50 value of the sample to be tested.
  • HEK293/GCGR/Ga15 cells in good culture status, discard the culture medium in the bottle, wash once with PBS buffer, add 0.05% TRYPSIN digestion solution for digestion, then add DMEM basic culture medium to terminate digestion, and centrifuge to collect the cells.
  • test data is processed using a four-parameter regression calculation method to calculate the EC50 value of the sample to be tested.
  • the EC50 values of the determination results of in vitro GLP-1 receptor binding activity and in vitro GCG receptor binding activity are shown in Table 3.
  • Ref1 is IBI362, a drug jointly developed by Innovent Biologics and Eli Lilly and Company that can treat obesity and diabetes at the same time.
  • the drug is a dual GLP-1 and GCG agonist.
  • each group of polypeptide derivatives of the present invention has the ability to bind to the dual receptors of GLP-1 and GCG, and has the binding activity to the dual receptors of GLP-1 and GCG, but it can also It can be seen that although the sequences are similar, the activities between the molecules are quite different (the level of receptor binding activity and the binding activity ratio of GLP-1 and GCG dual receptors, etc.). In particular, it can be seen from the above data that C-014, C-015, B-020 and B-024 all have excellent GLP-1 receptor and GCG receptor agonistic activities.
  • Example 4 Study on the hypoglycemic effect of OGTT in C57BL/6J mice
  • mice Healthy male mice were selected and adaptively raised for 7 days. The mice were randomly divided into groups based on body weight, with 10 mice in each group, including:
  • Blank control group including 10 mice, which were gavaged with pure water and blank solvent was injected subcutaneously into the abdomen;
  • Model control group including 10 mice, oral administration of glucose (2g/Kg) and subcutaneous injection of blank solvent into the abdomen;
  • mice Each group contains 10 mice, oral administration of glucose (2g/Kg), and subcutaneous injection of drugs into the abdomen.
  • mice in each experimental group determined above were administered according to the specific dosage shown in Table 4:
  • mice C57BL/6J (DIO) mice
  • mice are randomly divided into groups based on body weight, with 6 mice in each group. Only, divided into blank control group, semaglutide group, REF1 group and each experimental group;
  • the drug is administered once a day, and the route of administration is abdominal subcutaneous injection.
  • the time period of administration is fixed every day.
  • the first day of official administration is recorded as Day 1.
  • the specific dosage and other contents are shown in Table 9:
  • Body weight Measure body weight every time you dose
  • Food intake Test food intake after each dose
  • each group of peptide derivatives except C-017 has varying degrees of weight loss effect, and has a weight loss effect that is significantly better than that of semaglutide; the screened peptide derivatives Most products have more than 50% weight loss effect over semaglutide.
  • C-014, B-020 and B-024 even have nearly twice the weight loss effect without causing an excessive reduction in blood sugar. The effect is significant. . It can be expected that the polypeptide derivatives of the present invention will have broad application prospects in reducing blood sugar, especially in weight loss.
  • the GLP-1 receptor and GCG receptor co-agonist polypeptide derivatives disclosed in the present invention have dual GLP-1 and GCG receptor agonistic activities, and have prolonged action time. While effectively lowering blood sugar, they have significantly excellent weight loss effects. function and has strong industrial practicability.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Diabetes (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • General Chemical & Material Sciences (AREA)
  • Endocrinology (AREA)
  • Obesity (AREA)
  • Hematology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Emergency Medicine (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Child & Adolescent Psychology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)

Abstract

一种GLP-1受体和GCG受体共激动多肽衍生物及其应用，所述GLP-1受体和GCG受体共激动多肽衍生物具备GLP-1和GCG双受体激动活性，并具备延长的作用时效，在有效降低血糖的同时，具有显著优异的减重作用。

Description

一种GLP-1受体和GCG受体共激动多肽衍生物及其应用
本发明要求于2022年7月4日提交中国专利局、申请号为202210788637.6、发明名称为“一种GLP-1受体和GCG受体共激动剂及其应用”的中国专利申请的优先权，其全部内容通过引用结合在本发明中。
技术领域
本发明涉及多肽技术及其衍生物领域，尤其涉及一种GLP-1受体和GCG受体共激动多肽衍生物及其应用。
背景技术
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，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。
GLP-1(胰高血糖素样肽-1)是由末端空肠、回肠和结肠的L细胞所分泌的一种葡萄糖依赖性降血糖多肽激素，与GLP-1受体特异性结合后发挥降糖作用。GLP-1的主要优点是具有血糖依赖性的肠促胰岛素分泌作用，避免了糖尿病治疗中常存在的产生低血糖症的危险。除了调节血糖，GLP-1也可以阻止胰腺β细胞退化，刺激β细胞的增殖和分化，能从源头上改善糖尿病进程。此外，GLP-1还具有抑制胃酸分泌、延迟胃排空、抑制食欲等作用，具有部分减重效果。但是如果需要实现较好的体重减轻作用，一般需要加大给药剂量，而大剂量给予GLP-1衍生物容易产生胃肠道副作用，并且往往因耐受性差而导致治疗窗较 窄。
GCG(胰高血糖素)是在胰脏的α细胞中生成的激素，在机体寒冷、饥饿等应激状态下作用于肝脏，将肝脏中的糖原进行分解而提高血糖。实际上GCG在体内还具有促进脂肪降解、脂肪氧化、发热等作用(Diabetologia,2017,60,1851-1861)，长期给药可以通过增加能量代谢量而呈现出体重减轻的药效，但GCG这些对能量代谢的有益作用因其固有的升血糖作用而未能得以应用。因此，具备GLP-1受体和GCG受体激活活性的双靶点共激动剂，能够利用GCG在体内促进脂肪降解、脂肪氧化等功能，实现相比于GLP-1单靶点激动剂在减肥功效方面的显著增强。
目前，已研究发现胃泌酸调节素(OXM)具有良好的降糖减重效果，甚至明显优于现有的GLP-1类药物，如利拉鲁肽。胃泌酸调节素是肠上皮L-细胞分泌的一种短肽激素，其是由37个氨基酸组成的肽类激素，是胰高血糖素基因转录后加工的产物，在其前体第33-69位氨基酸区包括了胰高血糖素的全部序列，并在C末端延伸出一个8肽，因此又被成为“胰高血糖素-37”，其作为一种激活胰高血糖素样肽-1(GLP-1)和胰高血糖素(GCG)的双受体激动剂，虽然具有较好的降糖减重效果，但存在稳定性差、受体活性低等问题，导致其给药剂量大，很难达到控糖减重的最佳效果。
目前，关于多肽类GLP-1/GCG受体双重激动剂，已公开的专利文件有：CN201911103118.6、CN201780013643.1、CN201680021972.6、CN201580030150.X、CN201380048137.8、WO2008/071972、WO2008/101017、WO2009/155258、WO2010/096052、WO2010/096142、WO2011/075393、WO2008/152403等，但目前，尚无相关的双重激动剂上市。
因此，具有良好的降糖减重作用的GLP-1和GCG受体的共激动多肽及其衍生物目前仍存在巨大的临床需求，尤其是在具备更加优异的减重效果方面。
发明内容
因此，具有良好的降糖减重作用的GLP-1和GCG受体的共激动多肽及其衍生物目前仍存在巨大的临床需求。
为了解决上述问题，本发明提供了一种GLP-1受体和GCG受体共激动多肽衍生物及其应用。
本发明中，术语“索马鲁肽”是指一种GLP-1衍生物，其具有CAS登记号910463-68-2中的肽骨架和整体化合物结构。
本发明中，术语“GLP-1受体激动剂”可被定义为能够与GLP-1受体结合并能够将其活化的化合物。
本发明中，术语“GCG受体激动剂”可被定义为能够与GCG受体结合并能够将其活化的化合物。
本发明中，“GLP-1受体和GCG受体共激动多肽″能表现出相对于天然胰高血糖素针对胰高血糖素受体活性的至少约10％-约500％或更大，并亦表现出相对于天然GLP-1针对GLP-1受体活性的约至少10％-约200％或更大。
本发明中，术语“肽”包含3个或更多个氨基酸且通常小于50个氨基酸的序列，其中氨基酸为天然存在的或非天然存在的氨基酸。非天然存在的氨基酸指不在体内天然存在的氨基酸，但其可被引入到本文所述的肽结构中。
本发明中，有关肽(例如GLP-1或GCG)的术语“衍生物”表示经化学修饰(如共价修饰等)的肽或其类似物。典型的修饰包括酰胺、糖类、烷基、酰基、酯等。
本发明中，术语氨基酸“修饰”指氨基酸的取代、添加或缺失，包括用人蛋白质中常见的20种氨基酸以及非典型或非天然存在的氨基酸中的任何一种来取代或添加。
本发明中，术语“药学上可接受的盐”是指保留母体的生物活性的多肽或蛋白的盐。
本发明中，术语“药学上可接受的辅料”宽泛地指除活性治疗成分外的任何组分。辅料可以是惰性物质、无活性物质和/或非药学活性物质。
本发明中，术语“脂肪族二酸”包括直链或支链脂肪族二羧酸， 其具有至少两个碳原子并为饱和或不饱和的。脂肪族二酸的非限定实例为琥珀酸、己二酸、辛二酸、癸二酸、十二烷二酸、十四烷二酸、十六烷二酸、十七烷二酸、十八烷二酸和二十烷二酸。
第一方面，本发明提供了一种GLP-1受体和GCG受体共激动多肽衍生物或其药学上可接受的盐，其多肽具备GLP-1和GCG双受体的结合活性，本发明的多肽衍生物或其药学上可接受的盐的氨基酸序列如下式所示：
X1X2X3GTFTSDYSX12YLX15X16X17X18AX20X21FX23X24WLX27X28X29X30
其中：
X1选自H或Y；
X2选自V或Aib；
X3选自Q或H；
X12选自K、R或S；
X15选自E或D；
X16选自E、S或A；
X17选自K、R或Q；
X18选自K、R或A；
X20选自K、Q、R或H；
X21选自E或D；
X23选自V或I；
X24选自E或K；
X27选自L或E；
X28选自E、K或R；
X29选自G、A-NH2或A；
X30没有或选自GPSSG、PSG或GPSSG-NH2；
其中：
当X1为Y时，X30不为GPSSG-NH2；
当X2为V，X12为R时，X30不为GPSSG-NH2。
作为本发明的一种优选技术方案，所述共激动多肽的氨基酸序列选自：
YVQGTFTSDYSKYLEEKKAKEFVEWLLEGGPSSG(SEQ ID NO.1)；
YVQGTFTSDYSRYLEERRAQEFVKWLLEGGPSSG(SEQ ID NO.2)；
HVQGTFTSDYSRYLDERRAQEFVKWLLEGPSG(SEQ ID NO.3)；
HVQGTFTSDYSKYLDEKKAKEFVEWLLEGGPSSG-NH2(SEQ ID NO.4)；
HVQGTFTSDYSKYLEEKKAKEFVEWLLEGGPSSG-NH2(SEQ ID NO.5)；
H-Aib-QGTFTSDYSRYLEERRAQEFVKWLLEGGPSSG(SEQ ID NO.6)；
H-Aib-QGTFTSDYSRYLEERRAQEFVKWLLEGGPSSG-NH2(SEQ ID NO.7)；
H-Aib-QGTFTSDYSRYLEERRAQEFVKWLLEGGPSSG-NH2(SEQ ID NO.8)；
HVQGTFTSDYSRYLEERRAQEFVKWLLEGGPSSG(SEQ ID NO.9)；
HIQGTFTSDYSRYLEERRAQEFVKWLLEGGPSSG(SEQ ID NO.10)；
HVQGTFTSDYSKYLDEKKAKEFVEWLLEGGPSSG-NH2(SEQ ID NO.11)；
HVQGTFTSDYSKYLDEKAAKDFIEWLEKA-NH2(SEQ ID NO.12)；
HVQGTFTSDYSRYLEERAAREFVKWLERA(SEQ ID NO.13)；
H-Aib-HGTFTSDYSSYLDSQAAHEFVKWLLEGGPSSG(SEQ ID NO.14)；
H-Aib-HGTFTSDYSRYLDEQAARDFVKWLLEGGPSSG(SEQ ID NO.15)；或
H-Aib-HGTFTSDYSSYLDARAAHEFVKWLLEGGPSSG(SEQ ID NO.16)。
作为本发明的一种优选技术方案，所述衍生物通过所述多肽的第17位、第20位或第24位中的氨基酸K残基上的ε氨基与脂肪酸侧链连接，即所述多肽的K17、K20或K24的ε氨基与脂肪酸侧链连接。其中，“K数字”表示的是共激动多肽序列“数字”所表示位置的赖氨酸(K)，其ε-氨基与侧链进行连接；如“K20”表示为相应共激动多肽序列第20位的赖氨酸，并表示通过该赖氨酸的ε-氨基与相应脂肪酸侧链连接。
在本发明中，所述共激动多肽序列中尾端的“-NH2”表示在所述尾端，将尾端氨基酸羧基中的羟基替换为“-NH2”，即将尾端氨基酸的COOH修饰为CONH2。具体结构如下所示：
作为本发明的一种优选技术方案，所述脂肪酸侧链选自HOOC(CH2)nCO-γ-Glu-(AEEA)2-，n为14-22的任意整数。
作为本发明的一种优选技术方案，所述脂肪酸侧链选自HOOC(CH2)16CO-γ-Glu-(AEEA)2-或HOOC(CH2)18CO-γ-Glu-(AEEA)2-。
按照IUPAC命名法，HOOC(CH2)16CO-γ-Glu-(AEEA)2-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]”。
按照IUPAC命名法，HOOC(CH2)18CO-γ-Glu-(AEEA)2-可以被称为“[2-(2-[2-(2-[2-(2-)4-(19-羧基十九烷酰胺基)-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多肽的第17位、第20位或第24位的赖氨酸的ε氨基连接的脂肪酸侧链，优选所述脂肪酸侧链为HOOC(CH2)16CO-γ-Glu-(AEEA)2-或HOOC(CH2)18CO-γ-Glu-(AEEA)2-。
作为本发明的一种优选技术方案，本发明所述共激动多肽衍生物选自如下表1所示共激动多肽衍生物中的任意一种：
表1

进一步地，本发明所述共激动多肽衍生物选自如下表2所示共激动多肽衍生物中的任意一种：
表2

上述表中所述共激动多肽衍生物均由GLP-1受体和GCG受体共激动多肽(表中共激动多肽序列)连接脂肪酸侧链而成，其中，所述侧链连接至多肽序列指定位置的氨基酸K的ε-氨基。
本发明所述的多肽衍生物或其药学上可接受的盐可以通过化学合成的方法制备，也可以通过制备重组工程菌发酵表达并纯化修饰的方式制备。
第二方面，本发明提供了一种药物组合物，包括第一方面所述的多肽衍生物或其药学上可接受的盐，以及药学上可接受的辅料。所述药物组合物为注射液制剂、片剂、胶囊、糖浆、颗粒剂、块剂、乳剂或者复方制剂。
作为本发明的一种优选技术方案，本发明的药物组合物可以通过肠胃外给药，如皮下注射、皮内注射、静脉注射、肌肉注射、腹腔注射等。优选地，通过肠胃外给药的药物组合物为注射液制剂，以第一方面所述的多肽衍生物或其药学上可接受的盐为有效原料，再加上药学上可接受的辅料组成。
优选地，药学上可接受的辅料选自缓冲剂、渗透压调节剂、防腐剂、稳定剂、助溶剂、抗氧剂、pH值调节剂和抑菌剂中的一种或多种。
优选地，所述组合物的辅料为0.1-10mg/mL的缓冲剂、5-40mg/mL的渗透压调节剂和1-10mg/mL的防腐剂。
进一步地，所述缓冲剂的量为0.1-10mg/mL、0.5-5mg/mL或0.8-2mg/mL，如1mg/mL或1.5mg/mL；所述缓冲剂优选为磷酸氢二钠。
进一步地，所述渗透压调节剂的量为5-40mg/mL、8-30mg/mL或10-20mg/mL，如14mg/mL或17mg/mL；所述渗透压调节剂优选为丙二醇或甘油，更优选为丙二醇。
进一步地，所述防腐剂的量为1-10mg/mL、1-8mg/mL或1-6mg/mL，如2mg/mL或5mg/mL；所述防腐剂优选为苯酚或间甲酚，更优选为苯酚。
作为本发明的一种优选技术方案，本发明的药物组合物可以通过口服给药，如直接口服、掺入到饮水或食物中或灌胃等。
通过口服递送的药物组合物为固体组合物，以第一方面所述的多肽衍生物或其药学上可接受的盐为有效原料，再加上促渗剂等成分组成，这是由于口服药物在人体的吸收依赖于促渗剂的吸收促进作用。所述药物组合物包含表1中所述的任意一种共激动多肽衍生物或其药学上可接受的盐和N-(8-(2-羟基苯甲酰)氨基)辛酸盐，其中所述共激动多肽衍生物或其药学上可接受的盐的量为5-20mg，所述N-(8-(2-羟基苯甲酰)氨基)辛酸盐优选为N-(8-(2-羟基苯甲酰)氨基)辛酸钠(SNAC)，并且所述SNAC的量为200-400mg，所述口服制剂优选为口服片剂。
优选地，所述共激动多肽衍生物或其药学上可接受的盐的量为5mg、10mg、15mg或20mg；所述SNAC的量为250-350mg，更优选为 250mg、280mg、300mg、320mg或350mg。
优选地，所述药物组合物进一步包含药学上可接受的赋形剂，所述赋形剂选自粘合剂、填充剂、崩解剂、润滑剂和助流剂中的一种或多种。优选地，所述组合物的赋形剂为0.1-10％w/w的粘合剂、5-40％w/w的填充剂和0.1-10％w/w的润滑剂。进一步地，所述粘合剂的量为0.1-10％w/w，0.2-4％w/w，0.5-3％w/w，如1％w/w或2％w/w；所述粘合剂优选为聚维酮。所述填充剂的量为5-40％w/w，10-30％w/w或5-25％w/w，如10.9％w/w、18％w/w、19.5％w/w、或20.5％w/w；所述填充剂优选为微晶纤维素。所述润滑剂的量为0.1-10％w/w，0.5-5％w/w或1-3.5％w/w，如1％w/w；所述润滑剂优选为硬脂酸镁。
第三方面，本发明提供了第一方面所述的多肽衍生物或其药学上可接受的盐，或第二方面所述的药物组合物在制备治疗糖尿病的药物中的应用。
第四方面，本发明提供了第一方面所述的多肽衍生物或其药学上可接受的盐，或第二方面所述的药物组合物在制备减重的药物中的应用。
本发明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：
本发明提供的GLP-1受体和GCG受体共激动多肽衍生物具备GLP-1和GCG双受体激动活性，并具备延长的作用时效，在有效降低血糖的同时，具有显著优异的减重作用。
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，并不能限制本发明。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚，下面将对本发明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发明的一部分实施例，而不是全部的实施例。基于本发明中的实施例，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，都属于本发明保护的范围。
实施例1：GLP-1受体和GCG受体共激动多肽的制备
本实施例提供了多种GLP-1受体和GCG受体共激动多肽衍生物及其制备方法，本实施例为化学合成方法，制备方法如下(以C-004为例)：
1.C-004粗肽的合成
1.1树脂的溶胀去保护
称取担载量为0.3左右的Fmoc-Linker-MBHA树脂放入反应柱中，用DCM溶胀树脂30min，抽去溶液，用DMF清洗树脂2次，抽去溶液。加入适量的20％的哌啶/DMF溶液，在氮气鼓吹下反应5min，抽去溶液，再加入适量的20％的哌啶/DMF溶液，在氮气鼓吹下反应10min，接着用适量的DMF清洗树脂6次，得到脱除Fmoc保护基的树脂。
1.2接肽反应
称取一定量的Fmoc-Gly-OH保护氨基酸和缩合剂TBTU，加入反应柱，加入适量DMF溶剂，加入一定量的碱DIEA，在氮气鼓吹下，室温反应1-2h，用Kaiser试剂(茚三酮显色法)监测反应过程。反应结束后，用DMF清洗树脂3次。接着重复去保护步骤：加入适量的20％的哌啶/DMF溶液，在氮气鼓吹下反应5min，抽去溶液，再加入适量的20％的哌啶/DMF溶液，在氮气鼓吹下反应10min，接着用适量的DMF清洗树脂6次，按照序列从C端到N端的顺序，依次偶联。其中连接侧链使用的保护氨基酸为Fmoc-Lys(dde)-OH，最后一个保护氨基酸为Boc-His(Trt)-OH。
1.3侧链的修饰
偶联完所有主链保护氨基酸后，加入2％的水合肼/DMF溶液脱除Lys的侧链dde保护基，然后用DMF清洗树脂6-8次。按照主链偶联的方式，依次偶联Fmoc-AEEA-OH，Fmoc-AEEA-OH，Fmoc-Glu-OTBU，C20diacid-Otbu。
1.4多肽的裂解和干燥
将一定量比例为TFA:EDT:苯甲硫醚:苯酚:H2O＝88:4:3:3:2的裂解液加入试管中，冰浴冷却60min。将合成好的粗肽树脂慢慢加入预冷的裂解液中，在26℃左右条件下震荡或搅拌反应2h。可按1g肽树脂需要8mL裂解液，也可作适当调整。
将以上所得的裂解液抽滤得到清液，缓慢加入预冷无水乙醚中，搅拌、静置20min，用离心机离心得到白色固体，再用常温乙醚离心洗涤4次，干燥，得到白色粉末粗肽。
2.C-004的纯化
2.1分离
将粗肽洗脱，色谱条件为柱(30mm*250mm)，流动相1：0.05M醋酸铵(氨水调pH值约8.0)，流动相2：乙腈。洗脱梯度为乙腈在25分钟内从31％变化到38％，得到纯度大于95％的样品。
2.2旋蒸冻干
将得到的C-004溶液，用旋转蒸发仪旋去除乙腈，再用冷冻干燥机进行冻干，得到白色固体粉末。
其他尾端为-NH2的衍生物制备方法同C-004；而尾端为正常氨基酸、非尾端为-NH2的衍生物，在步骤1.1中选择王氏树脂(wang resin)代替Fmoc-Linker-MBHA树脂，其余步骤与本实施例相同。
实施例2：体外GLP-1受体结合活性测定
选取培养状态良好的HEK293/Luc/GLP1R细胞，弃去瓶中培养液，用PBS缓冲液洗1次，加入0.05％Trypsin消化液消化3min，然后加入DMEM基础培养基终止消化，离心收集细胞。用DMEM空白培养基调整细胞密度为8.0×105个/mL，50μL/孔接种于96孔细胞培养板中，于37℃、5％CO2条件下培养过夜。
用Fire-Lumi荧光素酶检测试剂盒检测共激动多肽衍生物的体外GLP-1受体结合活性：配制测定培养液，用测定培养液分步稀释样品至320nM，单次稀释倍数不超过10倍；之后在96孔板中按照进行5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，将稀释好的测定培养液加入细胞板中，每孔50μL，置于37℃、5％CO2条件下孵育6h。从培养箱中取出样品板，放置至室温。加入100μL Fire-Lumi检测液，反应5min，震荡10s，检测荧光强度。
试验数据采用四参数回归计算法进行处理，得到待测样品的EC50值。
实施例3：体外GCG受体结合活性测定
选取培养状态良好的HEK293/GCGR/Ga15细胞，弃去瓶中培养液，用PBS缓冲液洗1次，加入0.05％TRYPSIN消化液消化，然后加入DMEM基础培养液终止消化，离心收集细胞。用DMEM空白培养基调整细胞密度为8×105个/mL，5μL/孔接种于96孔细胞培养板中。
配制测定培养液(DMEM培养液添加0.5nM IBMX)，用测定培养液分步稀释样品至100nM，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，将稀释好的测定培养液加入细胞板中，37℃孵育30min；加入cAMP-d2工作液，每孔5μL，加入cAMP Eu-Cryptate抗体工作液，每孔5μL，96孔板覆盖封板膜，室温孵育60min；培养结束后，去除封板膜，在FRET模式下(665nm，620nm)，用HTRF读取器进行检测。
试验数据采用四参数回归计算法进行处理，可以计算出待测样品的EC50值。
体外GLP-1受体结合活性和体外GCG受体结合活性的测定结果EC50值如表3所示。其中，Ref1为信达生物制药与礼来制药合作研发的可以同时治疗肥胖和糖尿病的药物IBI362，该药物是一种GLP-1和GCG双重激动剂。
表3

由表中结果可知，除C-022以外，本发明各组多肽衍生物均具备与GLP-1和GCG双受体的结合能力，具备GLP-1和GCG双受体的结合活性，但也能看出，虽然序列相似，但各分子间活性差异较大(受体结合活性高低和GLP-1和GCG双受体的结合活性比等)。从上述数据尤其可以看出，其中C-014、C-015、B-020和B-024都具备非常优异的GLP-1受体和GCG受体激动活性。
实施例4：C57BL/6J小鼠OGTT降糖效果研究
(1)实验材料：
选取健康的雄性小鼠，适应性饲养7天，对小鼠依据体重随机分组，每组10只，其中：
空白对照组：包含10只小鼠，纯水灌胃，腹部皮下注射空白溶剂；
模型对照组：包含10只小鼠，口服葡萄糖(2g/Kg)，腹部皮下注射空白溶剂；
实验组：每组包含10只小鼠，口服葡萄糖(2g/Kg)，腹部皮下注射药物。
(2)实验方法
a.给药方式：
对上述确定的各实验组小鼠，按照表4所示的具体给药计量进行给药：
表4
b.血糖值检测：
血糖检测前禁食不禁水，同一天下午，将实验动物禁食不禁水， 在第二天早晨，禁食16h后获得禁食体重(以计算给药量及葡萄糖剂量)。在给药前测定给药前0h血糖，然后，皮下注射空白溶剂或药物溶液。在给药3h后对动物口服填喂葡萄糖(2g/Kg)，在口服葡萄糖后15min、30min、60min和120min检测血糖，通过血糖仪获得两个葡萄糖读数。在各时间点报告两个葡萄糖读数的平均值，绘制血糖变化曲线，计算AUC面积，结果见表5-8。
表5

注：“*”指的是，相对于模型对照组，p＜0.05；“**”指的是，
相对于模型对照组，p＜0.01。
表6


注：“*”指的是，相对于模型对照组，p＜0.05；“**”指的是，
相对于模型对照组，p＜0.01。
表7

注：“*”指的是，相对于模型对照组，p＜0.05；“**”指的是，
相对于模型对照组，p＜0.01。
表8


注：“*”指的是，相对于模型对照组，p＜0.05；“**”指的是，
相对于模型对照组，p＜0.01。
由实验结果可知，本发明各组多肽衍生物除了C-022外，其余多肽均具备显著的降糖作用，其中C-014、B-020、B-024效果最为显著，具备显著优于其他组的降糖作用。在对比研究中也证实，其降糖效果与索马鲁肽基本一致，甚至显著优于索马鲁肽。
实施例5：DIO小鼠减重效果及摄食量研究
(1)实验材料：C57BL/6J(DIO)小鼠
选取健康SPF级饲喂高脂饲料(60％脂肪)的雄性16-17周龄的C57小鼠，体重35-40g，适应性饲养14-17天；对小鼠依据体重随机分组，每组6只，分为空白对照组、索马鲁肽组、REF1组以及各实验组；
(2)实验方法：
a.给药方式：
每天给药一次，给药途径为腹部皮下注射，给药时固定每天给药的时间段，正式给药第一天记为Day1，具体给药剂量等内容见表9：
表9

b.检测指标：
体重：每次给药时对体重进行检测；
摄食量：每次给药后对摄食量进行检测；
(3)实验结果
本实施例测试结果如表10和表11所示：
表10
表11
由实验结果可知，在减重实验中，除C-017外其它各组多肽衍生物均具有不同程度的减重作用，且具备显著优于索马鲁肽的减重效果；筛选出的多肽衍生物多数具备超过索马鲁肽50％以上的减重效果，C-014、B-020和B-024等甚至有接近其两倍的减重效果，同时不会导致血糖的过分降低，效果显著。可以预期，本发明的多肽衍生物在降糖、尤其是减重方面，必然会具备广阔的应用前景。
工业实用性
本发明公开的GLP-1受体和GCG受体共激动多肽衍生物具备GLP-1和GCG双受体激动活性，并具备延长的作用时效，在有效降低血糖的同时，具有显著优异的减重作用，具有很强的工业实用性。

Claims (12)

  1. 一种GLP-1受体和GCG受体共激动多肽衍生物或其药学上可接受的盐，其特征在于，所述衍生物为酰化连接脂肪酸侧链的GLP-1和GCG受体共激动多肽，所述多肽的氨基酸序列如下式所示：
    X1X2X3GTFTSDYSX12YLX15X16X17X18AX20X21FX23X24WLX27X28X29X30
    其中：
    X1选自H或Y；
    X2选自V或Aib；
    X3选自Q或H；
    X12选自K、R或S；
    X15选自E或D；
    X16选自E、S或A；
    X17选自K、R或Q；
    X18选自K、R或A；
    X20选自K、Q、R或H；
    X21选自E或D；
    X23选自V或I；
    X24选自E或K；
    X27选自L或E；
    X28选自E、K或R；
    X29选自G、A-NH2或A；
    X30没有或选自GPSSG、PSG或GPSSG-NH2；
    其中：
    当X1为Y时，X30不为GPSSG-NH2；
    当X2为V，X12为R时，X30不为GPSSG-NH2。
  2. 根据权利要求1所述的共激动多肽衍生物或其药学上可接受的盐，其特征在于，所述多肽的氨基酸序列选自：
    YVQGTFTSDYSKYLEEKKAKEFVEWLLEGGPSSG；
    YVQGTFTSDYSRYLEERRAQEFVKWLLEGGPSSG；
    HVQGTFTSDYSRYLDERRAQEFVKWLLEGPSG；
    HVQGTFTSDYSKYLDEKKAKEFVEWLLEGGPSSG-NH2；
    HVQGTFTSDYSKYLEEKKAKEFVEWLLEGGPSSG-NH2；
    H-Aib-QGTFTSDYSRYLEERRAQEFVKWLLEGGPSSG；
    H-Aib-QGTFTSDYSRYLEERRAQEFVKWLLEGGPSSG-NH2；
    H-Aib-QGTFTSDYSRYLEERRAQEFVKWLLEGGPSSG-NH2；
    HVQGTFTSDYSRYLEERRAQEFVKWLLEGGPSSG；
    HIQGTFTSDYSRYLEERRAQEFVKWLLEGGPSSG；
    HVQGTFTSDYSKYLDEKKAKEFVEWLLEGGPSSG-NH2；
    HVQGTFTSDYSKYLDEKAAKDFIEWLEKA-NH2；
    HVQGTFTSDYSRYLEERAAREFVKWLERA；
    H-Aib-HGTFTSDYSSYLDSQAAHEFVKWLLEGGPSSG；
    H-Aib-HGTFTSDYSRYLDEQAARDFVKWLLEGGPSSG；或
    H-Aib-HGTFTSDYSSYLDARAAHEFVKWLLEGGPSSG。
  3. 根据权利要求1或2所述的共激动多肽衍生物或其药学上可接受的盐，其特征在于，所述衍生物通过所述多肽的第17位、第20位或第24位中的氨基酸K残基上的ε氨基与脂肪酸侧链连接。
  4. 根据权利要求1-3中任一项所述的共激动多肽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(CH2)nCO-γ-Glu-(AEEA)2-，n为14-22的任意整数。
  5. 根据权利要求4所述的共激动多肽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(CH2)16CO-γ-Glu-(AEEA)2-或HOOC(CH2)18CO-γ-Glu-(AEEA)2-。
  6. 根据权利要求4所述的共激动多肽衍生物或其药学上可接受的盐，其特征在于，所述共激动多肽衍生物选自如下所示共激动多肽衍生物中的任意一种：

  7. 根据权利要求6所述的共激动多肽衍生物或其药学上可接受的盐，其特征在于，所述共激动多肽衍生物选自如下所示共激动多肽衍生物中的任意一种：

  8. 一种药物组合物，其特征在于，包括权利要求1-7中任一项所述的共激动多肽衍生物或其药学上可接受的盐，以及药学上可接受的辅料。
  9. 根据权利要求8所述的药物组合物，其特征在于，所述药物组合物为注射液制剂、片剂、胶囊、糖浆、颗粒剂、块剂、乳剂或者复方制剂。
  10. 根据权利要求9所述的药物组合物，其特征在于，所述药物组合物为注射液制剂，所述辅料包括缓冲剂、渗透压调节剂和防腐剂。
  11. 权利要求1-7中任一项所述的共激动多肽衍生物或其药学上可接受的盐，或权利要求8-10中任一项所述的药物组合物在制备治疗糖尿病的药物中的应用。
  12. 权利要求1-7中任一项所述的共激动多肽衍生物或其药学上可接受的盐，或权利要求8-10中任一项所述的药物组合物在制备减重的药物中的应用。
PCT/CN2023/105659 2022-07-04 2023-07-04 一种glp-1受体和gcg受体共激动多肽衍生物及其应用 WO2024008058A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202210788637 2022-07-04
CN202210788637.6 2022-07-04

Publications (1)

Publication Number Publication Date
WO2024008058A1 true WO2024008058A1 (zh) 2024-01-11

Family

ID=84473191

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2023/105659 WO2024008058A1 (zh) 2022-07-04 2023-07-04 一种glp-1受体和gcg受体共激动多肽衍生物及其应用

Country Status (2)

Country Link
CN (3) CN115536739B (zh)
WO (1) WO2024008058A1 (zh)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
CN116410298B (zh) * 2023-06-06 2023-08-11 诺博泰科(成都)生物科技有限公司 一种三激动多肽化合物及其盐、药用组合物、药剂和用途

Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101790538A (zh) * 2007-02-15 2010-07-28 印第安纳大学研究与科技公司 胰高血糖素/glp-1受体共激动剂
CN103068841A (zh) * 2010-06-24 2013-04-24 西兰制药公司 胰高血糖素类似物
CN104583233A (zh) * 2012-06-21 2015-04-29 印第安纳大学研究及科技有限公司 表现出gip受体活性的胰高血糖素的类似物
CN104583232A (zh) * 2012-06-21 2015-04-29 印第安纳大学研究及科技有限公司 展现gip受体活性的胰高血糖素类似物
CN107735100A (zh) * 2015-06-22 2018-02-23 伊莱利利公司 胰高血糖素和glp‑1协同激动剂化合物
CN109745547A (zh) * 2017-11-06 2019-05-14 深圳市图微安创科技开发有限公司 Glp-1r/gcgr双激动剂多肽治疗胆汁性肝硬化
CN111372945A (zh) * 2017-11-06 2020-07-03 中山大学 基于胃泌酸调节素类似物glp-1r/gcgr双靶点激动剂多肽治疗特发性肺间质纤维化
CN111423506A (zh) * 2019-11-08 2020-07-17 成都奥达生物科技有限公司 一种glp-1化合物
CN111944055A (zh) * 2019-05-16 2020-11-17 浙江道尔生物科技有限公司 一种治疗代谢疾病的融合蛋白
CN115490760A (zh) * 2022-07-04 2022-12-20 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

Family Cites Families (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
PL2300035T3 (pl) * 2008-06-17 2016-04-29 Univ Indiana Res & Tech Corp Mieszani agoniści na bazie GIP do leczenia zaburzeń metabolicznych i otyłości
TW201625669A (zh) * 2014-04-07 2016-07-16 賽諾菲公司 衍生自艾塞那肽－４（Ｅｘｅｎｄｉｎ－４）之肽類雙重ＧＬＰ－１／升糖素受體促效劑
TW201625668A (zh) * 2014-04-07 2016-07-16 賽諾菲公司 作為胜肽性雙重ｇｌｐ－１／昇糖素受體激動劑之艾塞那肽－４衍生物
CN104031141B (zh) * 2014-05-30 2016-08-24 天津世传生物科技有限公司 用于治疗2型糖尿病的长效肠激素多肽类似物及应用
EP3204408B1 (en) * 2014-10-10 2020-05-06 Novo Nordisk A/S Stable glp-1 based glp-1/glucagon receptor co-agonists
CA3093383A1 (en) * 2014-10-24 2016-04-28 Merck Sharp & Dohme Corp. Co-agonists of the glucagon and glp-1 receptors
PH12017501222B1 (en) * 2014-12-30 2024-07-03 Hanmi Pharmaceutical Co Ltd Glucagon derivatives with improved stability
WO2016198604A1 (en) * 2015-06-12 2016-12-15 Sanofi Exendin-4 derivatives as dual glp-1 /glucagon receptor agonists
US10493125B2 (en) * 2015-12-09 2019-12-03 Merck Sharp & Dohme Corp. Co-agonists of the glucagon and GLP-1 receptors
CN109836503B (zh) * 2017-11-24 2022-09-16 浙江道尔生物科技有限公司 一种治疗代谢疾病的多重活性蛋白
WO2019140025A1 (en) * 2018-01-12 2019-07-18 Eli Lilly And Company Combination therapy
WO2019200594A1 (zh) * 2018-04-19 2019-10-24 杭州先为达生物科技有限公司 酰化的glp-1衍生物
CN111171135B (zh) * 2018-11-12 2023-04-07 天津药物研究院有限公司 具有双重受体激动作用的胰高血糖素衍生肽及其用途
KR20210091230A (ko) * 2018-11-12 2021-07-21 톈진 인스티튜트 오브 파마슈티컬 리서치 씨오., 엘티디. 글루카곤 유도 펩티드 및 이의 용도
CN110684082B (zh) * 2019-10-08 2021-12-10 江苏诺泰澳赛诺生物制药股份有限公司 Gip和glp-1双激动多肽化合物及药学上可接受的盐与用途
EP4148064A4 (en) * 2020-05-09 2024-06-12 Tianjin Institute Of Pharmaceutical Research Co., Ltd. POLYPEPTIDE DERIVATIVE WITH AGONISTIC DOUBLE RECEPTOR ACTIVITY AND USE THEREOF
CN114349828B (zh) * 2020-11-27 2023-12-08 江苏师范大学 Glp-1/胰高血糖素受体双重激动剂及其应用
CN115850438B (zh) * 2021-12-28 2023-05-12 北京惠之衡生物科技有限公司 一种长效glp-1衍生物

Patent Citations (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101790538A (zh) * 2007-02-15 2010-07-28 印第安纳大学研究与科技公司 胰高血糖素/glp-1受体共激动剂
CN103068841A (zh) * 2010-06-24 2013-04-24 西兰制药公司 胰高血糖素类似物
CN104583233A (zh) * 2012-06-21 2015-04-29 印第安纳大学研究及科技有限公司 表现出gip受体活性的胰高血糖素的类似物
CN104583232A (zh) * 2012-06-21 2015-04-29 印第安纳大学研究及科技有限公司 展现gip受体活性的胰高血糖素类似物
CN107735100A (zh) * 2015-06-22 2018-02-23 伊莱利利公司 胰高血糖素和glp‑1协同激动剂化合物
CN109745547A (zh) * 2017-11-06 2019-05-14 深圳市图微安创科技开发有限公司 Glp-1r/gcgr双激动剂多肽治疗胆汁性肝硬化
CN111372945A (zh) * 2017-11-06 2020-07-03 中山大学 基于胃泌酸调节素类似物glp-1r/gcgr双靶点激动剂多肽治疗特发性肺间质纤维化
CN111944055A (zh) * 2019-05-16 2020-11-17 浙江道尔生物科技有限公司 一种治疗代谢疾病的融合蛋白
CN111423506A (zh) * 2019-11-08 2020-07-17 成都奥达生物科技有限公司 一种glp-1化合物
CN115490760A (zh) * 2022-07-04 2022-12-20 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
CN115536739A (zh) * 2022-07-04 2022-12-30 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
CN116063455A (zh) * 2022-07-04 2023-05-05 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及其应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ZHAO FENGHUI, ZHOU QINGTONG, CONG ZHAOTONG, HANG KAINI, ZOU XINYU, ZHANG CHAO, CHEN YAN, DAI ANTAO, LIANG ANYI, MING QIANQIAN, WAN: "Structural insights into multiplexed pharmacological actions of tirzepatide and peptide 20 at the GIP, GLP-1 or glucagon receptors", NATURE COMMUNICATIONS, vol. 13, no. 1, XP093104614, DOI: 10.1038/s41467-022-28683-0 *

Also Published As

Publication number Publication date
CN116063455B (zh) 2023-10-24
CN116063455A (zh) 2023-05-05
CN115490760B (zh) 2023-04-14
CN115536739B (zh) 2023-04-14
CN115490760A (zh) 2022-12-20
CN115536739A (zh) 2022-12-30

Similar Documents

Publication Publication Date Title
CN114349828A (zh) Glp-1/胰高血糖素受体双重激动剂及其应用
WO2024008058A1 (zh) 一种glp-1受体和gcg受体共激动多肽衍生物及其应用
US9260503B2 (en) Multi-substituted insulins
AU2017371516B2 (en) Acylated GLP-1/GLP-2 dual agonists
JP2023011864A (ja) 肥満の治療のためのグルカゴン及びｇｌｐ－１コアゴニスト
JP2016519130A (ja) 治療ペプチド
WO2021227989A1 (zh) 具有双受体激动作用的多肽衍生物及其用途
WO2018104558A1 (en) Acylated glp-1/glp-2 dual agonists
WO2018103868A1 (en) Acylated glp-1/glp-2 dual agonists
JP7350851B2 (ja) グルカゴン由来ペプチド及びその用途
WO2021239115A1 (zh) Glp-1/胰高血糖素双重激动剂融合蛋白
WO2007000118A1 (fr) Fragments polypeptidiques d'exendine 4 et utilisation correspondante
CN116120425A (zh) 一种glp-1/gip受体双重激动剂及其应用
CN118234742A (zh) Glp-1和gip受体双重激动剂的药物组合物及其用途
CN110759991B (zh) 吉非罗齐-非洲爪蟾胰高血糖素样肽-1衍生物及其应用
CN112608378B (zh) 一类glp-1/胆囊收缩素-1受体双重激动剂及其应用
CN106084031A (zh) 一类glp‑1r/gcgr双重激动剂在用于降糖和减肥药物中的运用
CN115785249B (zh) 一类glp-1类似物及其应用
CN115960258B (zh) 一类GLP-1/glucagon/Y2受体三重激动剂及其应用
WO2024152259A1 (zh) 具有三重活性的融合蛋白及其应用
WO2023227133A1 (zh) 人胰淀素类似物、其衍生物及用途
CN115819619A (zh) 一类glp-1/y2受体双重激动剂及其应用
CN116947978A (zh) 一种glp-1和gip受体共激动多肽衍生物及其盐和制剂

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23834825

Country of ref document: EP

Kind code of ref document: A1