CN115785249A

CN115785249A - GLP-1 analogue and application thereof

Info

Publication number: CN115785249A
Application number: CN202211252991.3A
Authority: CN
Inventors: 韩京; 陈德
Original assignee: Jiangsu Normal University
Current assignee: Jiangsu Normal University
Priority date: 2022-10-13
Filing date: 2022-10-13
Publication date: 2023-03-14
Anticipated expiration: 2042-10-13
Also published as: CN115785249B; CN116514952B; CN116514952A

Abstract

The invention provides a GLP-1 analogue and application thereof, wherein the amino acid sequence of the GLP-1 analogue is one of the sequences in SEQ ID NO. 1-SEQ ID NO. 10. The GLP-1 analogue has strong agonistic activity to human GLP-1 receptor and high stability, and has pharmacokinetic characteristics of once-a-day or once-a-week administration. The invention is a variant designed based on a bullfrog GLP-1 sequence, and has excellent weight loss and lipid lowering effects while retaining the therapeutic effect of GLP-1 analogues on diabetes; the compound has great potential in the aspect of preparing medicines for treating metabolic syndrome, such as diabetes, obesity, dyslipidemia and the like.

Description

GLP-1 analogue and application thereof

Technical Field

The invention relates to the technical field of biological medicines, in particular to GLP-1 analogues and application thereof.

Background

Diabetes Mellitus (DM) is a group of metabolic diseases characterized by chronic hyperglycemia caused by various causes such as heredity and environment. Due to relative or absolute deficiency of insulin secretion; or insulin deficiency causes a series of metabolic disorders such as sugar, protein, fat, water and electrolytes. Persistent hyperglycemia is an essential feature. Prolonged illness can cause multiple system damage, leading to chronic complications of the eyes, kidneys, nerves, cardiovascular and cerebrovascular organs, lower limbs, and the like. The diabetes rate of Chinese adults is close to 10 percent. Diabetes is classified into type 1 diabetes (T1 DM), type 2 diabetes (T2 DM), gestational diabetes, and other specific types of diabetes. More than 90% of patients with T2DM are found. The current methods for treating diabetes mainly comprise oral hypoglycemic drugs and insulin treatment, but the methods can generate toxic and side effects in long-term treatment.

The Glucagon-Like Peptide-1 (Glucagon-Like Peptide-1, GLP-1) has the function of protecting beta cells, and the GLP-1 can act on the beta cells of the pancreatic islets, promote the transcription of insulin genes and the synthesis and secretion of insulin, stimulate the proliferation and differentiation of the beta cells of the pancreatic islets, inhibit the apoptosis of the beta cells of the pancreatic islets and increase the number of the beta cells of the pancreatic islets. In addition, GLP-1 can act on islet alpha cells to strongly inhibit the release of glucagon, and acts on islet delta cells to promote the secretion of somatostatin, and somatostatin can also be used as paracrine hormone to participate in inhibiting the secretion of glucagon. Research proves that GLP-1 can obviously improve the blood sugar condition of a T2DM animal model or a patient through a plurality of mechanisms, wherein the effects of promoting the regeneration and the repair of islet beta cells and increasing the number of the islet beta cells are particularly obvious, and a very good prospect is provided for the treatment of T2 DM. However, native GLP-1 has the limitation that it has a very short half-life and is degraded by dipeptidyl peptidase (DPP-IV) after 2 to 3 minutes of secretion, and is also rapidly degraded by DPP-IV even when GLP-1 is exogenously administered. In addition, endogenous GLP-1 is also rapidly filtered and metabolized by the kidney, and the in vivo half-life is only 2-3min. Therefore, there is a need to find new GLP-1 receptor agonists that have similar biological activity to GLP-1 but long-lasting effect in vivo.

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 (T2 DM), nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), dyslipidemia are closely related to obesity. GLP-1 can suppress appetite, delay gastric emptying and reduce body weight. Although GLP-1 has excellent hypoglycemic effect and certain weight-reducing effect, the weight-reducing effect is still relatively limited, and the lipid-regulating effect is also relatively weak. Thus, there remains a need for new GLP-1 analogs that are safer and more tolerable to obese and diabetic patients, are effective in weight loss, control blood glucose, and are effective in lipid regulation.

Disclosure of Invention

The invention aims to provide GLP-1 analogues and application thereof, wherein the GLP-1 analogues can have strong agonistic activity to human GLP-1 receptors and high stability, can have the pharmacokinetic characteristic of once-a-day or once-a-week administration, and can have excellent weight loss and lipid lowering effects on the treatment effect of diabetes; the compound has great potential in the aspect of preparing medicines for treating metabolic syndromes, such as diabetes, obesity, dyslipidemia and the like.

In order to achieve the above purpose, the technical scheme of the invention is as follows:

a GLP-1 analogue, wherein the amino acid sequence of the GLP-1 analogue is one of the following sequences:

(1)SEQ ID NO:1

(2)SEQ ID NO:2

(3)SEQ ID NO:3

(4)SEQ ID NO:4

(5)SEQ ID NO:5

(6)SEQ ID NO:6

(7)SEQ ID NO:7

(8)SEQ ID NO:8

(9)SEQ ID NO:9

(10)SEQ ID NO:10

the invention also provides pharmaceutically acceptable salts of the GLP-1 analogue.

Preferably, the salt is a salt of a GLP-1 analogue with one of the following compounds: hydrochloric acid, acetic acid, salicylic acid, lauric acid, cinnamic acid, citric acid, oxalic acid, lactic acid and succinic acid.

The invention also provides a medicament prepared from the GLP-1 analogue, wherein the medicament is any one of tablets, capsules, inhalants, sprays, injections, films, patches, emulsions or compound preparations in pharmaceutics, and the medicament consists of the GLP-1 analogue and pharmaceutically acceptable pharmaceutic adjuvants, carriers or diluents.

The invention also provides a pharmaceutical composition containing the GLP-1 analogue, which is prepared from any GLP-1 analogue as an effective raw material or pharmaceutically acceptable salt thereof as an effective raw material and a pharmaceutically acceptable carrier or diluent.

The invention also provides the use of a GLP-1 analogue of the invention or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, or a medicament thereof, for the manufacture of a medicament for the treatment of a metabolic disease or disorder. In particular aspects, the metabolic disease or disorder is diabetes, obesity, dyslipidemia. In a particular aspect, the diabetes is T1DM, T2DM, or gestational diabetes. In particular aspects, the medicament is for treating more than one metabolic disease or disorder, e.g., diabetes and obesity; obesity and dyslipidemia; diabetes and dyslipidemia; diabetes, dyslipidemia, and obesity.

Compared with the prior art, the invention has the beneficial effects that:

the GLP-1 analogue provided by the invention is a variant designed based on a bullfrog GLP-1 sequence, has unexpected excellent weight-losing and lipid-lowering effects while retaining the treatment effect of the GLP-1 analogue on diabetes, and has better blood glucose-lowering effect and remarkably improved weight-losing and lipid-lowering effects compared with the GLP-1 medicaments on the market, and has more potential in the treatment aspect of diseases such as diabetes, obesity, dyslipidemia and the like. In addition, the GLP-1 analogue provided by the invention has strong agonistic activity to a human GLP-1 receptor and stable chemical property, and has the pharmacokinetic characteristic of supporting once-a-day or once-a-week administration. The GLP-1 analogue provided by the invention has obviously better therapeutic effect on metabolic diseases such as T2DM, obesity, dyslipidemia and the like than the existing medicaments on the market. Therefore, the GLP-1 analogue provided by the invention is suitable to be used as an active ingredient of medicaments for treating metabolic diseases (such as diabetes, obesity, dyslipidemia and the like).

Drawings

FIG. 1 is a graph showing the results of acute hypoglycemic effects of a single administration of each test substance in ICR mice;

FIG. 2 is a graph showing the results of a long-lasting hypoglycemic effect on db/db mice given a single dose of each test substance;

FIG. 3 is a graph showing the results of percentage change in body weight of each subject after chronic administration for 21 days in DIO mice.

Detailed Description

The invention is described in further detail below with reference to the figures and specific examples.

Unless defined otherwise herein, scientific and technical terms used in the present application shall have the meanings that are commonly understood by one of ordinary skill in the art. Generally, the terms and methods used in connection with chemistry, biology, pharmacology, as described herein are well known and commonly used in the art.

In addition, the amino acids of the present invention are abbreviated as follows according to the nomenclature rules 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).

In addition, all amino acid residues in the polypeptide compounds of the present invention are preferably in the L configuration, unless explicitly indicated.

In addition, -NH at the C-terminus of the sequence ₂ "moiety indicates an amide group (-CONH) at C terminal ₂ )。

In addition, in the present invention, in addition to natural amino acids, unnatural amino acid α -aminoisobutyric acid (Aib) is used in the sequence.

The present invention is illustrated by the following examples, which should not be construed as limiting the scope of the invention in any way.

Example 1

Synthesis of polypeptide Compound of SEQ ID NO 1

(1) Swelling of the resin

0.278g (0.1 mmol equiv.) of Rink Amide MBHA resin with a loading of 0.36mmol/g was weighed into a 25mL reactor, and the resin was washed 1 time with 7mL of DCM and methanol alternately, 2 times with 7mL of DCM, then 1h with 7mL of DCM, and finally 3 times with 7mL of DMF.

(2) Removal of Fmoc protecting group of resin

Transferring the swelled resin into a PSI-200 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 for 2min each time to obtain the Rink resin with the Fmoc protecting group removed.

(3) Synthesis of Fmoc-Ala-Rink amide-MBHAResin

Fmoc-Ala-OH (0.4 mmol) is weighed, dissolved by 2mL DMF, added with 3mL DIC/HOBt (0.4 mmol/0.44 mmol) condensing agent, added into a reactor, shaken for reaction for 2h at room temperature, filtered to remove reaction liquid, and washed with 7mL DMF for 4 times, kaiser reagent is used to detect whether the reaction coupling is complete or not, if not, the coupling is 2 times.

(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. Wherein Lys at the side chain modification site employs Fmoc-Lys (Dde) -OH protection strategy, while Boc-His (Boc) -OH is used for N-terminal His.

(5) Modification of Lys side chains

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, adding 0.4mmol of Fmoc-Glu-OtBu,0.4mmol of DIC and 0.44mmol of HOBt after the Dde protecting group is removed, and carrying out oscillation reaction for 2 hours. After the Fmoc protecting group is removed, 0.4mmol of Fmoc-Glu-OtBu,0.4mmol of DIC and 0.44mmol of HOBt are added again, and the mixture is shaken for reaction for 2 hours. After Fmoc protecting group removal, 0.4mmol palmitic acid, 0.4mmol DIC and 0.44mmol HOBt were added for condensation reaction for 2h, and after completion of the reaction the resin was washed 4 times with 7mL DMF.

(6) Cleavage of polypeptides

The polypeptide-linked resin obtained above was transferred to a round-bottomed flask, and 5mL of the resin was cleaved using a cleavage agent Reagent R (TFA/thioanisole/phenol/EDT, 90, 3, V/V), reacted in an oil bath at a constant temperature of 30 ℃ for 2h, the cleavage solution was poured into 40mL of iced ether, the crude product was washed 3 times with 15mL of iced ether after refrigerated centrifugation, and finally dried with nitrogen to obtain a crude peptide.

(7) Purification and characterization 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.20mm, 12 μm); mobile phase A:0.1% tfa/water (V/V), mobile phase B: methanol (V/V); the flow rate is 8mL/min; the detection wavelength was 214nm. Eluting with linear gradient (20-70% B/30 min), collecting target peak, removing methanol, lyophilizing to obtain pure product 0.10g with purity higher than 98%, and determining target polypeptide molecular weight by LC-MS. The theoretical relative molecular mass is 3982.6.ESI-MS M/z calculated value [ M +3H ]] ³⁺ 1328.5,[M+4H] ⁴⁺ 996.6; observed value [ M +3H] ³⁺ 1328.0,[M+4H] ⁴⁺ 996.3。

Example 2

Synthesis of polypeptide Compound of SEQ ID NO. 2

The synthesis method is the same as example 1, the target peak is collected and freeze-dried to obtain 0.11g of a pure product, the purity is more than 98%, and the molecular weight of the target polypeptide is confirmed by MS. The theoretical relative molecular mass is 3983.5.ESI-MS M/z calculated value [ M +3H ]] ³⁺ 1328.8,[M+4H] ⁴⁺ 996.9; observed value [ M +3H] ³⁺ 1328.3,[M+4H] ⁴⁺ 996.5。

Example 3

Synthesis of polypeptide Compound of SEQ ID NO. 3

The synthesis method is the same as example 1, the target peak is collected and freeze-dried to obtain 0.09g of a pure product, the purity is higher than 98%, and the molecular weight of the target polypeptide is confirmed by MS. The theoretical relative molecular mass is 3982.6.ESI-MS M/z calculated value [ M +3H ]] ³⁺ 1328.5,[M+4H] ⁴⁺ 996.6; observed value [ M +3H] ³⁺ 1328.0,[M+4H] ⁴⁺ 996.3。

Example 4

Synthesis of polypeptide Compound of SEQ ID NO. 4

The synthesis method is the same as example 1, the target peak is collected and freeze-dried to obtain 0.10g of a pure product, the purity is more than 98%, and the molecular weight of the target polypeptide is confirmed by MS. The theoretical relative molecular mass is 3814.4.ESI-MS M/z calculated value [ M +3H ]] ³⁺ 1272.5,[M+4H] ⁴⁺ 954.6; observed value [ M +3H] ³⁺ 1272.0,[M+4H] ⁴⁺ 954.2。

Example 5

Synthesis of polypeptide Compound of SEQ ID NO 5

The synthesis method is the same as example 1, the target peak is collected and freeze-dried to obtain 0.11g of a pure product, the purity is more than 98%, and the molecular weight of the target polypeptide is confirmed by MS. The theoretical relative molecular mass is 3815.3.ESI-MS M/z calculated value [ M +3H ]] ³⁺ 1272.8,[M+4H] ⁴⁺ 954.8; observed value [ M +3H] ³⁺ 1272.3,[M+4H] ⁴⁺ 954.5。

Example 6

Synthesis of polypeptide Compound of SEQ ID NO. 6

(1) Swelling of the resin

0.262g (0.1 mmol 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 PSI-200 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 as to obtain the Rink resin with the Fmoc protecting group removed.

(3) Synthesis of Fmoc-Ala-Rink amide-MBHAResin

Fmoc-Ala-OH (0.4 mmol) was weighed, dissolved in 3mL 10% DMF/DMSO (v/v), 2mL DIC/HOBt (0.4 mmol/0.44 mmol) condensing agent was added, preactivated for 30min, activated amino acid was added to the reactor, reacted at room temperature with shaking for 2h, the reaction solution was filtered off and the resin was washed 4 times with 7mL DMF, and the reaction coupling was checked for completion using Kaiser reagent, if not complete, 2 couplings.

(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 Lys at position 12, fmoc-Lys (Alloc) -OH, fmoc-Lys (Dde) -OH, fmoc-Lys (Mtt) -OH, fmoc-Lys (ivDde) -OH or the like can be used. In this example, fmoc-Lys (Dde) -OH protection strategy was used, while Boc-His (Boc) -OH was used for N-terminal His.

(5) Modification of Lys side chain

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 12 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 hours. After removing the Fmoc protecting group, adding 0.4mmol of Fmoc-AEEA-OH,0.4mmol of DIC and 0.44mmol of HOBt again, and carrying out concussion condensation reaction for 2h. 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

(7) Purification and characterization 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.20mm, 12 μm); mobile phase A:0.1% tfa/water (V/V), mobile phase B: methanol (V/V); the flow rate is 8mL/min; the detection wavelength was 214nm. Eluting with linear gradient (20-80% B/30 min), collecting target peak, removing methanol, lyophilizing to obtain pure product 0.11g with purity of more than 98%, and determining the molecular weight of the target polypeptide by LC-MS. The theoretical relative molecular mass is 4330.9.ESI-MS M/z calculated value [ M +3H ]] ³⁺ 1444.6,[M+4H] ⁴⁺ 1083.7; observed value [ M +3H] ³⁺ 1444.1,[M+4H] ⁴⁺ 1083.3。

Example 7

Synthesis of polypeptide Compound of SEQ ID NO 7

The synthesis method is the same as example 6, the target peak is collected and freeze-dried to obtain 0.12g of a pure product, the purity is more than 98%, and the molecular weight of the target polypeptide is confirmed by MS. The theoretical relative molecular mass is 4331.9.ESI-MS M/z calculated value [ M +3H ]] ³⁺ 1445.0,[M+4H] ⁴⁺ 1084.0; observed value [ M +3H] ³⁺ 1444.4,[M+4H] ⁴⁺ 1083.5。

Example 8

Synthesis of polypeptide Compound of SEQ ID NO 8

The synthesis method is the same as example 6, the target peak is collected and freeze-dried to obtain 0.12g of a pure product, the purity is more than 98%, and the molecular weight of the target polypeptide is confirmed by MS. The theoretical relative molecular mass is 4330.9.ESI-MS M/z calculated value [ M +3H ]] ³⁺ 1444.6,[M+4H] ⁴⁺ 1083.7; observed value [ M +3H] ³⁺ 1444.1,[M+4H] ⁴⁺ 1083.3。

Example 9

Synthesis of polypeptide Compound of SEQ ID NO 9

The synthesis method is the same as example 6, the target peak is collected and freeze-dried to obtain 0.11g of a pure product, the purity is more than 98%, and the molecular weight of the target polypeptide is confirmed by MS. The theoretical relative molecular mass is 4162.7.ESI-MS M/z calculated value [ M +3H ]] ³⁺ 1388.6,[M+4H] ⁴⁺ 1041.7; observed value [ M +3H] ³⁺ 1388.0,[M+4H] ⁴⁺ 1041.3。

Example 10

Synthesis of polypeptide Compound of SEQ ID NO 10

The synthesis method is the same as that of example 6, the target peak is collected and freeze-dried to obtain 0.12g of a pure product, the purity is more than 98%, and the molecular weight of the target polypeptide is confirmed by MS. The theoretical relative molecular mass is 4163.7.ESI-MS M/z calculated value [ M +3H ]] ³⁺ 1388.9,[M+4H] ⁴⁺ 1041.9; observed value [ M +3H] ³⁺ 1388.4,[M+4H] ⁴⁺ 1041.5。

Example 11

Determination of agonistic Activity of polypeptide Compounds on human GLP-1 receptor

The agonistic effect of the polypeptide compound on the receptor is determined by a functional assay, GLP-1 receptor agonistic activity is determined by measuring the cAMP response of HEK-293 cell lines stably expressing the human GLP-1 receptor. Cells stably expressing the GLP-1 receptor were divided into T175 flasks and grown overnight in medium (DMEM/10% fbs) to near confluency, then the medium was removed and the cells were washed with calcium and magnesium free PBS and then treated with Accutase enzyme for protease. The detached cells were washed and resuspended in assay buffer (20mM HEPES,0.1% BSA,2mM IBMX,1 × HBSS) and the cell density determined, and 25. Mu.L aliquots were dispensed into wells of 96-well plates. For measurement, 25 μ Ι _ of a solution of the test polypeptide compound in assay buffer was added to the wells, followed by incubation for 30 minutes at room temperature. 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 30 minutes and then the fluorescence ratio at 665/620nm was measured. By measuring the concentration (EC) that causes 50% activation of the maximal response ₅₀ ) To quantify the in vitro potency of agonists.

The test data (nM) in the examples of the present patent application are shown in table 1 below, and although the test data are stated in terms of a certain number of significant figures, it should not be considered as indicating that the data have been determined to be the number of significant figures exactly.

Table 1: agonistic activity of polypeptide compounds at human GLP-1 receptor

As shown in Table 1, all of the polypeptide compounds showed strong agonistic activity to GLP-1 receptor, and at the same time, all of the polypeptide compounds showed better GLP-1 receptor agonistic activity than GLP-1.

Example 12

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 were dissolved in 1mL of solution in different buffer systems and gently stirred 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 solubility-derived supernatants were stored at 40 ℃ for 7 days, then the samples were centrifuged at 4500rpm for 20 minutes and the supernatants were subjected to HPLC analysis to determine peak areas. By comparing the peak areas (t) before the start of the stability experiment ₀ ) And the peak area (t) after 7 days of storage ₇ ) Get "% remaining peptide". Calculated according to the following formula: % remaining peptide = [ (peak area t) ₇ )×100]Area per peak t ₀ Stability is expressed as "% remaining peptide", and the results are shown in table 2 below.

Table 2: solubility and stability of polypeptide compounds

As shown in the results in Table 2, the polypeptide compound of the present invention has significantly improved solubility in pH conditions acceptable for injections in vivo, and has favorable formulation properties, as compared to native GLP-1. In addition, the polypeptide compound of the present invention has high stability under pH 4.5 and neutral pH conditions.

Example 13

Pharmacokinetic Properties of polypeptide Compounds in rat

SD rats were given 50nmol/kg of liraglutide, semaglutide, SEQ ID NO:2 and SEQ ID NO:7 by subcutaneous (s.c.) injection and blood samples were collected at 0.25h, 0.5h, 1h, 2h, 4h, 8h, 16h, 24h and 48h post-administration. 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 3).

Table 3: pharmacokinetic profile of polypeptide Compounds in rats

Sample (I)	T _1/2 (h)	C _max (ng/mL)
			Liraglutide	2.3	489
Semaglutide	9.2	519
			SEQ ID NO:2	5.0	452
SEQ ID NO:7	13.3	429

As shown in the results of table 3, the polypeptide compound of the present invention has a significantly prolonged in vivo half-life, superior to that of liraglutide or semaglutide, and has pharmacokinetic characteristics supporting once-daily administration or once-weekly administration.

Example 14

Acute hypoglycemic activity of polypeptide compound in mouse

Male ICR mice, randomly grouped, 6 per group. Only water was given and fasted overnight. The blank group was administered with normal saline (10 mg/kg) by intraperitoneal injection, the administration group was 3 groups, and the mice were administered with 30nmol/kg of semaglutide, SEQ ID NO:2 and SEQ ID NO:7 by intraperitoneal single injection in a non-fasting state. After 30 minutes, each group of mice was administered 3g/kg of glucose solution intraperitoneally. Measuring blood glucose level with blood glucose meter at-30 min, 0min, 15min, 30min, 60min, and 120 min.

As shown in the result of figure 1, an acute blood glucose reduction experiment in an ICR mouse shows that polypeptide compounds of SEQ ID NO. 2 and SEQ ID NO. 7 remarkably improve the glucose tolerance level of the mouse, have excellent blood glucose reduction effect, and the blood glucose reduction effect of the polypeptide compounds is also obviously superior to semaglutide.

Example 15

Long-acting hypoglycemic activity of polypeptide compound in mice

Male db/db mice were randomly divided into groups of 6 mice each. The blank group was injected subcutaneously with normal saline (10 mg/kg), the administration component was 2 groups, mice were fed and drunk freely during the experiment, and mice were injected subcutaneously with semaglutide and SEQ ID NO:7 in a single injection of 30nmol/kg subcutaneously in a non-fasting state, respectively. Blood glucose levels were measured with a glucometer at 0h before dosing, and at 4h, 6h, 24h, 48h and 72h after dosing for each group of mice.

As shown in the results of FIG. 2, the results of in vivo blood glucose lowering experiments in db/db mice show that the polypeptide compound of the present invention shows a long-lasting blood glucose lowering activity which is significantly superior to that of semaglutide, a positive control drug.

Example 16

Effect of polypeptide Compounds on blood ester and body weight in diet-induced obesity (DIO) mice

Male C57BL/6J mice, weighing about 22g, were fed with D12492 high-fat diet from Research Diets for 18 weeks to create DIO mouse models. Before the start of administration, groups of DIO mice were randomly divided by body weight into 4 groups of 6 mice each, namely, a physiological saline group (blank control group), a positive control group (semaglutide) and a test sample group (SEQ ID NO:2 and SEQ ID NO: 7). Each group of mice was injected subcutaneously once every two days with normal saline (10 mg/kg), semaglutide (30 nmol/kg), SEQ ID NO:7 (30 nmol/kg), and once daily with SEQ ID NO:2 (30 nmol/kg) for a 21-day administration cycle. Mice body weight changes were recorded daily. After the experiment, each group of mice was sacrificed, blood was taken to prepare serum, liver was taken to prepare homogenate, and Triglyceride (TG) and Total Cholesterol (TC) contents of the liver and serum were measured.

As shown in the results of FIG. 3, the polypeptide compounds of the present invention, SEQ ID NO 2 and SEQ ID NO 7, at a dose of 30nmol/kg, were able to reduce the body weight of mice by 28.8% and 31.6%, respectively, after continuous administration for 3 weeks in DIO mice. Semaglutide reduced the body weight of mice by only 16.0%. The above results indicate that SEQ ID NO 2 and SEQ ID NO 7 have excellent weight loss effects, which are significantly superior to semaglutide.

Table 4: serum Total Cholesterol (TC) and Triglyceride (TG) levels 3 weeks after DIO mice treatment

Sample (dosage)	Total cholesterol (mmol/L)	Triglyceride (mmol/L)
			Blank control (saline group)	9.15±0.56	1.98±0.22
Semaglutide(30nmol/kg)	7.98±0.42**	1.51±0.09*
			SEQ ID NO:2(30nmol/kg)	3.68±0.55***,###	0.63±0.18***,###
SEQ ID NO:7(30nmol/kg)	3.56±0.39***,###	0.52±0.12***,###

^* : comparison with blank control group P<0.05； ^** : comparison with blank control group P<0.01； ^*** : comparison with blank control group P<0.001； ^### : ratio to semaglutide P<0.001 (One-WayANOVA, tukeypost hoc test), results are expressed as mean. + -. SD of 6 mice per group.

Table 5: total liver cholesterol (TC) and Triglyceride (TG) levels 3 weeks after DIO mouse treatment

Sample (dosage)	Total cholesterol (mg/g)	Triglycerides (mg/g)
			Blank control (saline group)	17.33±1.67	102.56±9.26
Semaglutide(30nmol/kg)	14.33±1.22*	85.33±7.25**
			SEQ ID NO:2(30nmol/kg)	7.22±0.90***,###	46.89±7.28***,###
SEQ ID NO:7(30nmol/kg)	7.05±0.41***,###	42.25±5.33***,###

As shown in the results of Table 4 and Table 5, the polypeptide compounds of the present invention, SEQ ID NO:2 and SEQ ID NO:7, when administered continuously in DIO mice for 3 weeks, significantly reduced the serum and liver Triglyceride (TG) and Total Cholesterol (TC) contents of the mice, and the serum and liver blood lipids-reducing effects of SEQ ID NO:2 and SEQ ID NO:7 were significantly stronger than that of the positive control drug semaglutide. The results show that the SEQ ID NO. 2 and the SEQ ID NO. 7 have the effects of losing weight and reducing the blood fat of the liver and serum which are obviously superior to semaglutide, and the polypeptide compound has unexpected and excellent weight-losing and lipid-regulating effects.

Claims

1. A GLP-1 analog, characterized in that the amino acid sequence of the GLP-1 analog is one of the following sequences:

(1)SEQ ID NO:1

(2)SEQ ID NO:2

(3)SEQ ID NO:3

(4)SEQ ID NO:4

(5)SEQ ID NO:5

(6)SEQ ID NO:6

(7)SEQ ID NO:7

(8)SEQ ID NO:8

(9)SEQ ID NO:9

(10)SEQ ID NO:10

2. a pharmaceutically acceptable salt of a GLP-1 analog, characterized in that: the amino acid sequence of the GLP-1 analogue is one of the amino acid sequences described in claim 1.

3. The pharmaceutically acceptable salt of a GLP-1 analogue according to claim 2, wherein said salt is a salt of a GLP-1 analogue with one of the following compounds: hydrochloric acid, acetic acid, salicylic acid, lauric acid, cinnamic acid, citric acid, oxalic acid, lactic acid and succinic acid.

4. The medicament prepared from the GLP-1 analogues according to claim 1, wherein the medicament is any one of tablets, capsules, inhalants, sprays, injections, films, patches, emulsions or compound preparations in pharmaceutics, and the medicament consists of the GLP-1 analogues and pharmaceutically acceptable pharmaceutic adjuvants, carriers or diluents.

5. A pharmaceutical composition comprising a GLP-1 analog, wherein said pharmaceutical composition comprises the GLP-1 analog of claim 1 as an active ingredient, or a pharmaceutically acceptable salt of the GLP-1 analog of claim 2 or 3 as an active ingredient, in combination with a pharmaceutically acceptable carrier or diluent.

6. Use of a GLP-1 analogue or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, or a medicament thereof, according to any one of claims 1-5, for the manufacture of a medicament for the treatment of a metabolic disease or disorder.

7. Use according to claim 6, wherein the metabolic disease or disorder is diabetes, obesity and dyslipidemia.