CN117417431A - Polypeptides with agonistic activity on GLP-1, glucagon and GIP receptor and application thereof - Google Patents

Polypeptides with agonistic activity on GLP-1, glucagon and GIP receptor and application thereof Download PDF

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CN117417431A
CN117417431A CN202311316622.0A CN202311316622A CN117417431A CN 117417431 A CN117417431 A CN 117417431A CN 202311316622 A CN202311316622 A CN 202311316622A CN 117417431 A CN117417431 A CN 117417431A
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acid
polypeptide
glp
glucagon
agonistic activity
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廖世杰
刘静
刘云
韦庆军
罗晓婷
谢天裕
蒋能
李翡翠
唐海军
罗恺
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First Affiliated Hospital of Guangxi Medical University
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Abstract

The invention discloses a polypeptide with agonistic activity to GLP-1, glucagon and GIP receptor and application thereof, wherein the polypeptide can act on the GLP-1 receptor to increase insulin secretion and delay gastric emptying so as to reduce blood sugar; acting on glucagon receptor, producing beneficial effects on body weight, energy metabolism, lipid metabolism; acting on GIP receptor, promoting insulin secretion, improving sensitivity of islet cells to glucose, and reducing blood sugar. The polypeptide can improve the metabolic function, provide better effects of controlling blood sugar, losing weight and regulating fat by regulating the three receptors simultaneously and affecting a plurality of physiological paths, and has great potential in the aspect of medicaments for treating diabetes, obesity, non-alcoholic fatty liver disease, non-alcoholic fatty hepatitis, dyslipidemia and other diseases.

Description

Polypeptides with agonistic activity on GLP-1, glucagon and GIP receptor and application thereof
Technical Field
The invention relates to the technical field of biological medicines, in particular to a polypeptide with agonistic activity on GLP-1, glucagon and GIP receptors and application thereof.
Background
Obesity has become a global epidemic problem affecting human health and quality of life, and its prevalence has steadily increased since the mid 20 th century. Obesity is an important risk factor for many chronic metabolic diseases, including type 2 diabetes (T2 DM), hypertension, high cholesterol, cardiovascular disease, fatty liver, etc. Studies have shown that clinically 80-90% of T2DM patients are associated with overweight or obesity. The existing medicines for treating obesity are few in variety, limited in curative effect and remarkable in side effect.
Glucagon-like peptide-1 (GLP-1) is a peptide hormone secreted by the intestinal tract and mainly plays a role in regulating blood glucose. GLP-1 can protect islet cells, reduce damage and death of islet cells, help maintain functions and quantity of islet cells, and can stimulate islet cells to secrete insulin so as to reduce blood sugar level. GLP-1 also inhibits glucagon release, thereby reducing the amount of glucose released by the liver. In addition, GLP-1 also has the effects of delaying gastric emptying, suppressing appetite and the like, and has partial weight reduction effect. GLP-1 drugs increase the bioactivity of GLP-1 through different mechanisms, thereby regulating blood sugar, improving insulin resistance and promoting weight loss. Currently marketed drugs include liraglutide, semaglutide and the like, which are widely used in the treatment of T2DM and exhibit good therapeutic effects and safety. However, GLP-1 drugs have the disadvantages of short half-life, easy generation of adverse reactions in the gastrointestinal tract, and the like, and the drugs are usually required to be administered by subcutaneous injection or continuous infusion, so that the compliance of patients is poor. Thus, there is a need to develop more convenient and safe tolerating therapeutic agents.
Glucagon (glucon) is a hormone secreted by islet alpha cells. Under the condition of low blood sugar, the secretion of glucagon is increased, so that the liver is promoted to decompose glycogen and release glucose into blood, thereby recovering the blood sugar level and ensuring the energy supply of tissues such as brain of a body. In addition, glucopon has the effects of promoting lipolysis, fat oxidation, fever and the like in vivo, and long-term administration can exhibit a weight-reducing effect by increasing energy metabolism, but it may cause too rapid or too high blood sugar rise, causing hyperglycemia symptoms, and thus cannot be used.
Glucose-dependent insulin peptide (GIP), a peptide hormone secreted by small intestine K cells, belongs to incretins, has various roles in the human body, and is mainly involved in blood glucose regulation, fat metabolism, appetite regulation, and the like. GIP can stimulate islet beta cells to secrete insulin so as to reduce blood sugar, promote the utilization of glucose, protect islet cells, reduce damage and death of islet cells, and help to maintain the functions and quantity of islets. And GIP can promote fat metabolism, maintain balance of fat metabolism, and suppress appetite by affecting the central nervous system to affect the appetite center. However, GIP has weak biological activity and a relatively mild blood glucose regulating effect.
GLP-1, glucopon and GIP receptors all belong to the GPCR receptor family, with similar protein structures and binding mechanisms, making it possible to design multiple agonists for these three receptors. Multiple agonists of these three receptors can act on GLP-1 receptor, glucopon receptor and GIP receptor simultaneously, and can exert GLP-1, glucopon and GIP activities simultaneously. GLP-1 can reduce blood sugar, delay gastric emptying and inhibit appetite; glucopon can promote lipolysis and raise blood sugar, but the blood sugar-raising effect can be counteracted by the blood sugar-lowering activity of GLP-1; GIP can protect islet cells, stimulate insulin secretion, and promote glucose utilization. The three activities of the GLP-1/glucon/GIP receptor triple agonists are matched with each other, so that the control effect on blood sugar can be enhanced, fat can be decomposed, and the weight can be reduced. For the treatment of T2DM, obesity and its related metabolic syndrome, triple agonists targeting three receptors may provide better glycemic control and weight loss effects with significant advantages over GLP-1 analogues alone.
Disclosure of Invention
The invention provides a polypeptide with agonistic activity to GLP-1, glucagon and GIP receptors and application thereof, wherein the polypeptide can act on the GLP-1 receptors to increase insulin secretion and delay gastric emptying so as to reduce blood sugar; acting on glucagon receptor, producing beneficial effects on body weight, energy metabolism, lipid metabolism; acting on GIP receptor, promoting insulin secretion, improving sensitivity of islet cells to glucose, and reducing blood sugar; the polypeptide can improve the metabolic function, provide better blood sugar control and weight reduction effects by regulating the three receptors simultaneously and affecting a plurality of physiological paths, and can show potential clinical application prospects in the treatment of metabolic syndromes such as diabetes, obesity, non-alcoholic fatty liver disease, non-alcoholic fatty hepatitis, dyslipidemia and the like.
In order to achieve the above object, the present invention has the following technical scheme:
a polypeptide having agonistic activity to GLP-1, glucagon and GIP receptors, the amino acid sequence of said polypeptide having the general formula:
His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Met-Ser-Arg-Ala-Xaa 1 -Glu-Xaa 2 -Ile-Ala-Xaa 3 -Arg-Le u-Phe-Val-Asp-Trp-Leu-Ile-Glu-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2 wherein: xaa 1 Selected from Leu or Met; xaa 2 Selected from Lys-R1 or Lys-R2 with side chains chemically modified; xaa 3 Selected from Ala or Val;
the chemical structural formula of the Lys-R1 is as follows:
the chemical structure of the Lys-R2 is as follows:
preferably, the sequence structure of the polypeptide is selected from any one of the amino acid sequences shown in SEQ ID NO. 1-4:
the invention also provides pharmaceutically acceptable salts of the polypeptides having agonistic activity on GLP-1, glucagon and GIP receptors.
Further, the pharmaceutically acceptable salt is a salt of a polypeptide having agonistic activity to GLP-1, glucagon and GIP receptor with one of the following compounds; the following compounds include hydrochloric acid, formic acid, acetic acid, pyruvic acid, butyric acid, caproic acid, benzenesulfonic acid, pamoic acid, benzoic acid, salicylic acid, lauric acid, cinnamic acid, propionic acid, dodecylsulfuric acid, citric acid, ascorbic acid, wine stearic acid, oxalic acid, lactic acid, succinic acid, malonic acid, maleic acid, fumaric acid, aspartic acid, sulfosalicylic acid.
The invention also provides a medicament prepared from the polypeptide with agonistic activity on GLP-1, glucagon and GIP receptors, wherein the medicament comprises any one of tablets, capsules, syrup, tincture, inhalant, spray, injection, film, patch, powder, granule, emulsion, suppository or compound preparation.
The invention also provides a pharmaceutical composition prepared from a polypeptide having agonistic activity to GLP-1, glucagon and GIP receptors, the pharmaceutical composition comprising the polypeptide having agonistic activity to GLP-1, glucagon and GIP receptors, a pharmaceutically acceptable carrier or diluent; or the pharmaceutical composition comprises pharmaceutically acceptable salts, pharmaceutically acceptable carriers or diluents of the polypeptides having agonistic activity to GLP-1, glucagon and GIP receptors.
The invention also provides application of the polypeptide with agonistic activity to GLP-1, glucagon and GIP receptor or the pharmaceutically acceptable salt of the polypeptide with agonistic activity to GLP-1, glucagon and GIP receptor or the medicament or the pharmaceutical composition in preparing medicaments for treating metabolic diseases or symptoms; the metabolic disease or disorder includes diabetes, obesity, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis or dyslipidemia.
The polypeptide compound prepared by the invention has strong agonistic activity to GLP-1 receptor, and better agonistic activity to glucogon receptor, but has relatively weak agonistic activity to GIP receptor, realizes better hypoglycemic, weight-reducing and lipid-regulating effects, has lower gastrointestinal side effect, and provides a new idea for preparing the high-efficiency and low-toxicity multiple agonist.
Compared with the prior art, the invention has the beneficial technical effects that:
(1) The polypeptide provided by the invention has the obvious weight reduction and weight increase prevention effects while reducing blood sugar more effectively, and can be used for regulating lipid metabolism better;
(2) The polypeptide of the invention has a unique N-terminal 6-10 bit sequence structure (FTSDM) and a unique in-vitro GLP-1 receptor, glucagon receptor and GIP receptor agonism activity proportion, thereby bringing about remarkably improved weight reduction and lipid metabolism regulation effects, and having lower gastrointestinal side effects and unexpected beneficial effects;
(3) Compared with the reported GLP-1/glucopon/GIP receptor triple agonists, the polypeptide provided by the invention has stronger GLP-1 receptor and glucopon receptor agonistic activity and weaker GIP receptor agonistic activity, but the weight reduction, lipid metabolism regulation and hypoglycemic activity are obviously improved, the gastrointestinal side effects are obviously reduced, the polypeptide has more potential in the aspect of treating metabolic diseases, and a new thought is provided for the drug development of the multiple agonists;
(4) The polypeptide provided by the invention has stable chemical property and has pharmacokinetic characteristics for supporting at least once weekly administration; the polypeptide provided by the invention has better treatment effect on metabolic diseases such as T2DM, obesity, dyslipidemia and the like than the existing medicines on the market. Therefore, the polypeptide provided by the invention is suitable for being used as an active ingredient of medicines for treating metabolic diseases, such as diabetes, obesity, nonalcoholic fatty liver disease, nonalcoholic fatty hepatitis, dyslipidemia and the like.
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FIG. 1 shows the percentage change in body weight of each subject of the invention over 21 days of prolonged DIO mice administration.
Detailed Description
The present invention will be described in detail below with reference to the drawings and examples.
Unless defined otherwise herein, scientific and technical terms used in this specification shall have the meanings commonly understood by one of ordinary skill in the art. Generally, the terms and methods used in connection with chemistry, molecular biology, cell biology, pharmacology, according to the invention, are well known and commonly used in the art.
All combinations of the various elements disclosed herein are within the scope of the invention. Furthermore, the scope of the invention should not be limited by the specific disclosure provided below.
Further, the amino acids mentioned in the present invention may be abbreviated as follows according to the naming convention of IUPAC-IUB:
alanine (Ala, a); arginine (Arg, R); asparagine (Asn, N); aspartic acid (Asp, D); cysteine (Cys, C); glutamic acid (Glu, E); glutamine (Gln, Q); glycine (Gly, G); histidine (His, H); isoleucine (Ile, I); leucine (Leu, L); lysine (Lys, K); methionine (Met, M); phenylalanine (Phe, F); proline (Pro, P); serine (Ser, S); threonine (Thr, T); tryptophan (Trp, W); tyrosine (Tyr, Y); valine (Val, V).
Further, unless explicitly indicated, all amino acid residues in the polypeptides of the invention are preferably in the L configuration.
Further, "-NH on the C-terminus of the sequence 2 "part indicates an amide group (-CONH) at the C-terminus 2 )。
Further, in addition to natural amino acids, unnatural amino acids, alpha-aminoisobutyric acid (Aib) are used in the sequences of the invention.
Furthermore, the polypeptide compound can be synthesized by a polypeptide solid-phase synthesis method or can be produced by a genetic engineering technology.
The following specific embodiments are provided in order to illustrate the present invention in more detail, but the aspects of the present invention are not limited thereto.
Example 1
Synthesis of polypeptide Compound of SEQ ID NO. 1
(1) Swelling of the resin
0.338g (0.1 mmol equivalent) of RinkAmide MBHA resin with a loading of 0.296mmol/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 LDMF.
(2) Removal of Fmoc protecting groups from resin
Transferring the swelled resin into a PSI-200 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 20% piperidine/DMF (v/v) deprotection solvent, reacting with the resin for 15min, washing the resin for 4 times by 7mL of LDMF for 1.5min each time, and obtaining the Rink resin without Fmoc protecting groups.
(3) Synthesis of Fmoc-Ser-Rink amide-MBHAresin
Fmoc-Ser (tBu) -OH (0.4 mmoL) was weighed, 2mLHBTU/HOBt (0.4 mmoL/0.44 mmoL) condensing agent and 1mLDIPEA (0.8 mmoL) activated base were added, after pre-activation for 30min, the activated amino acid was added to the reactor, the reaction was stirred at room temperature in DMF for 2h, after the reaction solution was filtered off, the resin was washed 4 times with 7mLDMF, and the reaction coupling was checked for completion using Kaiser reagent, 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. As the Lys of the Lys site in which the side chain is modified, fmoc-Lys (Alloc) -OH, fmoc-Lys (Dde) -OH, fmoc-Lys (Mtt) -OH, fmoc-Lys (ivDde) -OH or the like can be used. Fmoc-Lys (Dde) -OH protection strategy was used in this example, while Boc-His (Boc) -OH was used for the N-terminal His.
(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 16-position Lys, and after the Dde protecting group is removed, 0.4mmol of Fmoc-AEEA-OH,0.4mmol of HBTU and 0.44mmol of HOBt are added to carry out oscillation condensation reaction for 2h. After removal of Fmoc protecting groups, 0.4mmol Fmoc-AEEA-OH,0.4mmol HBTU and 0.44mmol HOBt were added again and the reaction was performed with shaking for 2h. After removal of Fmoc protecting groups, 0.4mmol Fmoc-Glu-OtBu,0.4mmol HBTU 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 HBTU and 0.44mmol of HOBt were added for condensation reaction for 2h, and after the reaction was completed, the resin was washed 4 times with 7 mM LDMF.
(6) Cleavage of polypeptides
The resin with the polypeptide is transferred into a round bottom bottle, 5mL of a cutting agent (triisopropylsilane/water/TFA, 2.5:2.5:95, 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 diethyl ether, the crude product is washed 3 times by 15mL of glacial diethyl ether after refrigerated centrifugation, and finally the crude peptide is obtained by blowing with nitrogen.
(7) Purification of polypeptides
Dissolving the crude polypeptide product in water/methanol, filtering with 0.25 μm microporous membrane, and purifying with Shimadzu preparation type reversed phase HPLC system. Chromatographic conditions were C18 reverse phase preparation column (250 mm. Times.4.6 mm,5 μm); mobile phase a:0.1% TFA/water (V/V), mobile phase B: methanol (V/V); the flow rate is 0.8mL/min; the detection wavelength was 214nm. Eluting with linear gradient (50-90% B/15 min), collecting target peak, removing methanol, lyophilizing to obtain pure product with purity of 0.16g or more than 99%, and determining molecular weight of target polypeptide by MS. The theoretical relative molecular mass is 4874.6.ESI-MS M/z calculated [ M+3H] 3+ 1625.9,[M+4H] 4+ 1219.7; observed value [ M+3H] 3+ 1625.5,[M+4H] 4+ 1219.1。
Example 2
Synthesis of polypeptide compound of SEQ ID No. 2
The synthesis method is the same as that of example 1, 0.17g of the target peak is collected and freeze-dried to obtain a pure product, the purity is more than 99%, and the molecular weight of the target polypeptide is confirmed by MS. The theoretical relative molecular mass is 4828.5.ESI-MS M/z calculated [ M+3H] 3+ 1610.5,[M+4H] 4+ 1208.1; observed value [ M+3H] 3+ 1610.2,[M+4H] 4+ 1207.9。
Example 3
Synthesis of polypeptide compound of SEQ ID No. 3
The synthesis was carried out as in example 1, except that the modification of the Lys side chain was carried out by changing the mono-tert-butyl octadecanedioate to mono-tert-butyl eicosanedioate. And collecting target peaks, freeze-drying to obtain 0.18g of pure product with purity of more than 99%, and confirming the molecular weight of target polypeptide by MS. The theoretical relative molecular mass is 4902.7.ESI-MS M/z calculated [ M+3H] 3+ 1635.2,[M+4H] 4+ 1226.7; observed value [ M+3H] 3+ 1634.8,[M+4H] 4+ 1226.4。
Example 4
Synthesis of polypeptide compound of SEQ ID No. 4
The synthesis was carried out as in example 1, except that the modification of the Lys side chain was carried out by changing the mono-tert-butyl octadecanedioate to mono-tert-butyl eicosanedioate. Collecting target peak, freeze drying to obtain 0.17g of pure product with purity greater than 99%, and determining molecular weight of target polypeptide by MS. The theoretical relative molecular mass is 4856.6.ESI-MS M/z calculated [ M+3H] 3+ 1619.9,[M+4H] 4+ 1215.2; observed value [ M+3H] 3+ 1619.5,[M+4H] 4+ 1214.6。
Example 5
Determination of agonistic Activity of polypeptide Compounds on GLP-1 receptor, glucoago receptor and GIP receptor
Agonism of the receptor by the polypeptide compounds is determined by a functional assay that measures cAMP response of HEK-293 cell lines stably expressing human GLP-1 receptor, glucoon receptor or GIP receptor. Cells stably expressing the three receptors were each split into T175 flasks and grown in medium overnight to near confluency, after which the medium was removed and the cells were washed with calcium and magnesium free PBS and then protease treated with Accutase 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 pair agonistsIs quantified for in vitro efficacy.
The test data (nM) in the examples of this patent application are shown in Table 1 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 1: EC of polypeptide compounds to human GLP-1 receptor, glucagon receptor and GIP receptor 50 Value (expressed in nM)
As shown in Table 1, the agonistic activity of the polypeptide compound of the present invention to GLP-1 receptor is stronger than that of natural GLP-1 (about 2.6-4.4 times stronger), the agonistic activity of the polypeptide compound of the present invention to glucopon receptor is weaker than that of natural glucopon (about 1.4-8.7 times weaker), and the agonistic activity of the GIP receptor of the polypeptide compound of the present invention is weaker than that of natural GIP (about 25.6-38.4 times weaker), which indicates that the polypeptide compound of the present invention has strong GLP-1 receptor agonistic activity, better glucopon receptor agonistic activity and weaker GIP receptor agonistic activity, has a specific agonistic activity ratio to GLP-1 receptor, GIP receptor and glucopon receptor, and also conforms to the characteristics of triple agonists described in the present patent.
Example 6
Pharmacokinetic properties of polypeptide Compounds in rats
SD rats were given 50nmol/kg of subcutaneous (s.c.) injection and blood samples were collected 0.25h, 0.5h, 1h, 2h, 4h, 8h, 16h, 24h, 36h and 48h 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 2).
Table 2: pharmacokinetic profile of polypeptide Compounds in rats
Sample of T 1/2 (h) C max (ng/mL)
Semaglutide 9.4 498
SEQ ID NO:1 11.6 522
SEQ ID NO:2 13.1 519
As the results in table 2 show, the in vivo half-life of the polypeptide compounds of the present invention is significantly prolonged over once a week dosing semaglutine already on the market, demonstrating that the polypeptide compounds of the present invention have pharmacokinetic profiles supporting at least once a week dosing.
Example 7
Influence of polypeptide Compounds on blood lipid and body weight in diet-induced obese (DIO) mice
Male C57BL/6J mice, weighing about 21g, were kept on the D12492 high fat diet of Research Diets for 18 weeks to make DIO mouse models. Before the start of the administration, the DIO mice of each group were randomly grouped according to body weight, 6 in each group, respectively, physiological saline group (blank control group), positive control group (semaglutide, tirzepatide and SAR441255 (GLP-1/glucoon/GIP receptor triple agonist, cell metanolism, 2022,34,1-16)), and test sample group (SEQ ID NO: 2). Each group of mice was subcutaneously injected with normal saline (10 mg/kg), semaglutide (10 nmol/kg), tirzepatide (10 nmol/kg), SEQ ID NO:2 (10 nmol/kg), or SAR441255 (10 nmol/kg) twice daily for 21 days of the dosing period. The body weight changes of mice were recorded daily and Nuclear Magnetic Resonance (NMR) was used to measure body fat mass before and at the end of the experiment. At the end of the experiment, each group of mice was sacrificed and liver tissues were taken to measure liver Triglyceride (TG) and Total Cholesterol (TC) content. Blood serum was also collected and the serum glutamic-pyruvic transaminase (ALT), glutamic-oxaloacetic transaminase (AST), triglyceride (TG) and Total Cholesterol (TC) levels were measured.
Table 3: body weight and body fat changes in DIO mice over a 3 week dosing period
Sample (dose) Overall weight change (%) Body fat change (%)
Blank control (normal saline group) -1.2±3.3 -1.6±0.6
Semaglutide(10nmol/kg) -17.5±2.1 *** -23.8±3.8 ***
Tirzepatide(10nmol/kg) -24.5±3.3 *** -36.9±2.7 ***
SAR441255(10nmol/kg) -31.8±1.6 *** -40.5±4.6 ***
SEQ ID NO:2(10nmol/kg) -39.2±2.8 ***,### -54.6±5.0 ***,###
*** : p compared with the blank control group<0.001； ### : group ratio P to semaglutide, tirzepatide and SAR441255<0.001 (One-Way ANOVA, tukey post hoc test) the results are expressed as mean ± SD of 6 mice per group.
As shown in the results of fig. 1 and table 3, the polypeptide compound of the present invention, SEQ ID No. 2, was administered continuously in DIO mice for 3 weeks, and the weight and body fat content of the mice could be significantly reduced, and the weight and body fat reducing effects of the polypeptide compound of the present invention were significantly stronger than those of the positive control semaglutide, tirzepatide and SAR441255. Notably, the GLP-1 receptor agonistic activity of SAR441255 is similar to native GLP-1, with the glucopon receptor agonistic activity being about 2-fold lower than native glucopon and the GIP receptor agonistic activity being about 2-fold lower than native GIP (Cell metaolism, 2022,34,1-16). From this, it is clear that GLP-1 receptor agonism and glucogon receptor agonism of SAR441255 are similar to those of SEQ ID NO. 2, but GIP receptor agonism of SAR441255 is significantly stronger than that of SEQ ID NO. 2, but that the polypeptide compound of the invention, SEQ ID NO. 2, shows significantly better weight and body fat reducing activity than that of SAR441255.
Table 4: liver Triglyceride (TG) and Total Cholesterol (TC) levels 3 weeks after DIO mice treatment
Sample (dose) Total cholesterol (mg/g) Triglyceride (mg/g)
Blank control (normal saline group) 11.6±0.5 139.7±11.6
Semaglutide(10nmol/kg) 9.1±0.5 *** 78.9±6.2 ***
Tirzepatide(10nmol/kg) 8.9±0.4 *** 74.6±5.9 ***
SAR441255(10nmol/kg) 6.1±0.3 *** 56.6±3.6 ***
SEQ ID NO:2(10nmol/kg) 4.1±0.1 ***,### 35.4±2.1 ***,###
*** : p compared with the blank control group<0.001； ### : group ratio P to semaglutide, tirzepatide and SAR441255<0.001 (One-Way ANOVA, tukey post hoc test) the results are expressed as mean ± SD of 6 mice per group.
Table 5: serum glutamic pyruvic transaminase (ALT) and glutamic oxaloacetic transaminase (AST) levels after 3 weeks of DIO mice treatment
Sample (dose) Glutamic pyruvic transaminase (U/L) Glutamic-oxaloacetic transaminase (U/L)
Blank control (normal saline group) 456±51 415±39
Semaglutide(10nmol/kg) 214±26 *** 266±31 ***
Tirzepatide(10nmol/kg) 229±15 *** 278±24 ***
SAR441255(10nmol/kg) 186±32 *** 196±18 ***
SEQ ID NO:2(10nmol/kg) 116±12 ***,### 96±9 ***,###
*** : p compared with the blank control group<0.001； ### : group ratio P to semaglutide, tirzepatide and SAR441255<0.001 (One-Way ANOVA, tukey post hoc test) the results are expressed as mean ± SD of 6 mice per group.
As shown in tables 4 and 5, the polypeptide compound prepared in the embodiment of the invention is continuously administered in DIO mice for 3 weeks, so that the liver triglyceride and total cholesterol content of the mice can be obviously reduced, the serum glutamic pyruvic transaminase and glutamic oxaloacetic transaminase content can be obviously reduced, and the effect of the polypeptide compound of the invention is obviously stronger than that of positive control medicines semaglutide, tirzepatide and SAR441255, which indicates that the polypeptide compound of the invention has good prospect for treating non-alcoholic fatty liver disease and non-alcoholic steatohepatitis.
Table 6: serum Triglyceride (TG) and Total Cholesterol (TC) levels 3 weeks after DIO mice treatment
Sample (dose) Total cholesterol (mmol/L) Triglyceride (mmol/L)
Blank control (normal saline group) 13.6±2.2 2.0±0.2
Semaglutide(10nmol/kg) 9.5±0.6 *** 1.4±0.2 ***
Tirzepatide(10nmol/kg) 9.4±0.3 *** 1.4±0.3 ***
SAR441255(10nmol/kg) 6.0±0.4 *** 1.1±0.2 ***
SEQ ID NO:2(10nmol/kg) 4.3±0.3 ***,### 0.5±0.1 ***,###
*** : p compared with the blank control group<0.001； ### : group ratio P to semaglutide, tirzepatide and SAR441255<0.001 (One-Way ANOVA, tukey post hoc test) the results are expressed as mean ± SD of 6 mice per group.
As the results in table 6 show, the polypeptide compound of the present invention can significantly reduce serum triglyceride and total cholesterol content of mice by continuous administration in DIO mice for 3 weeks, and the effect of the polypeptide compound of the present invention for reducing serum lipid (triglyceride and cholesterol) content is significantly stronger than that of positive control semaglutide, tirzepatide and SAR441255.
Example 8
Effect of polypeptide Compounds on db/db mouse glycosylated hemoglobin (HbA 1 c) and blood glucose
Male db/db mice were randomly grouped, 6 per group. Physiological saline group (blank control group), positive control group (semaglutide, tirzepatide and SAR 441255) and test sample group (SEQ ID NO: 2), respectively. 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. Each group of mice was subcutaneously injected with normal saline (10 mg/kg), semaglutide (10 nmol/kg), tirzepatide (10 nmol/kg), SEQ ID NO:2 (10 nmol/kg), or SAR441255 (10 nmol/kg) twice daily for a period of 35 days. The mice were fasted overnight after the end of treatment and blood was taken to measure the fasting blood glucose values and HbA1c (%).
Table 7: hbA1c (%) change in db/db mice over a 35 day dosing period
Sample (dose) HbA1c% (before treatment) HbA1c% (after treatment)
Blank control (normal saline group) 6.5±0.1 7.1±0.4
Semaglutide(10nmol/kg) 6.6±0.2 6.0±0.2 ***
Tirzepatide(10nmol/kg) 6.7±0.1 5.9±0.2 ***
SAR441255(10nmol/kg) 6.5±0.3 5.9±0.3 ***
SEQ ID NO:2(10nmol/kg) 6.6±0.3 5.0±0.1 ***,###
*** : p compared with the blank control group<0.001； ### : group ratio P to semaglutide, tirzepatide and SAR441255<0.001 (One-Way ANOVA, tukey post hoc test) the results are expressed as mean ± SD of 6 mice per group.
As shown in the results of Table 7, the polypeptide compound of the present invention was administered continuously in db/db mice for 35 days, the HbA1c value of the mice could be significantly reduced, and the HbA1c value of the mice of the polypeptide compound group of the present invention after treatment was significantly lower than those of the positive controls semaglutide, tirzepatide and SAR441255, indicating that the polypeptide compound of the present invention had a good glycemic control.
Table 8: fasting blood glucose changes in db/db mice over a 35 day dosing period
Sample (dose) Fasting blood glucose (%)
Blank control (normal saline group) +4.6±0.7％
Semaglutide(10nmol/kg) -3.5±0.4％ ***
Tirzepatide(10nmol/kg) -4.1±0.3％ ***
SAR441255(10nmol/kg) -4.6±0.3％ ***
SEQ ID NO:2(10nmol/kg) -11.6±0.4％ ***,###
*** : p compared with the blank control group<0.001； ### : group ratio P to semaglutide, tirzepatide and SAR441255<0.001 (One-Way ANOVA, tukey post hoc test) the results are expressed as mean ± SD of 6 mice per group.
As shown in the results of Table 8, the polypeptide compound prepared in the embodiment of the invention can obviously reduce the fasting blood glucose of the db/db mice after being continuously administered in the db/db mice for 35 days, which indicates that the polypeptide compound has excellent blood glucose control effect and the blood glucose control effect of the polypeptide compound is obviously stronger than that of positive control medicines semaglutide, tirzepatide and SAR441255.
Example 9
Gastrointestinal side effects of polypeptide Compounds
Male SD rats (200-250 g) were randomly grouped and housed in a single cage, and each group of rats was given kaolin feed (Research Diets) in addition to normal feed 4 days prior to the experiment, and the kaolin feed was placed in separate compartments of a food funnel to allow the rats to become accustomed to the presence of kaolin feed in the cage. Rats were fasted for 12h prior to the experiment, 10% DMSO/water (blank), 3mg/kg cisplatin (control group to determine whether the model was successful) and 25nmol/kg,50nmol/kg and 100nmol/kg semaglutide, tirzepatide, SAR441255, SEQ ID NO:2 were intraperitoneally injected into each group of rats at 0 h. And then, quickly giving the common feed and the kaolin feed which are weighed in advance to each group of rats, recording the feeding amount of each group of rats in the common feed and the kaolin feed for 24 hours, and judging the strength of side effects caused by the compound according to the consumption amount of the common feed and the kaolin feed.
Table 9: normal diet and kaolin food intake by SD rats at 24 hours
Sample of Feed intake (g) of common feed Kaolin food intake (g)
Blank control 25.2±1.6g 0.2±0.1g
Cisplatin (cisplatin) 13.9±2.2g *** 3.3±0.5g ***
Semaglutide(25nmol/kg) 20.2±1.3g *** 0.7±0.2g ***
Tirzepatide(25nmol/kg) 18.3±2.0g *** 0.8±0.3g ***
SAR441255(25nmol/kg) 18.1±1.2g *** 0.9±0.2g ***
SEQ ID NO:2(25nmol/kg) 15.3±0.9g ***,### 0.2±0.1g ###
*** : p compared with the blank control group<0.001； ### : group ratio P to semaglutide, tirzepatide and SAR441255<0.001 (One-Way ANOVA, tukey post hoc test) the results are expressed as mean ± SD of 6 rats per group.
Table 10: normal diet and kaolin food intake by SD rats at 24 hours
*** : p compared with the blank control group<0.001； ### : group ratio P to semaglutide, tirzepatide and SAR441255<0.001 (One-Way ANOVA, tukey post hoc test) the results are expressed as mean ± SD of 6 rats per group.
Table 11: normal diet and kaolin food intake by SD rats at 24 hours
Sample of Feed intake (g) of common feed Kaolin food intake (g)
Blank control 25.2±1.6g 0.2±0.1g
Cisplatin (cisplatin) 13.9±2.2g *** 3.3±0.5g ***
Semaglutide(100nmol/kg) 18.1±1.9g *** 1.1±0.3g ***
Tirzepatide(100nmol/kg) 16.8±1.4g *** 1.2±0.3g ***
SAR441255(100nmol/kg) 16.2±1.6g *** 1.0±0.2g ***
SEQ ID NO:2(100nmol/kg) 13.0±0.3g ***,### 0.3±0.1g ###
*** : p compared with the blank control group<0.001； ### : group ratio P to semaglutide, tirzepatide and SAR441255<0.001 (One-Way ANOVA, tukey post hoc test) the results are expressed as mean ± SD of 6 rats per group.
As shown in the results of tables 9-11, the polypeptide compounds prepared in the examples of the present invention have good rat feeding inhibition effect at the dosages of 25nmol/kg,50nmol/kg and 100nmol/kg, and are superior to the positive controls semaglutide, tirzepatide and SAR441255. However, the polypeptide compounds prepared in the examples of the present invention did not cause significant kaolin feeding in rats at doses of 25nmol/kg,50nmol/kg, and 100nmol/kg compared to the blank, and the kaolin feeding in the polypeptide compound group prepared in the examples of the present invention was significantly lower than that in the positive control semaglutide, tirzepatide and SAR441255 rats, similar to the blank. This demonstrates that the polypeptide compounds prepared in the examples of the present invention do not cause gastrointestinal side effects in rats, which are significantly lower than those of positive controls semaglutide, tirzepatide and SAR441255.

Claims (8)

1. A polypeptide having agonistic activity to GLP-1, glucagon and GIP receptors, characterized in that the polypeptide has the amino acid sequence of formula:
His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Met-Ser-Arg-Ala-Xaa 1 -Glu-Xaa 2 -Ile-Ala-Xaa 3 -Arg-Le u-Phe-Val-Asp-Trp-Leu-Ile-Glu-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2 ，
wherein:
Xaa 1 selected from Leu or Met;
Xaa 2 selected from Lys-R1 or Lys-R2 with side chains chemically modified;
Xaa 3 selected from Ala or Val;
the chemical structure of the Lys-R1 is as follows:
the chemical structure of the Lys-R2 is as follows:
2. the polypeptide having agonistic activity to GLP-1, glucagon and GIP receptor according to claim 1, wherein the sequence structure of the polypeptide is selected from any one of the amino acid sequences shown in SEQ ID NOs 1 to 4:
SEQ ID NO:1
SEQ ID NO:2
SEQ ID NO:3
SEQ ID NO:4
3. a population of pharmaceutically acceptable salts of polypeptides having agonistic activity to GLP-1, glucagon and GIP receptors according to any one of claims 1-2.
4. A pharmaceutically acceptable salt of a polypeptide having agonistic activity to GLP-1, glucagon and GIP receptor according to claim 3, wherein said pharmaceutically acceptable salt is a salt of a polypeptide having agonistic activity to GLP-1, glucagon and GIP receptor with one of the following compounds; the following compounds include hydrochloric acid, formic acid, acetic acid, pyruvic acid, butyric acid, caproic acid, benzenesulfonic acid, pamoic acid, benzoic acid, salicylic acid, lauric acid, cinnamic acid, propionic acid, dodecylsulfuric acid, citric acid, ascorbic acid, wine stearic acid, oxalic acid, lactic acid, succinic acid, malonic acid, maleic acid, fumaric acid, aspartic acid, sulfosalicylic acid.
5. A pharmaceutical formulation prepared from a polypeptide having agonistic activity for GLP-1, glucagon and GIP receptors according to any one of claims 1-2, wherein said pharmaceutical formulation comprises any one of a pharmaceutical formulation comprising a tablet, capsule, syrup, tincture, inhalant, spray, injection, film, patch, powder, granule, emulsion, suppository or compound formulation.
6. A pharmaceutical composition prepared from a polypeptide having agonistic activity at GLP-1, glucagon and GIP receptors, characterized in that the pharmaceutical composition comprises a polypeptide having agonistic activity at GLP-1, glucagon and GIP receptors according to any one of claims 1-2, a pharmaceutically acceptable carrier or diluent; or the pharmaceutical composition comprises a pharmaceutically acceptable salt, pharmaceutically acceptable carrier or diluent of a polypeptide having agonistic activity at GLP-1, glucagon and GIP receptors as defined in any one of claims 3-4.
7. Use of a polypeptide having agonistic activity to GLP-1, glucagon and GIP receptors according to any one of claims 1-2 or a pharmaceutically acceptable salt of a polypeptide having agonistic activity to GLP-1, glucagon and GIP receptors according to any one of claims 3-4 or a pharmaceutical agent according to claim 5 or a pharmaceutical composition according to claim 6 for the preparation of a medicament for the treatment of a metabolic disease or disorder.
8. The use according to claim 7, wherein the metabolic disease or disorder is diabetes, obesity, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis or dyslipidemia.
CN202311316622.0A 2023-10-12 2023-10-12 Polypeptides with agonistic activity on GLP-1, glucagon and GIP receptor and application thereof Pending CN117417431A (en)

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