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CN112608378A - GLP-1/cholecystokinin-1 receptor dual agonists and application thereof - Google Patents

GLP-1/cholecystokinin-1 receptor dual agonists and application thereof Download PDF

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CN112608378A
CN112608378A CN202011428688.5A CN202011428688A CN112608378A CN 112608378 A CN112608378 A CN 112608378A CN 202011428688 A CN202011428688 A CN 202011428688A CN 112608378 A CN112608378 A CN 112608378A
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韩京
孟庆华
杨启萌
周凤
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Abstract

The invention provides a GLP-1/CCK-1 receptor dual-agonist polypeptide compound. The GLP-1/CCK-1 receptor dual-agonist polypeptide compound has the effects of reducing blood sugar more effectively, promoting weight loss, reversing insulin resistance and regulating lipid metabolism. The polypeptide compound of the present invention has higher GLP-1 receptor agonistic activity than its natural ligand, and has selective agonistic activity against CCK-1 receptor. The polypeptide compound provided by the invention has stable chemical properties and low side effects, does not cause the occurrence of acute pancreatitis, and is suitable to be used as an active ingredient of medicaments for treating metabolic diseases, such as diabetes, obesity, hyperlipidemia, NAFLD, NASH and the like.

Description

GLP-1/cholecystokinin-1 receptor dual agonists and application thereof
Technical Field
The invention relates to a biological medicine, in particular to a GLP-1/cholecystokinin-1 receptor dual agonist and application thereof.
Background
Obesity and its associated metabolic syndrome have become global public health problems, and the incidence and course of many metabolic syndromes such as type 2 diabetes (T2DM), non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), dyslipidemia are closely related to obesity. Studies have shown that clinically 80-90% of patients with T2DM 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). The ideal weight loss effect is generally difficult to achieve by only exercising and diet control to lose weight. The existing medicines for treating obesity have limited curative effect, and many medicines for treating obesity also have obvious side effects, such as mental symptoms caused by acting on central nerves, serious cardiovascular effects and the like. Currently, 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 co-transporter 2(SGLT2) inhibitors have better weight control effect (j.med.chem.,2018,61, 5580-. Bariatric surgery has significant therapeutic effects on obesity, but patients suffer from a greater surgical risk 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 that can safely and effectively control weight and has a therapeutic effect on primary diseases is an ideal choice.
GLP-1 is a glucose-dependent hypoglycemic polypeptide hormone secreted by L cells of the tail jejunum, ileum and colon, and has the function of reducing blood sugar after being specifically combined with GLP-1 receptors. The main advantage of GLP-1 is its blood sugar-dependent incretin secretion, avoiding the risk of hypoglycemia often present in diabetes treatment. In addition to regulating blood glucose, GLP-1 also prevents pancreatic beta cell degeneration, stimulates beta cell proliferation and differentiation, and can fundamentally improve the progression of diabetes. In addition, GLP-1 also has effects of inhibiting gastric acid secretion, delaying gastric emptying, and suppressing appetite, and has partial weight loss effect. A number of long acting GLP-1 class drugs are currently marketed, such as liraglutide, semaglutide and dulaglutide. Although GLP-1 drugs have safe hypoglycemic effects, if better weight loss is to be achieved, the dosage is generally increased, and the GLP-1 drugs are easily subjected to gastrointestinal side effects due to large dosage, and have poor tolerance, so that the treatment window is narrow. Thus, there remains a need for therapeutic agents that are safer and more tolerable, and that are effective in reducing weight and controlling blood glucose.
Cholecystokinin (CCK) is a gastrointestinal hormone secreted by intestinal I cells, and 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 action of CCK is achieved by agonizing CCK receptors, which belong to the family of G protein-coupled receptors, and have two subtypes, CCK-1 and CCK-2. The CCK-1 receptor is the primary receptor involved in food intake and satiety, while the CCK-2 receptor mediates 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 agonist activity at both the CCK-1 and CCK-2 receptors. Selective agonism of CCK-1 receptor is more beneficial in inhibiting food intake, and TW201716432A discloses a class of CCK-8 long-acting analogs (NN9056) having CCK-1 receptor selective agonistic activity. However, CCK-1 receptor agonism easily causes acute pancreatitis, which is a major obstacle faced by CCK-1 receptor as a weight loss drug targeting receptor, and at present, no weight loss drug acting on CCK-1 receptor is on the market.
The XenGLP-1 is GLP-1 analogues of animal sources found in Xenopus laevis bodies, and compared with natural GLP-1, the XenGLP-1 has better hypoglycemic activity and stability. In addition to being more resistant to degradation by dipeptidyl peptidase (DPP-IV), XenGLP-1 also appears to be much more stable to degradation by Neutral Endopeptidase (NEP) than GLP-1. XenGLP-1 is a potent agonist of the GLP-1 receptor, with many of the glucose-regulating effects observed with native GLP-1, and many preclinical studies have shown that XenGLP-1 has several beneficial anti-diabetic properties, including enhanced glucose-dependent insulin synthesis and secretion, slowed gastric emptying, decreased food intake and weight loss, and promotion of beta cell proliferation and restoration of islet function, among others (biochem. Pharmacol.,2017,142, 155-. These effects are beneficial not only for diabetics, but also for patients suffering from obesity. Patients with obesity have an increased risk of hypertension, hyperlipidemia, diabetes, NAFLD, NASH, musculoskeletal and cardiovascular diseases.
The effects of stimulating CCK-1 receptors to inhibit food intake and promote satiety can improve the weight loss effect of GLP-1 medicaments. Trevaskis et al describe the simultaneous combination of a CCK-8 analog and the GLP-1 receptor agonist exendin-4 (exendin-4) with significantly improved weight loss compared to exendin-4 alone (Diabetes obes. However, selective simultaneous agonism of the GLP-1 receptor and the CCK-1 receptor is essential to avoid the occurrence of side effects, especially severe side effects such as acute pancreatitis.
Disclosure of Invention
The invention aims to provide a novel polypeptide compound with GLP-1/CCK-1 receptor dual-agonist action, which is a variant designed based on a XenGLP-1 sequence, retains the therapeutic action of the XenGLP-1 on diabetes, has the beneficial action of selectively exciting a CCK-1 receptor on feeding, does not cause acute pancreatitis, and has more potential than a single receptor agonist in the aspect of preparing medicaments for treating 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:
a GLP-1/CCK-1 receptor dual-agonist polypeptide compound has an amino acid sequence general formula as follows:
His-Xaa1-Glu-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Thr-Glu-Tyr-Leu-Glu-Glu-Xaa2-Ala-Ala-Xaa3-Glu-Phe-Ile-Glu-Trp-Leu-Ile-Lys-Gly-Xaa4-Xaa5-Xaa6-Asp-Xaa7-Nle-Gly-Trp-Nle-DMeAsp-Xaa8-NH2
wherein:
Xaa1taken from Ala, D-Ala, Gly or Aib;
Xaa2taken from Glu, Lys or side chain quiltA modified Lys;
Xaa3is taken from Lys or Lys modified in side chain;
Xaa4is taken from Lys or Lys modified in side chain;
Xaa5is obtained from Ala or AEEA;
Xaa6is obtained from Ala or AEEA;
Xaa7is taken from Phe (4sm) or sTyr;
Xaa8is taken from Phe or MePhe;
wherein Lys having a modified side chain is selected from Lys (gamma-Glu-CO- (CH)2)n-CH3) Or Lys (AEEA-AEEA-gamma-Glu-CO- (CH)2)n-COOH)，
Lys(γ-Glu-CO-(CH2)n-CH3) The structural formula of (A) is shown as the following formula:
Figure BDA0002825765270000031
Lys(AEEA-AEEA-γ-Glu-CO-(CH2)n-COOH) is represented by the formula:
Figure BDA0002825765270000032
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-(CH2)14-CH3)-Ala-Ala-Lys-Glu-Phe-Ile-Glu-Trp-Leu-Ile-Lys-Gly-Lys-AEEA-AEEA-Asp-Phe(4sm)-Nle-Gly-Trp-Nle-DMeAsp-MePhe-NH2
(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-(CH2)14-CH3)-Ala-Ala-Lys-Glu-Phe-Ile-Glu-Trp-Leu-Ile-Lys-Gly-Lys-AEEA-AEEA-Asp-Phe(4sm)-Nle-Gly-Trp-Nle-DMeAsp-MePhe-NH2
(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-(CH2)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-NH2
(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-(CH2)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-NH2
(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-(CH2)14-CH3)-Glu-Phe-Ile-Glu-Trp-Leu-Ile-Lys-Gly-Lys-AEEA-AEEA-Asp-Phe(4sm)-Nle-Gly-Trp-Nle-DMeAsp-MePhe-NH2
(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-(CH2)14-CH3)-Glu-Phe-Ile-Glu-Trp-Leu-Ile-Lys-Gly-Lys-AEEA-AEEA-Asp-Phe(4sm)-Nle-Gly-Trp-Nle-DMeAsp-MePhe-NH2
(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-(CH2)16-COOH)-Glu-Phe-Ile-Glu-Trp-Leu-Ile-Lys-Gly-Lys-AEEA-AEEA-Asp-Phe(4sm)-Nle-Gly-Trp-Nle-DMeAsp-MePhe-NH2
(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-(CH2)16-COOH)-Glu-Phe-Ile-Glu-Trp-Leu-Ile-Lys-Gly-Lys-AEEA-AEEA-Asp-Phe(4sm)-Nle-Gly-Trp-Nle-DMeAsp-MePhe-NH2
(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-(CH2)14-CH3)-AEEA-AEEA-Asp-Phe(4sm)-Nle-Gly-Trp-Nle-DMeAsp-MePhe-NH2
(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-(CH2)14-CH3)-AEEA-AEEA-Asp-Phe(4sm)-Nle-Gly-Trp-Nle-DMeAsp-MePhe-NH2
(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-(CH2)16-COOH)-AEEA-AEEA-Asp-Phe(4sm)-Nle-Gly-Trp-Nle-DMeAsp-MePhe-NH2
(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-(CH2)16-COOH)-AEEA-AEEA-Asp-Phe(4sm)-Nle-Gly-Trp-Nle-DMeAsp-MePhe-NH2
the invention also provides pharmaceutically acceptable salts of the GLP-1/CCK-1 receptor dual agonist polypeptide compounds.
Preferably, the salt is formed by the GLP-1/CCK-1 receptor dual agonist polypeptide compound and 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, pectinic 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, ethanolic acid, lithospermic acid, oxalic acid, lactic acid, succinic acid, malonic acid, hemisulfuric acid, malic acid, maleic acid, malic 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 the GLP-1/CCK-1 receptor dual agonist polypeptide compound, which comprises: any GLP-1/CCK-1 receptor dual-agonist polypeptide compound or pharmaceutically acceptable salt thereof is taken as an effective raw material, and a pharmaceutically acceptable carrier or diluent is added.
The invention also provides a medicament containing the GLP-1/CCK-1 receptor dual-agonist polypeptide compound, wherein the medicament is any one of capsules, tablets, spraying agents, inhalants, injections, patches, emulsions, films, powders or compound preparations in pharmaceutics, and the medicament consists of the GLP-1/CCK-1 receptor dual-agonist polypeptide compound and pharmaceutically acceptable pharmaceutic adjuvants, carriers or diluents.
The invention also provides application of the GLP-1/CCK-1 receptor dual-agonist polypeptide compound, the pharmaceutically acceptable salt thereof, the pharmaceutical composition thereof or the medicament thereof in preparing a medicament for treating metabolic diseases or symptoms. In particular aspects, the metabolic disease or disorder is diabetes, NAFLD, NASH, hyperlipidemia, or obesity. In a particular aspect, the diabetes is type 1 diabetes, T2DM or gestational diabetes. In particular aspects, 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 dual-agonist polypeptide compound has the effects of reducing weight, preventing weight gain and regulating lipid metabolism while more effectively reducing blood sugar, and has unexpected beneficial effects compared with the existing medicament. The polypeptide compound of the invention has higher agonistic activity on GLP-1 receptor than natural ligand, and has selective agonistic activity on CCK-1 receptor and does not excite CCK-2 receptor. The polypeptide compound provided by the invention has stable chemical properties, is not easily degraded by DPP-IV and NEP in vivo, is not easily filtered by glomeruli, has obviously improved stability, and has the pharmacokinetic characteristic of supporting once-a-day administration or once-a-week 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 characteristics favorable for preparation. The polypeptide compound provided by the invention has low immunogenicity, and has better therapeutic 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 GLP-1 receptor agonists which are already on the market and CCK-1 receptor agonists which are reported, the polypeptide compound has lower side effects and can not cause the occurrence of acute pancreatitis. Therefore, the polypeptide compound provided by the invention is suitable for being used as an active ingredient of medicaments 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 unnatural amino acid definitions of polypeptide compounds of the invention
Figure BDA0002825765270000061
Drawings
FIG. 1 shows the long-lasting hypoglycemic effect of a single administration of each test substance in the non-fasting state of db/db mice.
Detailed Description
The following abbreviations are used throughout the 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 dimethyl formamide
Fmoc 9-fluorenylmethoxycarbonyl
Boc tert-butyloxycarbonyl group
DMSO dimethyl sulfoxide
DIC N, N' -diisopropylcarbodiimide
HOBT 1-hydroxy-benzotriazole
Alloc allyloxycarbonyl radical
Dde 1- (4, 4-dimethyl-2, 6-dioxocyclohexylidene) -ethyl
Mtt 4-Methyltriphenylmethyl
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 Dubecker's modified eagle'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-(CH2)14-CH3)-Ala-Ala-Lys-Glu-Phe-Ile-Glu-Trp-Leu-Ile-Lys-Gly-Lys-AEEA-AEEA-Asp-Phe(4sm)-Nle-Gly-Trp-Nle-DMeAsp-MePhe-NH2
(1) Swelling of the resin
0.262g (0.1mmol equiv.) of Rink Amide MBHA resin loaded at 0.382mmol/g was weighed into a 25mL reactor, the resin was washed 1 time with 7mL of DCM and methanol alternately, 2 times with 7mL of DCM, then the resin was swollen with 7mL of DCM for 1h, and finally 3 times with 7mL of DMF.
(2) Removal of Fmoc protecting group of resin
Transferring the swelled resin into a PSI200 polypeptide synthesizer, adding 7mL of 20% piperidine/DMF (v/v) to react for 5min at room temperature, filtering out the deprotection solution, washing the resin once with 7mL of DMF, adding 7mL of 20% piperidine/DMF (v/v) deprotection solvent to react with the resin for 15min, and finally washing the resin 4 times with 7mL of DMF, wherein each time lasts for 1.5min, so that the Rink resin with the Fmoc protecting group removed is obtained.
(3) Synthesis of Fmoc-MePhe-Rink amide-MBHA Resin
Weighing Fmoc-MePhe-OH (0.4mmol), dissolving with 3mL of 10% DMF/DMSO (v/v), adding 2mL of DIC/HOBt (0.4mmol/0.44mmol) condensing agent, pre-activating for 30min, adding the activated amino acid into a reactor, shaking for reaction for 2h at room temperature, filtering out the reaction solution, washing the resin with 7mL of DMF for 4 times, and detecting whether the reaction coupling is complete by using Kaiser reagent, if not complete, 2 times of coupling.
(4) Elongation of peptide chain
And repeating the deprotection and coupling steps according to the sequence of the peptide chain to connect corresponding amino acids in sequence until the synthesis of the peptide chain is finished. As the Lys at position 17, Fmoc-Lys (alloc) -OH, Fmoc-Lys (Dde) -OH, Fmoc-Lys (Mtt) -OH or Fmoc-Lys (ivDde) -OH, etc. can be used. In this example, Fmoc-Lys (Dde) -OH protection strategy was used, while the N-terminal His was Boc-His (Boc) -OH. Furthermore, Fmoc-Phe (4sm-TCE) -OH protected by side chain TCE was selected at Phe (4sm) at position 34.
(5) Modification of Lys side chain
After the peptide chain synthesis is finished, 7mL of 2% hydrazine hydrate/DMF (v/v) is added to selectively remove the Dde protecting group of Lys at the 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, and the mixture is shaken for reaction for 2 hours. After Fmoc protecting group was removed by the same method as above, 0.4mmol of palmitic acid, 0.4mmol of DIC and 0.44mmol of HOBt were added and the reaction was completed and the resin was washed 4 times with 7mL of DMF.
(6) Cleavage of polypeptides
Transferring the obtained resin connected with the polypeptide into a round-bottom bottle, cutting the resin by using 5mL of cutting agent Reagent R (TFA/thioanisole/phenol/EDT, 90:5:3:2, V/V), reacting for 2h in an oil bath at constant temperature of 30 ℃, pouring the cutting liquid into 40mL of ethyl acetate, washing a crude product for 3 times by using 15mL of ethyl acetate after refrigerated centrifugation, and finally drying by using nitrogen to obtain the crude peptide. The crude peptide was dissolved in acetic acid and the TCE protecting group of Phe (4sm) was removed using hydrochloric acid activated zinc dust.
(7) Purification of polypeptides
And dissolving the crude target polypeptide in water, filtering by using a 0.25 mu m microporous filter membrane, and purifying by using an Shimadzu preparative reversed-phase HPLC system. The chromatographic conditions were C18 reverse phase preparative columns (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 8 mL/min; the detection wavelength was 214 nm. Eluting by linear gradient (20% B-70% B/30min), collecting target peak, removing methanol, lyophilizing to obtain pure product 0.24g with purity greater than 98%, and determining target polypeptide molecular weight 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-(CH2)14-CH3)-Ala-Ala-Lys-Glu-Phe-Ile-Glu-Trp-Leu-Ile-Lys-Gly-Lys-AEEA-AEEA-Asp-Phe(4sm)-Nle-Gly-Trp-Nle-DMeAsp-MePhe-NH2
The synthesis method is the same as that of example 1, and the target peak is collected and lyophilized to obtain 0.23g of a 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-(CH2)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-NH2
(1) Swelling of the resin
0.262g (0.1mmol equiv.) of Rink Amide MBHA resin loaded at 0.382mmol/g was weighed into a 25mL reactor, the resin was washed 1 time with 7mL of DCM and methanol alternately, 2 times with 7mL of DCM, then the resin was swollen with 7mL of DCM for 1h, and finally 3 times with 7mL of DMF.
(2) Removal of Fmoc protecting group of resin
Transferring the swelled resin into a PSI200 polypeptide synthesizer, adding 7mL of 20% piperidine/DMF (v/v) to react for 5min at room temperature, filtering out the deprotection solution, washing the resin once with 7mL of DMF, adding 7mL of 20% piperidine/DMF (v/v) deprotection solvent to react with the resin for 15min, and finally washing the resin 4 times with 7mL of DMF, wherein each time lasts for 1.5min, so that the Rink resin with the Fmoc protecting group removed is obtained.
(3) Synthesis of Fmoc-MePhe-Rink amide-MBHA Resin
Weighing Fmoc-MePhe-OH (0.4mmol), dissolving with 3mL of 10% DMF/DMSO (v/v), adding 2mL of DIC/HOBt (0.4mmol/0.44mmol) condensing agent, pre-activating for 30min, adding the activated amino acid into a reactor, shaking for reaction for 2h at room temperature, filtering out the reaction solution, washing the resin with 7mL of DMF for 4 times, and detecting whether the reaction coupling is complete by using Kaiser reagent, if not complete, 2 times of coupling.
(4) Elongation of peptide chain
And repeating the deprotection and coupling steps according to the sequence of the peptide chain to connect corresponding amino acids in sequence until the synthesis of the peptide chain is finished. As the Lys at position 17, Fmoc-Lys (alloc) -OH, Fmoc-Lys (Dde) -OH, Fmoc-Lys (Mtt) -OH or Fmoc-Lys (ivDde) -OH, etc. can be used. In this example, Fmoc-Lys (Dde) -OH protection strategy was used, while the N-terminal His was Boc-His (Boc) -OH. Furthermore, Fmoc-Phe (4sm-TCE) -OH protected by side chain TCE was selected at Phe (4sm) at position 34.
(5) Modification of Lys side chain
And after the peptide chain synthesis is finished, adding 7mL of 2% hydrazine hydrate/DMF (v/v) to selectively remove the Dde protecting group of Lys at the 17 th position, adding 0.4mmol of Fmoc-AEEA-OH, 0.4mmol of DIC and 0.44mmol of HOBt after the Dde protecting group is removed, and carrying out concussion condensation reaction for 2 h. After Fmoc protecting group removal, 0.4mmol of Fmoc-AEEA-OH, 0.4mmol of DIC and 0.44mmol of HOBt are added again, and the mixture is subjected to concussion condensation reaction for 2 hours. After removing the Fmoc protecting group, 0.4mmol of Fmoc-Glu-OtBu, 0.4mmol of DIC and 0.44mmol of HOBt are added, and the mixture is subjected to concussion condensation reaction for 2 hours. After Fmoc protecting group removal, 0.4mmol of octadecanedioic acid mono-tert-butyl ester, 0.4mmol of DIC and 0.44mmol of HOBt were added for condensation reaction for 2 hours, and after the reaction was completed, the resin was washed 4 times with 7mL of DMF.
(6) Cleavage of polypeptides
Transferring the obtained resin connected with the polypeptide into a round-bottom bottle, cutting the resin by using 5mL of cutting agent Reagent R (TFA/thioanisole/phenol/EDT, 90:5:3:2, V/V), reacting for 2h in an oil bath at constant temperature of 30 ℃, pouring the cutting liquid into 40mL of ethyl acetate, washing a crude product for 3 times by using 15mL of ethyl acetate after refrigerated centrifugation, and finally drying by using nitrogen to obtain the crude peptide. The crude peptide was dissolved in acetic acid and the TCE protecting group of Phe (4sm) was removed using hydrochloric acid activated zinc dust.
(7) Purification of polypeptides
And dissolving the crude target polypeptide in water, filtering by using a 0.25 mu m microporous filter membrane, and purifying by using an Shimadzu preparative reversed-phase HPLC system. The chromatographic conditions were C18 reverse phase preparative columns (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 8 mL/min; the detection wavelength was 214 nm. Eluting by linear gradient (20% B-70% B/30min), collecting target peak, removing methanol, lyophilizing to obtain pure product 0.27g with purity greater than 98%, and determining target polypeptide molecular weight 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-(CH2)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-NH2
The synthesis method is the same as that of example 3, and the target peak is collected and lyophilized to obtain 0.25g of a 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-(CH2)14-CH3)-Glu-Phe-Ile-Glu-Trp-Leu-Ile-Lys-Gly-Lys-AEEA-AEEA-Asp-Phe(4sm)-Nle-Gly-Trp-Nle-DMeAsp-MePhe-NH2
The synthesis method is the same as example 1, and the target peak is collected and lyophilized 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-(CH2)14-CH3)-Glu-Phe-Ile-Glu-Trp-Leu-Ile-Lys-Gly-Lys-AEEA-AEEA-Asp-Phe(4sm)-Nle-Gly-Trp-Nle-DMeAsp-MePhe-NH2
The synthesis method is the same as that of example 1, and the target peak is collected and lyophilized to obtain 0.18g of a 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-(CH2)16-COOH)-Glu-Phe-Ile-Glu-Trp-Leu-Ile-Lys-Gly-Lys-AEEA-AEEA-Asp-Phe(4sm)-Nle-Gly-Trp-Nle-DMeAsp-MePhe-NH2
The synthesis method is the same as that of example 3, and the target peak is collected and lyophilized to obtain 0.26g of a 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-(CH2)16-COOH)-Glu-Phe-Ile-Glu-Trp-Leu-Ile-Lys-Gly-Lys-AEEA-AEEA-Asp-Phe(4sm)-Nle-Gly-Trp-Nle-DMeAsp-MePhe-NH2
The synthesis method is the same as that of example 3, and the target peak is collected and lyophilized to obtain 0.23g of a 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-(CH2)14-CH3)-AEEA-AEEA-Asp-Phe(4sm)-Nle-Gly-Trp-Nle-DMeAsp-MePhe-NH2
The synthesis method is the same as that of example 1, and the target peak is collected and lyophilized to obtain 0.17g of a 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-(CH2)14-CH3)-AEEA-AEEA-Asp-Phe(4sm)-Nle-Gly-Trp-Nle-DMeAsp-MePhe-NH2
The synthesis method is the same as that of example 1, and the target peak is collected and lyophilized to obtain 0.25g of a 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-(CH2)16-COOH)-AEEA-AEEA-Asp-Phe(4sm)-Nle-Gly-Trp-Nle-DMeAsp-MePhe-NH2
The synthesis method is the same as that of example 3, and the target peak is collected and lyophilized to obtain 0.23g of a 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-(CH2)16-COOH)-AEEA-AEEA-Asp-Phe(4sm)-Nle-Gly-Trp-Nle-DMeAsp-MePhe-NH2
The synthesis method is the same as that of example 3, and the target peak is collected and lyophilized to obtain 0.27g of a 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 polypeptide compound at the receptor is determined by a functional assay that measures the cAMP response of HEK-293 cell lines stably expressing the human GLP-1 receptor. Cells stably expressing the GLP-1 receptor were split into T175 flasks and grown overnight in media (DMEM/10% FBS) to near confluency, then the media was removed and the cells were washed with calcium and magnesium free PBS and then treated with Accutase enzyme for protease. Detached cells were washed and resuspended in assay buffer (20mM HEPES, 0.1% BSA,2mM IBMX,1 × HBSS) and cell density determined and 25 μ L aliquots dispensed into wells of a 96-well plate. Is composed ofFor measurement, 25 μ Ι _ of a solution of the test polypeptide compound in assay buffer was added to the wells, followed by incubation at room temperature for 30 minutes. Cell cAMP levels were determined based on Homogeneous Time Resolved Fluorescence (HTRF) using the Cisbio kit. After addition of HTRF reagent diluted in lysis buffer (kit components), the plates were incubated for 1 hour and then the fluorescence ratio at 665/620nm was measured. By measuring the concentration (EC) that causes 50% activation of the maximal response50) To quantify the in vitro potency of agonists.
1321-N1 cells stably expressing CCK-1 receptor or CCK-2 receptor were cultured with DMEM-31966 (containing 10% FBS, 1% sodium pyruvate, 1% penicillin, 1% streptomycin). One day before the assay, cells were transferred to 384-well plates and the compound was dissolved in IP-One buffer (containing 10mmol/L HEPES,1mmol/L CaCl)24.2mmol/L KCl,146mmol/L NaCl,5.5mmol/L glucose, 50mmol/L LiCl) and diluted and added to 384 well plates. After 1 hour incubation at 37 ℃, intracellular inositol 1-phosphate concentrations were determined using the IP-One HTRF Assay kit by detecting the concentration (EC) that caused 50% activation of the maximal response50) To quantify the in vitro potency of agonists.
The test data (nM) in the examples of this patent application are shown in table 2 below, and although the test data are stated in terms of a number of significant figures, it should not be considered as indicating that the data have been determined to be exactly the number of significant figures.
Table 2: EC of polypeptide compounds on GLP-1 receptor, CCK-1 receptor and CCK-2 receptor50Values (in nM)
Sample (I) EC50(GLP-1 receptor) EC50(CCK-1 receptor) EC50(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, the agonistic activity of all the polypeptide compounds on GLP-1 receptor is higher than that of natural GLP-1, and the agonistic activity of most polypeptide compounds on CCK-1 receptor is also higher than that of CCK-8 and NN9056, and all the polypeptide compounds show high agonistic selectivity on CCK-1 receptor and higher agonistic selectivity on CCK-1 receptor than NN9056, while CCK-8 has no agonistic selectivity on CCK-1 receptor.
Example 14
Solubility and stability testing of polypeptide Compounds
Prior to testing the solubility and stability of a polypeptide compound, its purity is first determined using HPLC. Then, based on the determined% purity, 10mg of the polypeptide compound was dissolved in 1mL of solution in different buffer systems with gentle stirring for 2 hours. After centrifugation at 4500rpm for 20 minutes, the supernatant was analyzed by HPLC to determine peak area. And then comparing with the corresponding sample standard solution, and calculating to obtain the relative concentration of the tested sample solution. For stability testing, aliquots of the supernatants obtained from solubility were stored at 40 ℃ for 7 days, then the samples were centrifuged at 4500rpm for 20 minutes and the supernatants were analyzed by HPLC to determine peak areas. By comparing the peak areas (t) before the start of the stability experiment0) And peak area (t) after 7 days of storage7) Get "% remaining peptide". Calculated according to the following formula: % remaining peptide ═ area of peak t7)×100]Area per peak t0Stability is expressed as "% remaining peptide", and the results are shown in table 3 below.
Table 3: solubility and stability of polypeptide compounds
Figure BDA0002825765270000151
Figure BDA0002825765270000161
As shown in the results in Table 3, compared with the natural GLP-1 and CCK-8, the polypeptide compound of the invention has greatly improved solubility under the condition of acceptable pH of injection in a body and has the characteristic of being beneficial to preparation. The polypeptide compounds of the invention also have high solubility at pH 4.5, a property that may allow co-formulation for combination therapy with insulin or insulin derivatives. In addition, the polypeptide compound of the present invention also has high stability under the conditions of pH 4.5 and neutral pH.
Example 15
Stability of polypeptide Compounds to DPP-IV and NEP enzymes
The test sample was incubated with purified human DPP-IV or NEP enzyme at 37 ℃ for 0, 2, 4, and 8 hours, and the peak area of the residual sample in the solution at each time point was measured by HPLC to calculate the half-life of the sample, and the results are shown in Table 4.
Table 4: half-life of polypeptide Compounds in DPP-IV enzyme or NEP enzyme System (denoted by h)
Sample (I) Half-life (DPP-IV middle) 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 Table 4, the polypeptide compounds of the present invention have a half-life of more than 8 hours in both the DPP-IV enzyme-containing solution and NEP enzyme-containing solution, indicating that the polypeptide compounds can effectively resist the degradation by DPP-IV and NEP enzymes.
Example 16
Pharmacokinetic Properties of polypeptide Compounds in rat
Rats were given a subcutaneous (s.c.) injection of 50nmol/kg and blood samples were collected at 0.25, 0.5, 1, 2, 4, 8, 16, 24, 36 and 48 hours post-dose. After precipitation of the protein using acetonitrile, plasma samples were analyzed by LC-MS. Pharmacokinetic parameters and half-lives were calculated using WinonLin 5.2.1 (non-compartmental model) (table 5).
Table 5: pharmacokinetic profile of polypeptide Compounds in rats
Sample (I) T1/2(h) Cmax(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 shown in the results of table 5, the polypeptide compound of the present invention has a significantly prolonged in vivo half-life and pharmacokinetic profile supporting once-a-day administration or once-a-week administration.
Example 17
Effect of polypeptide Compounds on blood glucose in diabetes model mice (db/db mice)
Male db/db mice, randomly grouped, 6 per group. The blank group was given physiological saline (10mg/kg) by subcutaneous injection, the administration group was 6 groups, mice had free food and water during the experiment, and mice were given a single subcutaneous injection of 25nmol/kg of liraglutide, semaglutide, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 12, respectively, in a non-fasting state. Blood glucose levels were measured with a glucometer 0h before dosing, and 4, 6, 24 and 48h after dosing for each group of mice.
As shown in the results of FIG. 1, the results of in vivo blood glucose lowering experiments in db/db mice indicate that the polypeptide compound of the present invention shows a long-lasting blood glucose lowering activity superior to that of the positive control drug, namely, liraglutide or semagllutide.
Example 18
Effect of polypeptide Compounds on blood glucose and body weight in diet-induced obese (DIO) mice
Male C57BL/6J mice, weighing about 22g, were fed on high-fat D12492 diet from Research Diets for 18 weeks to create DIO mouse models. The blank control group 6 was fed with standard mouse feed only (control standard diet group). Before the start of administration, groups of DIO mice were randomly divided by body weight into 8 groups of 6 mice each, namely, a saline solution group (control high fat diet group), a positive control group (omaglutide and NN9056), and a test sample group (SEQ ID NOs: 4, 6, 8, 12). The normal saline (10mg/kg) was injected subcutaneously twice a day in the control standard diet group and the control high fat diet group, and the liraglutide and SEQ ID NO:6 were injected subcutaneously twice a day (25nmol/kg), semaglutide, NN9056, SEQ ID NO:4, SEQ ID NO:8, and SEQ ID NO:12 were injected subcutaneously once a day (25nmol/kg) for 21 days. Changes in body weight 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 (dosage) Overall body weight change (%) Body fat change (%)
Reference standard diet +1.5％(±0.3％) +6.0％(±2.6％)
Control high fat diet +0.6％(±0.3％) +2.5％(±0.9％)
Liraglutide (25nmol/kg twice daily) -15.1％(±3.2％)*** -30.8％(±4.2％)***
SEQ ID NO 6(25nmol/kg twice daily) -31.3％(±4.9％)***,### -56.1％(±5.4％)***,###
Semaglutide (25nmol/kg once a day) -15.9％(±2.1％)*** -32.4％(±4.8％)***
NN9056(25nmol/kg once a day) -11.3％(±2.0％)*** -20.4％(±2.2％)***
SEQ ID NO 4(25nmol/kg twice daily) -32.9％(±3.9％)***,### -57.2％(±5.0％)***,###
SEQ ID NO:8(25nmol/kg once a day) -33.5％(±3.5％)***,### -54.1％(±4.2％)***,###
12(25nmol/kg once daily) -32.2％(±3.5％)***,### -56.2％(±3.1％)***,###
***: p compared to control high fat diet group<0.001；###: in comparison with Liraglutide, NN9056 and semagllutide group P<0.001
As shown in table 6, the results of the polypeptide compound of the present invention administered continuously in DIO mice for 3 weeks significantly reduced the body weight and body fat content of the mice, and the effect of the polypeptide compound of the present invention was significantly stronger than that of the positive control drugs, liraglutide, NN9056 and semagllutide.
Example 19
Effect of polypeptide Compounds on glycated hemoglobin (HbA1c) and fasting plasma glucose in db/db mice
Male db/db mice, randomly grouped, 6 per group. One week after acclimatization, tail bleeds were performed to measure initial HbA1c values and fasting plasma glucose values prior to initiation of treatment. The blank group was given physiological saline (10mg/kg) by subcutaneous injection twice daily, and the administration groups were 7 groups, and 25nmol/kg of liraglutide (twice daily), semaglutide (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 period was 5 weeks, and fasting blood glucose values were measured after overnight fasting of mice after the end of treatment, while HbA1c (%) values were measured from blood (tables 7, 8).
Table 7: HbA1c (%) change in db/db mice over a 5-week dosing period
Figure BDA0002825765270000181
Figure BDA0002825765270000191
As shown in the results of Table 7, the polypeptide compound of the present invention, administered continuously in db/db mice for 5 weeks, could lower the value of HbA1c, indicating that it has a superior glycemic control effect over liraglutide and semaglutide. NN9056 failed to control the increase in HbA1c values, and showed no significant glycemic control.
Table 8: fasting plasma glucose change (%)/db mice over a 5 week dosing period
Sample (dosage) Fasting blood glucose change (%)
Physiological saline +7.6％(±1.1％)
Liraglutide (25nmol/kg twice daily) -2.4％(±0.5％)***
SEQ ID NO 6(25nmol/kg twice daily) -8.9％(±0.7％)***,###
NN9056(25nmol/kg once a day) +6.8％(±0.8％)
Semaglutide (25nmol/kg once a day) -3.0％(±0.5％)***
SEQ ID NO. 4(25nmol/kg once a day) -9.4％(±0.7％)***,###
SEQ ID NO:8(25nmol/kg once a day) -10.1％(±1.3％)***,###
12(25nmol/kg once daily) -9.6％(±0.8％)***,###
***: comparison with physiological saline group P<0.001；###: in comparison with Liraglutide, NN9056 and semagllutide group P<0.001
As shown in the results in Table 8, the polypeptide compound of the present invention can significantly reduce fasting plasma glucose in db/db mice after continuous administration for 5 weeks in db/db mice, which indicates that the polypeptide compound has a very high blood glucose control effect, and the effect of the polypeptide compound of the present invention is significantly stronger than that of the positive control drugs NN9056, liraglutide and semagllutide.
Example 20
Evaluation of side effects of polypeptide Compounds
The 7-week-old male C57BL/6J mice 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 liraglutide, semaglutide, NN9056, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8 and SEQ ID NO. 12, respectively. Before and 48 hours after the administration, blood samples were taken to determine the values of serum amylase (amylase) and lipase (lipase).
Table 9: values of serum amylase and lipase (in U/L) before and after administration to C57BL/6J mice
Sample (I) 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 at a large dose in C57BL/6J mice did not result in an increase in the serum amylase and lipase values, indicating that acute pancreatitis did not occur. The values of serum amylase and lipase are increased to different degrees after NN9056, liraglutide and semagllutide are administered, and the side effect of acute pancreatitis is caused.
Finally, it should be noted that the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting, 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 or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.
Sequence listing
<110> university of Jiangsu profession
<120> GLP-1/cholecystokinin-1 receptor dual agonists 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 (10)

1. A GLP-1/CCK-1 receptor dual-agonistic polypeptide compound is characterized in that the general formula of the amino acid sequence is as follows: His-Xaa1-Glu-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Thr-Glu-Tyr-Leu-Glu-Glu-Xaa2-Ala-Ala-Xaa3-Glu-Phe-Ile-Glu-Trp-Leu-Ile-Lys-Gly-Xaa4-Xaa5-Xaa6-Asp-Xaa7-Nle-Gly-Trp-Nle-DMeAsp-Xaa8-NH2
Wherein:
Xaa1is taken from Ala, D-Ala, Gly or Aib;
Xaa2from Glu, Lys or Lys modified in the side chain;
Xaa3is taken from Lys or Lys modified in side chain;
Xaa4is taken from Lys or Lys modified in side chain;
Xaa5is obtained from Ala or AEEA;
Xaa6is obtained from Ala or AEEA;
Xaa7is taken from Phe (4sm) or sTyr;
Xaa8is taken from Phe or MePhe;
wherein Lys having a modified side chain is selected from Lys (gamma-Glu-CO- (CH)2)n-CH3) Or Lys (AEEA-AEEA-gamma-Glu-CO- (CH)2)n-COOH)，
Lys(γ-Glu-CO-(CH2)n-CH3) The structural formula of (A) is shown as the following formula:
Figure FDA0002825765260000011
Lys(AEEA-AEEA-γ-Glu-CO-(CH2)n-COOH) is represented by the formula:
Figure FDA0002825765260000012
wherein n is a natural number, and n is more than or equal to 12 and less than or equal to 20.
2. The class of GLP-1/CCK-1 receptor dual agonist polypeptide compounds of claim 1, wherein said n is 14, 16, 18 or 20.
3. The GLP-1/CCK-1 receptor dual agonist polypeptide compound of claim 1, wherein the amino acid sequence of said polypeptide compound is one of the following:
(1)
His-Ala-Glu-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Thr-Glu-Tyr-Leu-Glu-Glu-Lys(γ-Glu-CO-(CH2)14-CH3)-Ala-Ala-Lys-Glu-Phe-Ile-Glu-Trp-Leu-Ile-Lys-Gly-Lys-AEEA-AEEA-Asp-Phe(4sm)-Nle-Gly-Trp-Nle-DMeAsp-MePhe-NH2
(2)
His-Aib-Glu-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Thr-Glu-Tyr-Leu-Glu-Glu-Lys(γ-Glu-CO-(CH2)14-CH3)-Ala-Ala-Lys-Glu-Phe-Ile-Glu-Trp-Leu-Ile-Lys-Gly-Lys-AEEA-AEEA-Asp-Phe(4sm)-Nle-Gly-Trp-Nle-DMeAsp-MePhe-NH2
(3)
His-Ala-Glu-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Thr-Glu-Tyr-Leu-Glu-Glu-Lys(AEEA-AEEA-γ-Glu-CO-(CH2)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-NH2
(4)
His-Aib-Glu-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Thr-Glu-Tyr-Leu-Glu-Glu-Lys(AEEA-AEEA-γ-Glu-CO-(CH2)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-NH2
(5)
His-Ala-Glu-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Thr-Glu-Tyr-Leu-Glu-Glu-Glu-Ala-Ala-Lys(γ-Glu-CO-(CH2)14-CH3)-Glu-Phe-Ile-Glu-Trp-Leu-Ile-Lys-Gly-Lys-AEEA-AEEA-Asp-Phe(4sm)-Nle-Gly-Trp-Nle-DMeAsp-MePhe-NH2
(6)
His-Aib-Glu-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Thr-Glu-Tyr-Leu-Glu-Glu-Glu-Ala-Ala-Lys(γ-Glu-CO-(CH2)14-CH3)-Glu-Phe-Ile-Glu-Trp-Leu-Ile-Lys-Gly-Lys-AEEA-AEEA-Asp-Phe(4sm)-Nle-Gly-Trp-Nle-DMeAsp-MePhe-NH2
(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-(CH2)16-COOH)-Glu-Phe-Ile-Glu-Trp-Leu-Ile-Lys-Gly-Lys-AEEA-AEEA-Asp-Phe(4sm)-Nle-Gly-Trp-Nle-DMeAsp-MePhe-NH2
(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-(CH2)16-COOH)-Glu-Phe-Ile-Glu-Trp-Leu-Ile-Lys-Gly-Lys-AEEA-AEEA-Asp-Phe(4sm)-Nle-Gly-Trp-Nle-DMeAsp-MePhe-NH2
(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-(CH2)14-CH3)-AEEA-AEEA-Asp-Phe(4sm)-Nle- Gly-Trp-Nle-DMeAsp-MePhe-NH2
(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-(CH2)14-CH3)-AEEA-AEEA-Asp-Phe(4sm)-Nle-Gly-Trp-Nle-DMeAsp-MePhe-NH2
(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-(CH2)16-COOH)-AEEA-AEEA-Asp-Phe(4sm)-Nle-Gly-Trp-Nle-DMeAsp-MePhe-NH2
(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-(CH2)16-COOH)-AEEA-AEEA-Asp-Phe(4sm)-Nle-Gly-Trp-Nle-DMeAsp-MePhe-NH2。
4. a pharmaceutically acceptable salt of a GLP-1/CCK-1 receptor dual agonist polypeptide compound of claim 1.
5. The salt of claim 4, wherein the salt is a GLP-1/CCK-1 receptor dual agonist polypeptide compound and 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, pectinic 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, ethanolic acid, lithospermic acid, oxalic acid, lactic acid, succinic acid, malonic acid, hemisulfuric acid, malic acid, maleic acid, malic acid, alginic acid, fumaric acid, D-gluconic acid, glycerophosphoric acid, glucoheptonic acid, aspartic acid, thiocyanic acid or sulfosalicylic acid.
6. A pharmaceutical composition comprising a GLP-1/CCK-1 receptor dual agonist polypeptide compound of claim 1, wherein the pharmaceutical composition comprises: a GLP-1/CCK-1 receptor dual agonist polypeptide compound or a pharmaceutically acceptable salt of claim 4, and a pharmaceutically acceptable carrier or diluent.
7. A medicament comprising the pharmaceutical composition of claim 6, wherein said medicament is a pharmaceutically acceptable capsule, tablet, spray, inhalant, injection, patch, emulsion, film, powder, or combination thereof.
8. 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.
9. The use of claim 8, wherein the metabolic disease or disorder is at least one of diabetes, NAFLD, NASH, hyperlipidemia, obesity.
10. The use according to claim 9, wherein the diabetes is type 1 diabetes, type 2 diabetes or gestational diabetes.
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