CN112608378B - GLP-1/cholecystokinin-1 receptor dual agonist and application thereof - Google Patents
GLP-1/cholecystokinin-1 receptor dual agonist and application thereof Download PDFInfo
- Publication number
- CN112608378B CN112608378B CN202011428688.5A CN202011428688A CN112608378B CN 112608378 B CN112608378 B CN 112608378B CN 202011428688 A CN202011428688 A CN 202011428688A CN 112608378 B CN112608378 B CN 112608378B
- Authority
- CN
- China
- Prior art keywords
- acid
- glu
- lys
- gly
- thr
- Prior art date
- Legal status (The legal status 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 status listed.)
- Active
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
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
-
- 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
- A61P3/06—Antihyperlipidemics
-
- 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
Abstract
The present invention provides a class of GLP-1/CCK-1 receptor dual agonistic polypeptide compounds. The GLP-1/CCK-1 receptor double-excited polypeptide compound has the effects of promoting weight reduction, reversing insulin resistance and regulating lipid metabolism while more effectively reducing blood sugar. The polypeptide compound of the invention has higher agonistic activity to GLP-1 receptor than natural ligand and has selective agonistic activity to CCK-1 receptor. The polypeptide compound provided by the invention has stable chemical property and low side effect, does not cause acute pancreatitis, and is suitable for being used as an active ingredient of medicines for treating metabolic diseases such as diabetes, obesity, hyperlipidemia, NAFLD, NASH and the like.
Description
Technical Field
The invention relates to biological medicine, in particular to a GLP-1/cholecystokinin-1 receptor dual agonist and application thereof.
Background
Obesity and its related metabolic syndrome have become global public health problems, and the incidence and progression of many metabolic syndromes such as type 2 diabetes (T2 DM), nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), dyslipidemia are closely related to obesity. Studies have shown that clinically 80-90% of T2DM patients are associated with overweight or obesity, and the use of weight-loss therapy is beneficial in preventing and controlling disease conditions, including controlling blood glucose, reducing morbidity and disability (mortality), etc. Weight loss is generally difficult to achieve by exercise and diet control alone. The current medicines for treating obesity have limited curative effects, and many medicines for treating obesity have obvious side effects, such as mental symptoms caused by acting on central nerves, serious cardiovascular effects and the like. Only a few drugs alone can achieve 5-10% weight loss, and among the drugs for treating T2DM, only glucagon-like peptide (GLP-1) receptor agonists and sodium-glucose cotransporter 2 (SGLT 2) inhibitors have a better weight control effect (j.med.chem., 2018,61,5580-5593). Bariatric surgery has significant therapeutic effects on obesity, but patients suffer from a greater risk of surgery and the long-term effects of surgery are still unclear. Therefore, there is still a great clinical need for a drug for weight control, and a drug capable of safely and effectively controlling weight and having a primary disease treatment effect is an ideal choice.
GLP-1 is a glucose-dependent hypoglycemic polypeptide hormone secreted by L cells of the terminal jejunum, ileum and colon, and plays a hypoglycemic role after specific binding with GLP-1 receptor. The main advantage of GLP-1 is the glucose-dependent incretin secretion effect, avoiding the risk of hypoglycemia often present in diabetes treatment. In addition to regulating blood glucose, GLP-1 can also prevent pancreatic beta cell degeneration, stimulate beta cell proliferation and differentiation, and can improve diabetes progression from the source. In addition, GLP-1 also has the effects of inhibiting gastric acid secretion, delaying gastric emptying, inhibiting appetite and the like, and has partial weight reduction effect. Several long acting GLP-1 drugs, such as liraglutide, semaglutide and dulaglutine, etc., have been marketed. Although GLP-1 drugs have safe hypoglycemic effect, if better weight loss effect is required, the administration dosage is generally increased, and large-dose GLP-1 drugs are easy to produce gastrointestinal side effects and have poor tolerance, so that the treatment window is narrower. Thus, there remains a need for therapeutic agents that are more safely tolerated, and that are effective in reducing body weight and controlling blood glucose.
Cholecystokinin (CCK), a gastrointestinal hormone secreted by cells of the intestinal tract, the most important physiological role of CCK is to regulate energy metabolism by increasing satiety. In addition, CCK also has the effects of inhibiting gastric emptying, increasing islet beta cell area, and enhancing insulin secretion. The physiological effects of CCK are achieved by agonizing CCK receptors, which belong to the family of G protein-coupled receptors, and are of the two subtypes CCK-1 and CCK-2. CCK-1 receptors are the primary receptors involved in food intake and satiety, while CCK-2 receptors mediate other effects including central nervous system effects and increased islet beta cell area, among others. CCK-8 is an 8-peptide analog of CCK which has agonistic activity at both the CCK-1 receptor and the CCK-2 receptor. Selective agonism of CCK-1 receptors is more beneficial in inhibiting food intake and patent TW201716432A discloses a class of CCK-8 long-acting analogs (NN 9056) with CCK-1 receptor selective agonism. However, CCK-1 receptor agonism is easier to cause acute pancreatitis, which is a major obstacle faced by CCK-1 receptor as a targeting receptor of weight-reducing drugs, and no weight-reducing drugs acting on CCK-1 receptor are currently marketed.
XenGLP-1 is a class of animal-derived GLP-1 analogues found in Xenopus laevis, and XenGLP-1 has better hypoglycemic activity and stability than natural GLP-1. XenGLP-1 has been shown to be much more stable to degradation by Neutral Endopeptidase (NEP) than GLP-1, in addition to being more resistant to degradation by dipeptidyl peptidase (DPP-IV). XenGLP-1 is a potent agonist of the GLP-1 receptor, with many glucose regulatory effects observed with native GLP-1, and many preclinical studies have shown that XenGLP-1 has several beneficial antidiabetic properties, including enhanced glucose-dependent insulin synthesis and secretion, slow gastric emptying, reduced food intake and body weight, and promotion of beta cell proliferation and restoration of islet function, among others (biochem. Phacol., 2017,142,155-167; FASEB J.,2019,33,7113-7125). These effects are beneficial not only for diabetics, but also for patients suffering from obesity. Patients with obese subjects have a higher risk of developing hypertension, hyperlipidemia, diabetes, NAFLD, NASH, musculoskeletal and cardiovascular disease.
The effect of inhibiting ingestion and promoting satiety by exciting CCK-1 receptor can improve the weight reduction effect of GLP-1 medicines. L.trevaskis et al describe the simultaneous use of a CCK-8 analogue and the GLP-1 receptor agonist exendin-4 (exendin-4) which significantly improves weight loss compared to exendin-4 alone (Diabetes obes.metab.,2015,17,61-73). However, selective simultaneous agonism of the GLP-1 receptor and the CCK-1 receptor is critical to avoid the occurrence of side effects, particularly severe side effects such as acute pancreatitis.
Disclosure of Invention
The object of the present invention is to provide a novel polypeptide compound having a dual GLP-1/CCK-1 receptor agonism, which is a variant designed based on the XenGLP-1 sequence, retains the therapeutic effect of XenGLP-1 on diabetes while having a beneficial effect of selectively agonising the CCK-1 receptor on ingestion, and does not cause acute pancreatitis, and which has a potential more than a single receptor agonist in the preparation of a medicament for the treatment of metabolic syndrome such as obesity, diabetes, NAFLD, NASH and the like.
In order to achieve the above purpose, the technical scheme of the invention is as follows:
the GLP-1/CCK-1 receptor double-excited polypeptide compound has an amino acid sequence general formula:
His-Xaa 1 -Glu-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Thr-Glu-Tyr-Leu-Glu-Glu-Xaa 2 -Ala-Ala-Xaa 3 -Glu-Phe-Ile-Glu-Trp-Leu-Ile-Lys-Gly-Xaa 4 -Xaa 5 -Xaa 6 -Asp-Xaa 7 -Nle-Gly-Trp-Nle-DMeAsp-Xaa 8 -NH 2
wherein:
Xaa 1 is selected from Ala, D-Ala, gly or Aib;
Xaa 2 lys from Glu, lys or side chain modified;
Xaa 3 lys from Lys or modified side chain;
Xaa 4 lys from Lys or modified side chain;
Xaa 5 from Ala or AEEA;
Xaa 6 from Ala or AEEA;
Xaa 7 from Phe (4 sm) or sTyr;
Xaa 8 from Phe or MePhe;
wherein the side chain modified Lys is selected from Lys (gamma-Glu-CO- (CH) 2 ) n -CH 3 ) Or Lys (AEEA-AEEA-gamma-Glu-CO- (CH) 2 ) n -COOH)，
Lys(γ-Glu-CO-(CH 2 ) n -CH 3 ) The structural formula of (2) is shown as follows:
Lys(AEEA-AEEA-γ-Glu-CO-(CH 2 ) n -COOH) is represented by the formula:
wherein n is a natural number, and n is more than or equal to 12 and less than or equal to 20.
Preferably, n is 14, 16, 18 or 20.
Preferably, the amino acid sequence of the polypeptide compound is one of the following sequences:
(1)SEQ ID NO:1
His-Ala-Glu-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Thr-Glu-Tyr-Leu-Glu-Glu-Lys(γ-Glu-CO-(CH 2 ) 14 -CH 3 )-Ala-Ala-Lys-Glu-Phe-Ile-Glu-Trp-Leu-Ile-Lys-Gly-Lys-AEEA-AEEA-Asp-Phe(4sm)-Nle-Gly-Trp-Nle-DMeAsp-MePhe-NH 2
(2)SEQ ID NO:2
His-Aib-Glu-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Thr-Glu-Tyr-Leu-Glu-Glu-Lys(γ-Glu-CO-(CH 2 ) 14 -CH 3 )-Ala-Ala-Lys-Glu-Phe-Ile-Glu-Trp-Leu-Ile-Lys-Gly-Lys-AEEA-AEEA-Asp-Phe(4sm)-Nle-Gly-Trp-Nle-DMeAsp-MePhe-NH 2
(3)SEQ ID NO:3
His-Ala-Glu-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Thr-Glu-Tyr-Leu-Glu-Glu-Lys(AEEA-AEEA-γ-Glu-CO-(CH 2 ) 16 -COOH)-Ala-Ala-Lys-Glu-Phe-Ile-Glu-Trp-Leu-Ile-Lys-Gly-Lys-AEEA-AEEA-Asp-Phe(4sm)-Nle-Gly-Trp-Nle-DMeAsp-MePhe-NH 2
(4)SEQ ID NO:4
His-Aib-Glu-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Thr-Glu-Tyr-Leu-Glu-Glu-Lys(AEEA-AEEA-γ-Glu-CO-(CH 2 ) 16 -COOH)-Ala-Ala-Lys-Glu-Phe-Ile-Glu-Trp-Leu-Ile-Lys-Gly-Lys-AEEA-AEEA-Asp-Phe(4sm)-Nle-Gly-Trp-Nle-DMeAsp-MePhe-NH 2
(5)SEQ ID NO:5
His-Ala-Glu-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Thr-Glu-Tyr-Leu-Glu-Glu-Glu-Ala-Ala-Lys(γ-Glu-CO-(CH 2 ) 14 -CH 3 )-Glu-Phe-Ile-Glu-Trp-Leu-Ile-Lys-Gly-Lys-AEEA-AEEA-Asp-Phe(4sm)-Nle-Gly-Trp-Nle-DMeAsp-MePhe-NH 2
(6)SEQ ID NO:6
His-Aib-Glu-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Thr-Glu-Tyr-Leu-Glu-Glu-Glu-Ala-Ala-Lys(γ-Glu-CO-(CH 2 ) 14 -CH 3 )-Glu-Phe-Ile-Glu-Trp-Leu-Ile-Lys-Gly-Lys-AEEA-AEEA-Asp-Phe(4sm)-Nle-Gly-Trp-Nle-DMeAsp-MePhe-NH 2
(7)SEQ ID NO:7
His-Ala-Glu-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Thr-Glu-Tyr-Leu-Glu-Glu-Glu-Ala-Ala-Lys(AEEA-AEEA-γ-Glu-CO-(CH 2 ) 16 -COOH)-Glu-Phe-Ile-Glu-Trp-Leu-Ile-Lys-Gly-Lys-AEEA-AEEA-Asp-Phe(4sm)-Nle-Gly-Trp-Nle-DMeAsp-MePhe-NH 2
(8)SEQ ID NO:8
His-Aib-Glu-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Thr-Glu-Tyr-Leu-Glu-Glu-Glu-Ala-Ala-Lys(AEEA-AEEA-γ-Glu-CO-(CH 2 ) 16 -COOH)-Glu-Phe-Ile-Glu-Trp-Leu-Ile-Lys-Gly-Lys-AEEA-AEEA-Asp-Phe(4sm)-Nle-Gly-Trp-Nle-DMeAsp-MePhe-NH 2
(9)SEQ ID NO:9
His-Ala-Glu-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Thr-Glu-Tyr-Leu-Glu-Glu-Glu-Ala-Ala-Lys-Glu-Phe-Ile-Glu-Trp-Leu-Ile-Lys-Gly-Lys(γ-Glu-CO-(CH 2 ) 14 -CH 3 )-AEEA-AEEA-Asp-Phe(4sm)-Nle-Gly-Trp-Nle-DMeAsp-MePhe-NH 2
(10)SEQ ID NO:10
His-Aib-Glu-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Thr-Glu-Tyr-Leu-Glu-Glu-Glu-Ala-Ala-Lys-Glu-Phe-Ile-Glu-Trp-Leu-Ile-Lys-Gly-Lys(γ-Glu-CO-(CH 2 ) 14 -CH 3 )-AEEA-AEEA-Asp-Phe(4sm)-Nle-Gly-Trp-Nle-DMeAsp-MePhe-NH 2
(11)SEQ ID NO:11
His-Ala-Glu-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Thr-Glu-Tyr-Leu-Glu-Glu-Glu-Ala-Ala-Lys-Glu-Phe-Ile-Glu-Trp-Leu-Ile-Lys-Gly-Lys(AEEA-AEEA-γ-Glu-CO-(CH 2 ) 16 -COOH)-AEEA-AEEA-Asp-Phe(4sm)-Nle-Gly-Trp-Nle-DMeAsp-MePhe-NH 2
(12)SEQ ID NO:12
His-Aib-Glu-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Thr-Glu-Tyr-Leu-Glu-Glu-Glu-Ala-Ala-Lys-Glu-Phe-Ile-Glu-Trp-Leu-Ile-Lys-Gly-Lys(AEEA-AEEA-γ-Glu-CO-(CH 2 ) 16 -COOH)-AEEA-AEEA-Asp-Phe(4sm)-Nle-Gly-Trp-Nle-DMeAsp-MePhe-NH 2
the invention also provides pharmaceutically acceptable salts of GLP-1/CCK-1 receptor dual agonist polypeptide compounds.
Preferably, the salt is a salt of a GLP-1/CCK-1 receptor dual agonist polypeptide compound with one of the following compounds: hydrobromic acid, hydrochloric acid, methanesulfonic acid, phosphoric acid, ethanesulfonic acid, formic acid, p-toluenesulfonic acid, acetic acid, acetoacetic acid, pyruvic acid, pectate acid, butyric acid, caproic acid, benzenesulfonic acid, heptanoic acid, undecanoic acid, benzoic acid, salicylic acid, lauric acid, 2- (4-hydroxybenzoyl) benzoic acid, cinnamic acid, camphoric acid, cyclopentanepropionic acid, 3-hydroxy-2-naphthoic acid, camphorsulfonic acid, digluconic acid, nicotinic acid, pamoic acid, propionic acid, persulfuric acid, picric acid, 3-phenylpropionic acid, pivalic acid, itaconic acid, 2-hydroxyethanesulfonic acid, sulfamic acid, dodecylsulfuric acid, trifluoromethanesulfonic acid, naphthalenedisulfonic acid, 2-naphthalenesulfonic acid, citric acid, mandelic acid, ascorbic acid, wine stearic acid, lithonic acid, oxalic acid, lactic acid, succinic acid, malonic acid, hemisulfuric acid, malic acid, maleic acid, alginic acid, fumaric acid, D-gluconic acid, glycerophosphoric acid, glucoheptonic acid, aspartic acid, thiocyanic acid, or sulfosalicylic acid.
The invention also provides a pharmaceutical composition of a GLP-1/CCK-1 receptor dual agonist polypeptide compound, the pharmaceutical composition comprising: the compound is prepared from any GLP-1/CCK-1 receptor double-excited polypeptide compound or pharmaceutically acceptable salt thereof serving as an effective raw material and a pharmaceutically acceptable carrier or diluent.
The invention also provides a medicament containing the GLP-1/CCK-1 receptor double-excited polypeptide compound, wherein the medicament is any one of capsules, tablets, sprays, inhalants, injections, patches, emulsions, films, powder or compound preparations which are pharmaceutically acceptable pharmaceutical excipients, carriers or diluents.
The invention also provides application of the GLP-1/CCK-1 receptor double-excited polypeptide compound, pharmaceutically acceptable salt thereof, pharmaceutical composition thereof or medicament thereof in preparing medicaments for treating metabolic diseases or symptoms. In a particular aspect, the metabolic disease or disorder is diabetes, NAFLD, NASH, hyperlipidemia, or obesity. In particular aspects, the diabetes is type 1 diabetes, T2DM or gestational diabetes. In a particular aspect, the medicament is for treating more than one metabolic disease or disorder, e.g., diabetes and NAFLD, NASH or obesity; obesity and NASH or NAFLD; diabetes, NASH, and obesity; diabetes, NAFLD and obesity; or diabetes and obesity.
Compared with the prior art, the invention has the beneficial effects that:
compared with the existing GLP-1 receptor agonist, the GLP-1/CCK-1 receptor double-excited polypeptide compound has the effects of promoting weight reduction and preventing weight gain, regulating lipid metabolism and has unexpected beneficial effects compared with the existing medicines while more effectively reducing blood sugar. The polypeptide compound provided by the invention has higher agonistic activity to GLP-1 receptor than natural ligand, and simultaneously has selective agonistic activity to CCK-1 receptor, and can not agonize CCK-2 receptor. The polypeptide compound provided by the invention has stable chemical properties, is not easy to be degraded by DPP-IV and NEP in vivo, is not easy to be filtered by glomerulus, has obviously improved stability, and has pharmacokinetic characteristics for supporting once-daily administration or once-weekly administration. The polypeptide compound provided by the invention has improved biophysical properties, has higher solubility than natural GLP-1 and CCK-8 at neutral pH and pH 4.5, and has the property of being beneficial to preparation. The polypeptide compound provided by the invention has low immunogenicity, and has better treatment effect on metabolic diseases such as T2DM, obesity, NAFLD, NASH, hyperlipidemia and the like than the existing marketed drugs. The GLP-1 part of the polypeptide compound provided by the invention is selected from XenGLP-1, and compared with the GLP-1 receptor agonist which is marketed and the CCK-1 receptor agonist which is reported, the side effect is lower, and the occurrence of acute pancreatitis is avoided. Therefore, the polypeptide compound provided by the invention is suitable for being used as an active ingredient of medicines for treating metabolic diseases, such as diabetes, obesity, hyperlipidemia, NAFLD, NASH and the like.
The unnatural amino acids of the polypeptide compounds of the invention are defined in Table 1 below.
TABLE 1 definition of unnatural amino acids in polypeptide Compounds of the invention
Drawings
FIG. 1 shows the long-acting hypoglycemic effect of single administration of each subject in the non-fasted state of db/db mice.
Detailed Description
The following abbreviations are used throughout this specification:
english abbreviation Chinese
Gly glycine
Ser serine
Ala alanine
Thr threonine
Val valine
Ile isoleucine
Leu leucine
Tyr tyrosine
Phe-phenylalanine
His histidine
Pro proline
Asp aspartic acid
Met methionine
Glu glutamic acid
Trp tryptophan
Lys lysine
Arg arginine
Asn asparagine
Gln glutamine
Cys cysteine
Aib alphA-Aminoisobutyric acid
AEEA 8-amino-3, 6 dioxaoctanoic acid
DCM dichloromethane
DMF dimethylformamide
Fmoc 9-fluorenylmethoxycarbonyl
Boc
DMSO dimethyl sulfoxide
DIC N, N' -diisopropylcarbodiimide
HOBT 1-hydroxy-benzotriazoles
Alloc allyloxycarbonyl
Dde 1- (4, 4-dimethyl-2, 6-dioxocyclohexylidene) -ethyl
Mtt 4-methyltrityl
ivDde 1- (4, 4-dimethyl-2, 6-dioxocyclohexylidene) 3-methyl-butyl
TCE trichloroethylene
TFA trifluoroacetic acid
EDT dimercaptoethane
HPLC high performance liquid chromatography
LC-MS liquid chromatography-mass spectrometry
DMEM Du Beike modified IgE's medium
FBS fetal bovine serum
PBS phosphate buffered saline
HEPES 2- [4- (2-hydroxyethyl) piperazin-1-yl ] ethanesulfonic acid
BSA bovine serum albumin
IBMX 3-isobutyl-1-methylxanthine
HBSS Hanks' balanced salt solution
Example 1
Synthesis of polypeptide compound of SEQ ID No. 1
His-Ala-Glu-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Thr-Glu-Tyr-Leu-Glu-Glu-Lys(γ-Glu-CO-(CH 2 ) 14 -CH 3 )-Ala-Ala-Lys-Glu-Phe-Ile-Glu-Trp-Leu-Ile-Lys-Gly-Lys-AEEA-AEEA-Asp-Phe(4sm)-Nle-Gly-Trp-Nle-DMeAsp-MePhe-NH 2
(1) Swelling of the resin
0.262g (0.1 mmol equivalent) of Rink Amide MBHA resin with a loading of 0.382mmol/g was weighed into a 25mL reactor, the resin was alternately washed 1 time with 7mL of DCM and methanol, 2 times with 7mL of DCM, then the resin was swollen with 7mL of DCM for 1h, and finally the resin was washed 3 times with 7mL of DMF.
(2) Removal of Fmoc protecting groups from resin
Transferring the swelled resin into a PSI200 polypeptide synthesizer, adding 7mL of 20% piperidine/DMF (v/v) for reaction at room temperature for 5min, filtering off the deprotection solution, washing the resin once by 7mL of DMF, adding 7mL of 20% piperidine/DMF (v/v) for reaction with the resin for 15min, and washing the resin for 4 times by 7mL of DMF for 1.5min each time to obtain the Rink resin without Fmoc protecting groups.
(3) Synthesis of Fmoc-MePhe-Rink amide-MBHA Resin
Fmoc-MePhe-OH (0.4 mmol) was weighed, dissolved in 3mL 10% DMF/DMSO (v/v), 2mL DIC/HOBt (0.4 mmol/0.44 mmol) condensing agent was added, pre-activated for 30min, the activated amino acid was added to the reactor, the reaction was allowed to proceed with shaking at room temperature for 2h, the reaction solution was filtered off, the resin was washed 4 times with 7mL DMF, and the Kaiser reagent was used to determine if the reaction coupling was complete, and if not, 2 times.
(4) Extension of peptide chain
And (3) according to the sequence of the peptide chain, repeating the deprotection and coupling steps to sequentially connect corresponding amino acids until the peptide chain is synthesized. Wherein, the Lys at 17-position can be Fmoc-Lys (Alloc) -OH, fmoc-Lys (Dde) -OH, fmoc-Lys (Mtt) -OH or Fmoc-Lys (ivDde) -OH, etc. Fmoc-Lys (Dde) -OH protection strategy was used in this example, while Boc-His (Boc) -OH was used for the N-terminal His. Furthermore, selected at position 34 Phe (4 sm) is Fmoc-Phe (4 sm-TCE) -OH protected by the side chain TCE.
(5) Modification of Lys side chains
After the peptide chain synthesis is completed, 7mL of 2% hydrazine hydrate/DMF (v/v) is added to selectively remove Dde protecting group of Lys at 17 th position, after the Dde protecting group is removed, 0.4mmol of Fmoc-Glu-OtBu,0.4mmol of DIC and 0.44mmol of HOBt are added to carry out oscillation reaction for 2h. Then, after Fmoc protecting group was removed by the same method as described above, 0.4mmol of palmitic acid, 0.4mmol of DIC and 0.44mmol of HOBt were added for condensation reaction for 2 hours, and after completion of the reaction, the resin was washed with 7mL of DMF for 4 times.
(6) Cleavage of polypeptides
The obtained resin connected with the polypeptide is transferred into a round bottom bottle, 5mL of a cutting agent Reagent R (TFA/benzyl sulfide/phenol/EDT, 90:5:3:2, V/V) is used for cutting the resin, the resin is reacted for 2 hours in an oil bath at the constant temperature of 30 ℃, the cutting fluid is poured into 40mL of glacial ethyl ether, the crude product is washed 3 times by 15mL of glacial ethyl ether after refrigerated centrifugation, and finally the crude peptide is obtained by drying with nitrogen. The crude peptide was dissolved in acetic acid and the TCE protecting group of Phe (4 sm) was removed using hydrochloric acid activated zinc powder.
(7) Purification of polypeptides
Dissolving the target polypeptide crude product in water, filtering with 0.25 μm microporous membrane, and purifying with island jin preparation type reversed phase HPLC system. Chromatographic conditions were C18 reverse phase preparation column (250 mm. Times.20 mm,12 μm); mobile phase a:0.1% TFA/water (V/V), mobile phase B: methanol (V/V); the flow rate is 8mL/min; the detection wavelength was 214nm. Eluting with linear gradient (20-70% B/30 min), collecting target peak, removing methanol, lyophilizing to obtain pure product with purity of 0.24g or more than 98%, and determining molecular weight of target polypeptide by LC-MS.
Example 2
Synthesis of polypeptide compound of SEQ ID No. 2
His-Aib-Glu-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Thr-Glu-Tyr-Leu-Glu-Glu-Lys(γ-Glu-CO-(CH 2 ) 14 -CH 3 )-Ala-Ala-Lys-Glu-Phe-Ile-Glu-Trp-Leu-Ile-Lys-Gly-Lys-AEEA-AEEA-Asp-Phe(4sm)-Nle-Gly-Trp-Nle-DMeAsp-MePhe-NH 2
The synthesis method is the same as that of example 1, and the target peak is collected and freeze-dried to obtain 0.23g of pure product.
Example 3
Synthesis of polypeptide compound of SEQ ID No. 3
His-Ala-Glu-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Thr-Glu-Tyr-Leu-Glu-Glu-Lys(AEEA-AEEA-γ-Glu-CO-(CH 2 ) 16 -COOH)-Ala-Ala-Lys-Glu-Phe-Ile-Glu-Trp-Leu-Ile-Lys-Gly-Lys-AEEA-AEEA-Asp-Phe(4sm)-Nle-Gly-Trp-Nle-DMeAsp-MePhe-NH 2
(1) Swelling of the resin
0.262g (0.1 mmol equivalent) of Rink Amide MBHA resin with a loading of 0.382mmol/g was weighed into a 25mL reactor, the resin was alternately washed 1 time with 7mL of DCM and methanol, 2 times with 7mL of DCM, then the resin was swollen with 7mL of DCM for 1h, and finally the resin was washed 3 times with 7mL of DMF.
(2) Removal of Fmoc protecting groups from resin
Transferring the swelled resin into a PSI200 polypeptide synthesizer, adding 7mL of 20% piperidine/DMF (v/v) for reaction at room temperature for 5min, filtering off the deprotection solution, washing the resin once by 7mL of DMF, adding 7mL of 20% piperidine/DMF (v/v) for reaction with the resin for 15min, and washing the resin for 4 times by 7mL of DMF for 1.5min each time to obtain the Rink resin without Fmoc protecting groups.
(3) Synthesis of Fmoc-MePhe-Rink amide-MBHA Resin
Fmoc-MePhe-OH (0.4 mmol) was weighed, dissolved in 3mL 10% DMF/DMSO (v/v), 2mL DIC/HOBt (0.4 mmol/0.44 mmol) condensing agent was added, pre-activated for 30min, the activated amino acid was added to the reactor, the reaction was allowed to proceed with shaking at room temperature for 2h, the reaction solution was filtered off, the resin was washed 4 times with 7mL DMF, and the Kaiser reagent was used to determine if the reaction coupling was complete, and if not, 2 times.
(4) Extension of peptide chain
And (3) according to the sequence of the peptide chain, repeating the deprotection and coupling steps to sequentially connect corresponding amino acids until the peptide chain is synthesized. Wherein, the Lys at 17-position can be Fmoc-Lys (Alloc) -OH, fmoc-Lys (Dde) -OH, fmoc-Lys (Mtt) -OH or Fmoc-Lys (ivDde) -OH, etc. Fmoc-Lys (Dde) -OH protection strategy was used in this example, while Boc-His (Boc) -OH was used for the N-terminal His. Furthermore, selected at position 34 Phe (4 sm) is Fmoc-Phe (4 sm-TCE) -OH protected by the side chain TCE.
(5) Modification of Lys side chains
After the peptide chain synthesis is completed, 7mL of 2% hydrazine hydrate/DMF (v/v) is added to selectively remove Dde protecting group of Lys at 17 th position, after the Dde protecting group is removed, 0.4mmol of Fmoc-AEEA-OH,0.4mmol of DIC and 0.44mmol of HOBt are added, and oscillation condensation reaction is carried out for 2h. After removal of Fmoc protecting groups, 0.4mmol Fmoc-AEEA-OH,0.4mmol DIC and 0.44mmol HOBt were added again and the reaction was performed by shaking for 2h. After removal of Fmoc protecting groups, 0.4mmol Fmoc-Glu-OtBu,0.4mmol DIC and 0.44mmol HOBt were added and the reaction was performed by shaking for 2h. After Fmoc protecting groups were removed, 0.4mmol of mono-tert-butyl octadecanedioate, 0.4mmol of DIC and 0.44mmol of HOBt were added for condensation reaction for 2h, and after completion of the reaction the resin was washed 4 times with 7mL of DMF.
(6) Cleavage of polypeptides
The obtained resin connected with the polypeptide is transferred into a round bottom bottle, 5mL of a cutting agent Reagent R (TFA/benzyl sulfide/phenol/EDT, 90:5:3:2, V/V) is used for cutting the resin, the resin is reacted for 2 hours in an oil bath at the constant temperature of 30 ℃, the cutting fluid is poured into 40mL of glacial ethyl ether, the crude product is washed 3 times by 15mL of glacial ethyl ether after refrigerated centrifugation, and finally the crude peptide is obtained by drying with nitrogen. The crude peptide was dissolved in acetic acid and the TCE protecting group of Phe (4 sm) was removed using hydrochloric acid activated zinc powder.
(7) Purification of polypeptides
Dissolving the target polypeptide crude product in water, filtering with 0.25 μm microporous membrane, and purifying with island jin preparation type reversed phase HPLC system. Chromatographic conditions were C18 reverse phase preparation column (250 mm. Times.20 mm,12 μm); mobile phase a:0.1% TFA/water (V/V), mobile phase B: methanol (V/V); the flow rate is 8mL/min; the detection wavelength was 214nm. Eluting with linear gradient (20-70% B/30 min), collecting target peak, removing methanol, lyophilizing to obtain pure product with purity greater than 98%, and determining molecular weight of target polypeptide by LC-MS.
Example 4
Synthesis of polypeptide compound of SEQ ID No. 4
His-Aib-Glu-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Thr-Glu-Tyr-Leu-Glu-Glu-Lys(AEEA-AEEA-γ-Glu-CO-(CH 2 ) 16 -COOH)-Ala-Ala-Lys-Glu-Phe-Ile-Glu-Trp-Leu-Ile-Lys-Gly-Lys-AEEA-AEEA-Asp-Phe(4sm)-Nle-Gly-Trp-Nle-DMeAsp-MePhe-NH 2
The synthesis method is the same as that of example 3, and the target peak is collected and freeze-dried to obtain 0.25g of pure product.
Example 5
Synthesis of polypeptide compound of SEQ ID No. 5
His-Ala-Glu-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Thr-Glu-Tyr-Leu-Glu-Glu-Glu-Ala-Ala-Lys(γ-Glu-CO-(CH 2 ) 14 -CH 3 )-Glu-Phe-Ile-Glu-Trp-Leu-Ile-Lys-Gly-Lys-AEEA-AEEA-Asp-Phe(4sm)-Nle-Gly-Trp-Nle-DMeAsp-MePhe-NH 2
The synthesis method is the same as that of example 1, and the target peak is collected and freeze-dried to obtain 0.19g of pure product.
Example 6
Synthesis of polypeptide compound of SEQ ID No. 6
His-Aib-Glu-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Thr-Glu-Tyr-Leu-Glu-Glu-Glu-Ala-Ala-Lys(γ-Glu-CO-(CH 2 ) 14 -CH 3 )-Glu-Phe-Ile-Glu-Trp-Leu-Ile-Lys-Gly-Lys-AEEA-AEEA-Asp-Phe(4sm)-Nle-Gly-Trp-Nle-DMeAsp-MePhe-NH 2
The synthesis method is the same as that of example 1, and the target peak is collected and freeze-dried to obtain 0.18g of pure product.
Example 7
Synthesis of polypeptide compound of SEQ ID No. 7
His-Ala-Glu-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Thr-Glu-Tyr-Leu-Glu-Glu-Glu-Ala-Ala-Lys(AEEA-AEEA-γ-Glu-CO-(CH 2 ) 16 -COOH)-Glu-Phe-Ile-Glu-Trp-Leu-Ile-Lys-Gly-Lys-AEEA-AEEA-Asp-Phe(4sm)-Nle-Gly-Trp-Nle-DMeAsp-MePhe-NH 2
The synthesis method is the same as that of example 3, and the target peak is collected and freeze-dried to obtain 0.26g of pure product.
Example 8
Synthesis of polypeptide compound of SEQ ID No. 8
His-Aib-Glu-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Thr-Glu-Tyr-Leu-Glu-Glu-Glu-Ala-Ala-Lys(AEEA-AEEA-γ-Glu-CO-(CH 2 ) 16 -COOH)-Glu-Phe-Ile-Glu-Trp-Leu-Ile-Lys-Gly-Lys-AEEA-AEEA-Asp-Phe(4sm)-Nle-Gly-Trp-Nle-DMeAsp-MePhe-NH 2
The synthesis method is the same as that of example 3, and the target peak is collected and freeze-dried to obtain 0.23g of pure product.
Example 9
Synthesis of polypeptide compound of SEQ ID No. 9
His-Ala-Glu-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Thr-Glu-Tyr-Leu-Glu-Glu-Glu-Ala-Ala-Lys-Glu-Phe-Ile-Glu-Trp-Leu-Ile-Lys-Gly-Lys(γ-Glu-CO-(CH 2 ) 14 -CH 3 )-AEEA-AEEA-Asp-Phe(4sm)-Nle-Gly-Trp-Nle-DMeAsp-MePhe-NH 2
The synthesis method is the same as that of example 1, and the target peak is collected and freeze-dried to obtain 0.17g of pure product.
Example 10
Synthesis of polypeptide compound of SEQ ID No. 10
His-Aib-Glu-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Thr-Glu-Tyr-Leu-Glu-Glu-Glu-Ala-Ala-Lys-Glu-Phe-Ile-Glu-Trp-Leu-Ile-Lys-Gly-Lys(γ-Glu-CO-(CH 2 ) 14 -CH 3 )-AEEA-AEEA-Asp-Phe(4sm)-Nle-Gly-Trp-Nle-DMeAsp-MePhe-NH 2
The synthesis method is the same as that of example 1, and the target peak is collected and freeze-dried to obtain 0.25g of pure product.
Example 11
Synthesis of polypeptide compound of SEQ ID NO. 11
His-Ala-Glu-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Thr-Glu-Tyr-Leu-Glu-Glu-Glu-Ala-Ala-Lys-Glu-Phe-Ile-Glu-Trp-Leu-Ile-Lys-Gly-Lys(AEEA-AEEA-γ-Glu-CO-(CH 2 ) 16 -COOH)-AEEA-AEEA-Asp-Phe(4sm)-Nle-Gly-Trp-Nle-DMeAsp-MePhe-NH 2
The synthesis method is the same as that of example 3, and the target peak is collected and freeze-dried to obtain 0.23g of pure product.
Example 12
Synthesis of polypeptide compound of SEQ ID NO 12
His-Aib-Glu-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Thr-Glu-Tyr-Leu-Glu-Glu-Glu-Ala-Ala-Lys-Glu-Phe-Ile-Glu-Trp-Leu-Ile-Lys-Gly-Lys(AEEA-AEEA-γ-Glu-CO-(CH 2 ) 16 -COOH)-AEEA-AEEA-Asp-Phe(4sm)-Nle-Gly-Trp-Nle-DMeAsp-MePhe-NH 2
The synthesis method is the same as that of example 3, and the target peak is collected and freeze-dried to obtain 0.27g of pure product.
Example 13
Determination of agonistic Activity of polypeptide Compounds on GLP-1 receptor, CCK-1 receptor and CCK-2 receptor
Agonism of the receptor by the polypeptide compound is determined by a functional assay that measures cAMP response of HEK-293 cell lines stably expressing human GLP-1 receptor. Cells stably expressing the GLP-1 receptor were split into T175 flasks and grown in medium (DMEM/10% FBS) overnight to near confluence, then the medium was removed, and the cells were washed with calcium and magnesium free PBS and then protease treated with Actuase enzyme. The detached cells were washed and resuspended in assay buffer (20mM HEPES,0.1%BSA,2mM IBMX,1 ×hbss) and cell density was determined and 25 μl aliquots were dispensed into wells of 96-well plates. For measurement, 25 μl of a solution of the test polypeptide compound in the assay buffer was added to the wells, followed by incubation at room temperature for 30 minutes. The cAMP content of cells was determined based on Homogeneous Time Resolved Fluorescence (HTRF) using the Cisbio kit. After addition of HTRF reagents diluted in lysis buffer (kit components), the plates were incubated for 1 hour, and then the fluorescence ratio at 665/620nm was measured. By detecting the concentration that caused 50% of activation of the maximal response (EC 50 ) To quantify the in vitro potency of the agonist.
1321-N1 cells stably expressing the CCK-1 receptor or CCK-2 receptor were cultured with DMEM-31966 (containing 10% FBS,1% sodium pyruvate, 1% penicillin, 1% streptomycin). The day before the assay, cells were transferred to 384 well plates and the compounds were dissolved in IP-One buffer (containing 10mmol/L HEPES,1mmol +.L CaCl 2 4.2mmol/L KCl,146mmol/L NaCl,5.5mmol/L glucose, 50mmol/L LiCl) and diluted and added to 384 well plates. After incubation for 1 hour at 37 ℃, the intracellular inositol 1-phosphate concentration was determined using IP-One HTRF Assay kit by detecting the concentration that caused 50% of the maximum response to activation (EC 50 ) To quantify the in vitro potency of the agonist.
The test data (nM) in the examples of this patent application are shown in Table 2 below, and although the test data is stated in terms of a number of significant digits, it should not be considered to indicate that the data has been determined to be exactly a significant digit.
Table 2: EC of polypeptide compounds to GLP-1 receptor, CCK-1 receptor and CCK-2 receptor 50 Value (expressed in nM)
| Sample of | EC 50 (GLP-1 receptor) | EC 50 (CCK-1 receptor) | EC 50 (CCK-2 receptor) |
| GLP-1 | 0.082 | >100000 | >100000 |
| CCK-8 | >100000 | 0.95 | 0.98 |
| NN9056 | >100000 | 0.88 | 5598 |
| SEQ ID NO:1 | 0.041 | 0.68 | >100000 |
| SEQ ID NO:2 | 0.036 | 0.85 | >100000 |
| SEQ ID NO:3 | 0.053 | 0.76 | >100000 |
| SEQ ID NO:4 | 0.045 | 0.81 | >100000 |
| SEQ ID NO:5 | 0.035 | 0.58 | >100000 |
| SEQ ID NO:6 | 0.026 | 0.62 | >100000 |
| SEQ ID NO:7 | 0.031 | 0.52 | >100000 |
| SEQ ID NO:8 | 0.022 | 0.60 | >100000 |
| SEQ ID NO:9 | 0.068 | 0.93 | >100000 |
| SEQ ID NO:10 | 0.059 | 0.86 | >100000 |
| SEQ ID NO:11 | 0.062 | 0.80 | >100000 |
| SEQ ID NO:12 | 0.057 | 0.91 | >100000 |
As shown in Table 2, all polypeptide compounds had agonistic activity at GLP-1 receptor higher than native GLP-1 and most of the polypeptide compounds had agonistic activity at CCK-1 receptor higher than CCK-8 and NN9056, while all the polypeptide compounds showed high selectivity of CCK-1 receptor agonism and agonism selectivity at CCK-1 receptor higher than NN9056, and CCK-8 had no agonism selectivity at CCK-1 receptor.
Example 14
Solubility and stability test of polypeptide Compounds
Before testing the solubility and stability of polypeptide compounds, the purity was first determined using HPLC. Then, based on the determined% purity, 10mg of polypeptide compound was dissolved in 1mL of solution in a different buffer system, and gently stirred for 2 hours. After centrifugation at 4500rpm for 20 minutes, the supernatant was analyzed by HPLC to determine the peak area. And then comparing the sample solution with the corresponding sample standard solution, and calculating the relative concentration of the sample solution. For stability testing, an aliquot of the supernatant obtained from solubility was stored at 40 ℃ for 7 days, then the sample was centrifuged at 4500rpm for 20 minutes, and the supernatant was analyzed by HPLC to determine peak area. By comparing the peak areas (t 0 ) And the peak area (t) 7 ) The "% remaining peptide" was obtained. Calculated according to the following formula: % residual peptide= [ (peak area t) 7 )×100]Peak area t 0 Stability was expressed as "% remaining peptide", and the results of the calculation are shown in table 3 below.
Table 3: solubility and stability of polypeptide compounds
As shown in the results of Table 3, the polypeptide compound of the present invention has a significantly improved solubility in the pH condition of an injection acceptable to the body, compared with natural GLP-1 and CCK-8, and has the property of being advantageous for preparation. The polypeptide compounds of the invention also have high solubility at pH 4.5, a property which may allow co-formulations for combination therapy with insulin or insulin derivatives. In addition, the polypeptide compounds of the invention also have high stability at pH 4.5 and neutral pH.
Example 15
Stability of polypeptide Compounds against DPP-IV and NEP enzymes
The sample was incubated with purified human DPP-IV or NEP enzyme at 37℃for 0,2,4,8 hours, the peak area of the residual sample in the solution was measured at each time point by HPLC, and the half-life of the sample was calculated, and the results are shown in Table 4.
Table 4: half-life (expressed as h) of polypeptide compounds in DPP-IV enzyme or NEP enzyme systems
| Sample of | Half-life (DPP-IV) | Half-life (NEP middle) |
| GLP-1 | 1.5 | 1.9 |
| CCK-8 | 2.3 | 3.9 |
| SEQ ID NO:2 | >8 | >8 |
| SEQ ID NO:5 | >8 | >8 |
| SEQ ID NO:6 | >8 | >8 |
| SEQ ID NO:7 | >8 | >8 |
| SEQ ID NO:8 | >8 | >8 |
| SEQ ID NO:10 | >8 | >8 |
| SEQ ID NO:12 | >8 | >8 |
As shown in the results of Table 4, the half-life of the polypeptide compounds of the present invention in both DPP-IV enzyme-containing solution and NEP enzyme-containing solution systems is more than 8 hours, indicating that the degradation of DPP-IV and NEP enzymes can be effectively tolerated.
Example 16
Pharmacokinetic properties of polypeptide Compounds in rats
Rats were given 50nmol/kg of subcutaneous (s.c.) injection and blood samples were collected 0.25,0.5,1,2,4,8, 16, 24, 36 and 48 hours after administration. After precipitation of the proteins using acetonitrile, plasma samples were analyzed by LC-MS. The pharmacokinetic parameters and half-life were calculated using WinnLin 5.2.1 (non-compartmental model) (Table 5).
Table 5: pharmacokinetic profile of polypeptide Compounds in rats
| Sample of | T 1/2 (h) | C max (ng/mL) |
| Liraglutide | 2.2 | 503 |
| Semaglutide | 9.3 | 535 |
| NN9056 | 8.5 | 568 |
| SEQ ID NO:4 | 10.2 | 489 |
| SEQ ID NO:6 | 3.9 | 559 |
| SEQ ID NO:8 | 10.6 | 568 |
| SEQ ID NO:12 | 10.4 | 578 |
As the results in table 5 show, the in vivo half-life of the polypeptide compounds of the present invention is significantly prolonged, with pharmacokinetic profiles supporting once daily or once weekly dosing.
Example 17
Effect of polypeptide Compounds on blood glucose in diabetes model mice (db/db mice)
Male db/db mice were randomly grouped, 6 per group. Saline (10 mg/kg) was subcutaneously administered in blank groups, 6 groups of administration groups, free feeding and drinking water were administered during the mice experiments, and 25nmol/kg of liraglutide, semaglutinide, SEQ ID NO:4,SEQ ID NO:6,SEQ ID NO:8,SEQ ID NO:12, was subcutaneously administered in a single injection, respectively, in a non-fasting state. Blood glucose levels were measured with a glucometer at 0h before dosing, and at 4,6, 24 and 48h after dosing for each group of mice.
As shown in the results of FIG. 1, the results of the hypoglycemic experiments in db/db mice indicate that the polypeptide compound of the present invention shows a long-acting hypoglycemic activity superior to that of the positive control drug liraglutide or semaglutinide.
Example 18
Effects of polypeptide Compounds on diet-induced obesity (DIO) mice blood glucose and body weight
Male C57BL/6J mice, weighing about 22g, were fed with D12492 high fat diet from Research Diets for 18 weeks to make DIO mouse models. The placebo group 6 was fed with standard murine diet (control standard diet group). Before the start of the administration, DIO mice in each group were randomly grouped according to body weight, and 6 mice in each group were divided into 8 groups, namely, physiological saline group (control high fat diet group), positive control group (liraglutide, semaglutinide and NN 9056) and test sample group (SEQ ID NOs: 4,6, 8, 12). The control standard diet group and the control high-fat diet group were subcutaneously injected twice daily with physiological saline (10 mg/kg), the group of liraglutide and SEQ ID NO:6 was subcutaneously injected twice daily (25 nmol/kg), the group of semaglutinide, NN9056 and SEQ ID NO:4,SEQ ID NO:8,SEQ ID NO:12 was subcutaneously injected once daily (25 nmol/kg), and the administration period was 21 days. The body weight changes of mice were recorded daily and body fat mass was measured using Nuclear Magnetic Resonance (NMR) before and at the end of the experiment (table 6).
Table 6: body weight and body fat changes in DIO mice over a 3 week dosing period
| Sample (dose) | Overall weight change (%) | Body fat change (%) |
| Reference diet | +1.5％(±0.3％) | +6.0％(±2.6％) |
| Control high fat diet | +0.6％(±0.3％) | +2.5％(±0.9％) |
| Liraglutide (25 nmol/kg twice daily) | -15.1％(±3.2％) *** | -30.8％(±4.2％) *** |
| SEQ ID NO. 6 (25 nmol/kg twice daily) | -31.3％(±4.9％) ***,### | -56.1％(±5.4％) ***,### |
| Semaglutide (25 nmol/kg once daily) | -15.9％(±2.1％) *** | -32.4％(±4.8％) *** |
| NN9056 (25 nmol/kg once daily) | -11.3％(±2.0％) *** | -20.4％(±2.2％) *** |
| SEQ ID NO. 4 (25 nmol/kg twice daily) | -32.9％(±3.9％) ***,### | -57.2％(±5.0％) ***,### |
| SEQ ID NO. 8 (25 nmol/kg once daily) | -33.5％(±3.5％) ***,### | -54.1％(±4.2％) ***,### |
| SEQ ID NO. 12 (25 nmol/kg once daily) | -32.2％(±3.5％) ***,### | -56.2％(±3.1％) ***,### |
*** : p compared to the control high fat diet group<0.001； ### : ratio to group liraglutide, NN, 9056 and semaglutide P<0.001
As the results in table 6 show, the continuous administration of the polypeptide compound of the present invention in DIO mice for 3 weeks can significantly reduce the body weight and body fat content of the mice, and the effect of the polypeptide compound of the present invention is significantly stronger than that of the positive control drugs liraglutide, NN9056 and semaglutide.
Example 19
Effect of polypeptide Compounds on db/db mouse glycosylated hemoglobin (HbA 1 c) and fasting blood glucose
Male db/db mice were randomly grouped, 6 per group. After one week of adaptive feeding, the tail bleed measures the initial HbA1c values and fasting blood glucose values before the start of the treatment. Saline (10 mg/kg) was subcutaneously injected twice daily in the blank group, and the administration composition was 7 groups, and 25nmol/kg of liraglutide (twice daily), semaglutinide (once daily), NN9056 (once daily), SEQ ID NO:6 (twice daily), SEQ ID NO:4 (once daily), SEQ ID NO:8 (once daily), and SEQ ID NO:12 (once daily) were subcutaneously injected respectively. The treatment cycle was 5 weeks, and the mice were fasted overnight after the end of the treatment to measure fasting blood glucose values, while taking blood to measure HbA1c (%) values (tables 7, 8).
Table 7: hbA1c (%) change in db/db mice over a 5 week dosing period
As shown in the results of Table 7, the polypeptide compound of the present invention was continuously administered in db/db mice for 5 weeks, and the HbA1c value was reduced, indicating that the polypeptide compound has a good glycemic control effect, which is superior to liraglutide and semaglutinide. NN9056 was unable to control the increase in HbA1c value without significant glycemic control.
Table 8: fasting blood glucose changes (%)
| Sample (dose) | Fasting blood glucose change (%) |
| Physiological saline | +7.6％(±1.1％) |
| Liraglutide (25 nmol/kg twice daily) | -2.4％(±0.5％) *** |
| SEQ ID NO. 6 (25 nmol/kg twice daily) | -8.9％(±0.7％) ***,### |
| NN9056 (25 nmol/kg once daily) | +6.8％(±0.8％) |
| Semaglutide (25 nmol/kg once daily) | -3.0％(±0.5％) *** |
| SEQ ID NO. 4 (25 nmol/kg once daily) | -9.4％(±0.7％) ***,### |
| SEQ ID NO. 8 (25 nmol/kg once daily) | -10.1％(±1.3％) ***,### |
| SEQ ID NO. 12 (25 nmol/kg once daily) | -9.6％(±0.8％) ***,### |
*** : p compared with physiological saline group<0.001； ### : ratio to group liraglutide, NN, 9056 and semaglutide P<0.001
As shown in the results of Table 8, the continuous administration of the polypeptide compound of the present invention in db/db mice for 5 weeks can significantly reduce the fasting blood glucose of db/db mice, indicating that the polypeptide compound of the present invention has a very high glycemic control effect, and the effect of the polypeptide compound of the present invention is significantly stronger than that of positive control drugs NN9056, liraglutide and semaglutinide.
Example 20
Evaluation of side Effect of polypeptide Compounds
Male C57BL/6J mice at 7 weeks of age were divided into 8 groups of 6 mice each, and after one week of adaptive feeding, each group was subcutaneously injected with physiological saline, 100nmol/kg of liraglutide, semaglutinide, NN9056, SEQ ID NO:4,SEQ ID NO:6,SEQ ID NO:8 and SEQ ID NO:12. Blood samples were taken before and 48 hours after dosing to determine the serum amylase (amylase) and lipase (lipase) values.
Table 9: serum amylase and lipase values (U/L) before and after administration of C57BL/6J mice
| Sample of | Amylase (before administration) | Amylase(48h) | Lipase (before administration) | Lipase(48h) |
| Physiological saline | 2115±289 | 2058±315 | 6.2±0.5 | 6.2±0.8 |
| Liraglutide | 2256±305 | 2658±412 | 6.0±0.3 | 6.9±0.4 |
| Semaglutide | 2154±326 | 2789±348 | 6.6±0.7 | 7.8±0.5 |
| NN9056 | 2298±411 | 3428±512 | 6.4±0.2 | 8.5±0.9 |
| SEQ ID NO:4 | 2145±287 | 2010±354 | 6.1±0.4 | 5.7±0.3 |
| SEQ ID NO:6 | 2199±315 | 1989±158 | 6.7±0.5 | 6.0±0.3 |
| SEQ ID NO:8 | 2354±305 | 2014±325 | 6.4±0.4 | 5.9±0.5 |
| SEQ ID NO:12 | 2248±198 | 2004±158 | 6.3±0.4 | 6.1±0.3 |
As shown in the results of Table 9, the administration of the polypeptide compound of the present invention in a large dose in C57BL/6J mice did not result in an increase in serum amylase and lipase values, indicating that acute pancreatitis did not occur. The NN9056, liraglutide and semaglutinide all had various levels of elevated serum amylase and lipase values, with side effects leading to acute pancreatitis.
Finally, it should be noted that the above-mentioned embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same, and although the present invention has been described in detail with reference to examples, it should be understood by those skilled in the art that modifications and equivalents may be made to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention, and all such modifications and equivalents are intended to be encompassed in the scope of the claims of the present invention.
Sequence listing
<110> university of Jiangsu teachers and universities
<120> GLP-1/cholecystokinin-1 receptor dual agonist and application thereof
<160> 12
<170> SIPOSequenceListing 1.0
<210> 1
<211> 40
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 1
His Ala Glu Gly Thr Tyr Thr Asn Asp Val Thr Glu Tyr Leu Glu Glu
1 5 10 15
Lys Ala Ala Lys Glu Phe Ile Glu Trp Leu Ile Lys Gly Lys Xaa Xaa
20 25 30
Asp Xaa Xaa Gly Trp Xaa Xaa Xaa
35 40
<210> 2
<211> 40
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 2
His Xaa Glu Gly Thr Tyr Thr Asn Asp Val Thr Glu Tyr Leu Glu Glu
1 5 10 15
Lys Ala Ala Lys Glu Phe Ile Glu Trp Leu Ile Lys Gly Lys Xaa Xaa
20 25 30
Asp Xaa Xaa Gly Trp Xaa Xaa Xaa
35 40
<210> 3
<211> 40
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 3
His Ala Glu Gly Thr Tyr Thr Asn Asp Val Thr Glu Tyr Leu Glu Glu
1 5 10 15
Lys Ala Ala Lys Glu Phe Ile Glu Trp Leu Ile Lys Gly Lys Xaa Xaa
20 25 30
Asp Xaa Xaa Gly Trp Xaa Xaa Xaa
35 40
<210> 4
<211> 40
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 4
His Xaa Glu Gly Thr Tyr Thr Asn Asp Val Thr Glu Tyr Leu Glu Glu
1 5 10 15
Lys Ala Ala Lys Glu Phe Ile Glu Trp Leu Ile Lys Gly Lys Xaa Xaa
20 25 30
Asp Xaa Xaa Gly Trp Xaa Xaa Xaa
35 40
<210> 5
<211> 40
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 5
His Ala Glu Gly Thr Tyr Thr Asn Asp Val Thr Glu Tyr Leu Glu Glu
1 5 10 15
Glu Ala Ala Lys Glu Phe Ile Glu Trp Leu Ile Lys Gly Lys Xaa Xaa
20 25 30
Asp Xaa Xaa Gly Trp Xaa Xaa Xaa
35 40
<210> 6
<211> 40
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 6
His Xaa Glu Gly Thr Tyr Thr Asn Asp Val Thr Glu Tyr Leu Glu Glu
1 5 10 15
Glu Ala Ala Lys Glu Phe Ile Glu Trp Leu Ile Lys Gly Lys Xaa Xaa
20 25 30
Asp Xaa Xaa Gly Trp Xaa Xaa Xaa
35 40
<210> 7
<211> 40
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 7
His Ala Glu Gly Thr Tyr Thr Asn Asp Val Thr Glu Tyr Leu Glu Glu
1 5 10 15
Glu Ala Ala Lys Glu Phe Ile Glu Trp Leu Ile Lys Gly Lys Xaa Xaa
20 25 30
Asp Xaa Xaa Gly Trp Xaa Xaa Xaa
35 40
<210> 8
<211> 40
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 8
His Xaa Glu Gly Thr Tyr Thr Asn Asp Val Thr Glu Tyr Leu Glu Glu
1 5 10 15
Glu Ala Ala Lys Glu Phe Ile Glu Trp Leu Ile Lys Gly Lys Xaa Xaa
20 25 30
Asp Xaa Xaa Gly Trp Xaa Xaa Xaa
35 40
<210> 9
<211> 40
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 9
His Ala Glu Gly Thr Tyr Thr Asn Asp Val Thr Glu Tyr Leu Glu Glu
1 5 10 15
Glu Ala Ala Lys Glu Phe Ile Glu Trp Leu Ile Lys Gly Lys Xaa Xaa
20 25 30
Asp Xaa Xaa Gly Trp Xaa Xaa Xaa
35 40
<210> 10
<211> 40
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 10
His Xaa Glu Gly Thr Tyr Thr Asn Asp Val Thr Glu Tyr Leu Glu Glu
1 5 10 15
Glu Ala Ala Lys Glu Phe Ile Glu Trp Leu Ile Lys Gly Lys Xaa Xaa
20 25 30
Asp Xaa Xaa Gly Trp Xaa Xaa Xaa
35 40
<210> 11
<211> 40
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 11
His Ala Glu Gly Thr Tyr Thr Asn Asp Val Thr Glu Tyr Leu Glu Glu
1 5 10 15
Glu Ala Ala Lys Glu Phe Ile Glu Trp Leu Ile Lys Gly Lys Xaa Xaa
20 25 30
Asp Xaa Xaa Gly Trp Xaa Xaa Xaa
35 40
<210> 12
<211> 40
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 12
His Xaa Glu Gly Thr Tyr Thr Asn Asp Val Thr Glu Tyr Leu Glu Glu
1 5 10 15
Glu Ala Ala Lys Glu Phe Ile Glu Trp Leu Ile Lys Gly Lys Xaa Xaa
20 25 30
Asp Xaa Xaa Gly Trp Xaa Xaa Xaa
35 40
Claims (7)
1. A GLP-1/CCK-1 receptor dual agonism polypeptide compound, wherein the amino acid sequence of said polypeptide compound is one of the following sequences:
(4)
His-Aib-Glu-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Thr-Glu-Tyr-Leu-Glu-Glu-Lys(AEEA-AEEA-γ-Gl u-CO-(CH 2 ) 16 -COOH)-Ala-Ala-Lys-Glu-Phe-Ile-Glu-Trp-Leu-Ile-Lys-Gly-Lys-AEEA-AEEA-As p-Phe(4sm)-Nle-Gly-Trp-Nle-DMeAsp-MePhe-NH 2
(8)
His-Aib-Glu-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Thr-Glu-Tyr-Leu-Glu-Glu-Glu-Ala-Ala-Lys(AEE A-AEEA-γ-Glu-CO-(CH 2 ) 16 -COOH)-Glu-Phe-Ile-Glu-Trp-Leu-Ile-Lys-Gly-Lys-AEEA-AEEA-Asp-Phe(4sm)-Nle-Gly-Trp-Nle-DMeAsp-MePhe-NH 2
(12)
His-Aib-Glu-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Thr-Glu-Tyr-Leu-Glu-Glu-Glu-Ala-Ala-Lys-Glu-Phe-Ile-Glu-Trp-Leu-Ile-Lys-Gly-Lys(AEEA-AEEA-γ-Glu-CO-(CH 2 ) 16 -COOH)-AEEA-AEEA-As p-Phe(4sm)-Nle-Gly-Trp-Nle-DMeAsp-MePhe-NH 2
2. a pharmaceutically acceptable salt of a GLP-1/CCK-1 receptor double agonist polypeptide compound of the type defined in claim 1.
3. The salt of claim 2, wherein the salt is a salt of a GLP-1/CCK-1 receptor dual agonist polypeptide compound with one of the following compounds: hydrobromic acid, hydrochloric acid, methanesulfonic acid, phosphoric acid, ethanesulfonic acid, formic acid, p-toluenesulfonic acid, acetic acid, acetoacetic acid, pyruvic acid, pectate acid, butyric acid, caproic acid, benzenesulfonic acid, heptanoic acid, undecanoic acid, benzoic acid, salicylic acid, lauric acid, 2- (4-hydroxybenzoyl) benzoic acid, cinnamic acid, camphoric acid, cyclopentanepropionic acid, 3-hydroxy-2-naphthoic acid, camphorsulfonic acid, digluconic acid, nicotinic acid, pamoic acid, propionic acid, persulfuric acid, picric acid, 3-phenylpropionic acid, pivalic acid, itaconic acid, 2-hydroxyethanesulfonic acid, sulfamic acid, dodecylsulfuric acid, trifluoromethanesulfonic acid, naphthalenedisulfonic acid, 2-naphthalenesulfonic acid, citric acid, mandelic acid, ascorbic acid, wine stearic acid, lithonic acid, oxalic acid, lactic acid, succinic acid, malonic acid, hemisulfuric acid, malic acid, maleic acid, alginic acid, D-gluconic acid, glycerophosphoric acid, glucoheptoic acid, aspartic acid, thiocyanic acid, or sulfosalicylic acid.
4. A pharmaceutical composition comprising a GLP-1/CCK-1 receptor dual agonist polypeptide compound of claim 1, characterized in that the pharmaceutical composition comprises: a GLP-1/CCK-1 receptor dual agonism polypeptide compound or a pharmaceutically acceptable salt as described in claim 2, and a pharmaceutically acceptable carrier or diluent.
5. A pharmaceutical formulation comprising the pharmaceutical composition of claim 4, wherein the pharmaceutical formulation is a pharmaceutically acceptable capsule, tablet, spray, inhalant, injection, patch, emulsion, film or powder.
6. Use of a GLP-1/CCK-1 receptor dual agonist polypeptide compound of claim 1, a pharmaceutically acceptable salt thereof, a pharmaceutical composition thereof or a medicament thereof for the manufacture of a medicament for the treatment of a metabolic disease or disorder;
the metabolic disease or disorder is at least one of diabetes, NAFLD, NASH, hyperlipidemia, and obesity.
7. The use according to claim 6, wherein the diabetes is type 1 diabetes, type 2 diabetes or gestational diabetes.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011428688.5A CN112608378B (en) | 2020-12-09 | 2020-12-09 | GLP-1/cholecystokinin-1 receptor dual agonist and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011428688.5A CN112608378B (en) | 2020-12-09 | 2020-12-09 | GLP-1/cholecystokinin-1 receptor dual agonist and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112608378A CN112608378A (en) | 2021-04-06 |
| CN112608378B true CN112608378B (en) | 2023-09-08 |
Family
ID=75229390
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011428688.5A Active CN112608378B (en) | 2020-12-09 | 2020-12-09 | GLP-1/cholecystokinin-1 receptor dual agonist and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112608378B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116712530B (en) * | 2023-02-03 | 2024-03-08 | 江苏师范大学 | Long-acting GLP-1/glucon/GIP receptor triple agonist and application thereof |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2007305430A1 (en) * | 2006-10-03 | 2008-04-10 | Merck Sharp & Dohme Corp. | Glucagon receptor antagonist compounds, compositions containing such compounds and methods of use |
| CN103980235A (en) * | 2008-12-18 | 2014-08-13 | 赛马拜制药公司 | GPR120 receptor agonists and uses thereof |
| CN106432471A (en) * | 2016-12-14 | 2017-02-22 | 江苏师范大学 | Novel peptide conjugate of African clawed frog GLP-1 (glucagon like peptide-1), as well as application thereof |
| AU2015335828A1 (en) * | 2014-10-24 | 2017-05-11 | Merck Sharp & Dohme Llc | Co-agonists of the glucagon and GLP-1 receptors |
| CN107698677A (en) * | 2017-11-24 | 2018-02-16 | 江苏师范大学 | Peptide and its application is conjugated in cholic acid Africa xenopus glucagon-like peptide 1 |
| CN107987152A (en) * | 2017-11-24 | 2018-05-04 | 江苏师范大学 | Peptide and application thereof is conjugated in mycophenolic acid-Africa xenopus glucagon-like-peptide-1 |
| CN108359005A (en) * | 2017-11-24 | 2018-08-03 | 江苏师范大学 | Africa xenopus GLP-1 analogs and application thereof |
| CN110759991A (en) * | 2019-12-09 | 2020-02-07 | 江苏师范大学 | Gemfibrozil-xenopus laevis glucagon-like peptide-1 derivative and application thereof |
| CN111670042A (en) * | 2017-12-07 | 2020-09-15 | 阿道恰公司 | Compositions in the form of injectable aqueous solutions comprising human glucagon and a polyamino acid copolymer |
-
2020
- 2020-12-09 CN CN202011428688.5A patent/CN112608378B/en active Active
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2007305430A1 (en) * | 2006-10-03 | 2008-04-10 | Merck Sharp & Dohme Corp. | Glucagon receptor antagonist compounds, compositions containing such compounds and methods of use |
| CN103980235A (en) * | 2008-12-18 | 2014-08-13 | 赛马拜制药公司 | GPR120 receptor agonists and uses thereof |
| AU2015335828A1 (en) * | 2014-10-24 | 2017-05-11 | Merck Sharp & Dohme Llc | Co-agonists of the glucagon and GLP-1 receptors |
| CN106432471A (en) * | 2016-12-14 | 2017-02-22 | 江苏师范大学 | Novel peptide conjugate of African clawed frog GLP-1 (glucagon like peptide-1), as well as application thereof |
| CN107698677A (en) * | 2017-11-24 | 2018-02-16 | 江苏师范大学 | Peptide and its application is conjugated in cholic acid Africa xenopus glucagon-like peptide 1 |
| CN107987152A (en) * | 2017-11-24 | 2018-05-04 | 江苏师范大学 | Peptide and application thereof is conjugated in mycophenolic acid-Africa xenopus glucagon-like-peptide-1 |
| CN108359005A (en) * | 2017-11-24 | 2018-08-03 | 江苏师范大学 | Africa xenopus GLP-1 analogs and application thereof |
| CN111670042A (en) * | 2017-12-07 | 2020-09-15 | 阿道恰公司 | Compositions in the form of injectable aqueous solutions comprising human glucagon and a polyamino acid copolymer |
| CN110759991A (en) * | 2019-12-09 | 2020-02-07 | 江苏师范大学 | Gemfibrozil-xenopus laevis glucagon-like peptide-1 derivative and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| Peptide-based multi-agonists: a new paradigm in metabolic pharmacology;S.J.Brandt et al;《Journal of Internal Medicine》;全文 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112608378A (en) | 2021-04-06 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN114349828B (en) | GLP-1/glucagon receptor dual agonist and application thereof | |
| KR102351313B1 (en) | GIP/GLP1 co-agonist compounds | |
| US9764004B2 (en) | Glucagon receptor agonists | |
| US20230220033A1 (en) | Glp-1 agonist polypeptide compound and salt thereof, synthesis method therefor and use thereof | |
| EP2718317A1 (en) | Glucose dependent insulinotropic polypeptide analogs, pharmaceutical compositions and use thereof | |
| CN116143884B (en) | Long-acting GLP-1/glucon/GIP receptor triple agonist and application thereof | |
| WO2023000240A1 (en) | Long-acting glp-1 polypeptide analogue, and preparation method therefor and use thereof | |
| CN112608378B (en) | GLP-1/cholecystokinin-1 receptor dual agonist and application thereof | |
| KR20230008846A (en) | Polypeptide derivatives with dual receptor agonism and their uses | |
| JP2020531442A (en) | Acylated oxyntomodulin peptide analog | |
| CN110759991B (en) | Gemfibrozil-xenopus laevis glucagon-like peptide-1 derivative and application thereof | |
| CN112759640B (en) | GLP-1/gastrin receptor dual agonist and application thereof | |
| CN116120425A (en) | GLP-1/GIP receptor dual agonist and application thereof | |
| CN114437200A (en) | GLP-1/gastrin receptor dual agonist and application thereof | |
| CN115785249B (en) | GLP-1 analogues and application thereof | |
| CN116589536B (en) | Long-acting GLP-1/GIP receptor dual agonist and application thereof | |
| WO2016090628A1 (en) | Oxyntomodulin (oxm) analogs, synthesis and use thereof | |
| CN115960258B (en) | GLP-1/glucon/Y 2 Receptor triple agonists and uses thereof | |
| CN117624333A (en) | GLP-1 receptor, glucagon receptor and GIP receptor tri-excitation polypeptide compound and application thereof | |
| CN117186189A (en) | GLP-1/CCK-1 receptor double-excitation polypeptide with hypoglycemic and weight-reducing effects and application thereof | |
| CN117417430A (en) | Bullfrog GLP-1 analogues with agonistic activity on GLP-1 and glucagon receptor and application thereof | |
| CN115819551A (en) | GLP-1/glucagon/gastrin receptor triple agonist with site-specific modification and application thereof | |
| CN115819619A (en) | GLP-1/Y 2 Receptor dual agonist and application thereof | |
| CN117417431A (en) | Polypeptides with agonistic activity on GLP-1, glucagon and GIP receptor and application thereof | |
| CN117756913A (en) | Novel long-acting polypeptide compound, composition and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |