CN114621339B

CN114621339B - Long-acting GLP-1 derivative

Info

Publication number: CN114621339B
Application number: CN202210113945.9A
Authority: CN
Inventors: 曹海燕; 林兆生; 朱志伟; 王建宇; 辛瑞; 曹丙洲
Original assignee: Jilin Huisheng Biopharmaceutical Co ltd; Beijing Huizhiheng Biological Technology Co Ltd
Current assignee: Jilin Huisheng Biopharmaceutical Co ltd; Beijing Huizhiheng Biological Technology Co Ltd
Priority date: 2021-12-28
Filing date: 2022-01-30
Publication date: 2022-09-23
Anticipated expiration: 2042-01-30
Also published as: CN114874314A; CN114796462B; WO2023124847A1; CN115850438A; CN114621339A; CN114796462A; CN115850438B; CN114874314B

Abstract

The invention relates to a GLP-1 polypeptide analogue, a long-acting GLP-1 derivative of the polypeptide analogue modified by fatty acid acylation. The long-acting GLP-1 derivative has good binding affinity with a GLP-1 receptor (GLP-1R), and compared with other GLP-1 derivatives on the market, the long-acting GLP-1 derivative has more obvious and excellent blood sugar reducing capacity and weight losing capacity.

Description

Long-acting GLP-1 derivative

Technical Field

The invention relates to the field of polypeptide technology and derivatives thereof, in particular to a long-acting GLP-1(7-37) derivative, and a preparation method, a pharmaceutical composition and medical application thereof.

Background

Diabetes is a group of metabolic disorders such as carbohydrates, proteins, fats and the like caused by the absolute or relative insufficient secretion and/or utilization disorder of insulin, takes hyperglycemia as a main marker, and can be caused by various factors such as heredity, environment and the like. Diabetes is one of three major death diseases of human beings, and the death rate of the diabetes is second to cardiovascular and cerebrovascular diseases and cancers.

Diabetes is largely classified into type 1 diabetes and type 2diabetes, with the majority of patients being type 2diabetes patients (statistically, about 90%). Type 2diabetes (diabetes mellitus type2, T2DM), old called non-insulin dependent diabetes mellitus (NIDDM) or adult-onset diabetes (adult-onset diabetes), patients are characterized by hyperglycemia, relative lack of insulin, insulin resistance, etc. At present, clinically used medicaments for treating type 2diabetes mainly comprise biguanides, sulfonylureas, thiazolidinediones, DPP-4 receptor inhibitors, SGLT-2 receptor inhibitors and GLP-1 derivatives. Among them, GLP-1 derivatives have a similar hypoglycemic effect to insulin but almost no hypoglycemic risk, and have a weight-loss effect and a cardiovascular protection function, and thus are becoming major therapeutic drugs and research hotspots for type2 diabetes.

Glucagon-like peptide 1(GLP-1) is a secretin from intestinal L cells, and has effects of promoting insulin secretion, inhibiting glucagon release, stimulating pancreatic β cell proliferation, inducing pancreatic β cell regeneration, preventing pancreatic β cell apoptosis, improving insulin sensitivity, and increasing glucose utilization. Thus, GLP-1 and its analogs and derivatives play an important role in the development and progression of type 1 and 2 diabetes.

The amino acid sequences of GLP-1 analogues and glucagon are nearly half the same, and the GLP-1 analogues and the glucagon have multiple functions of promoting insulin secretion and biosynthesis with glucose dependence, inhibiting glucagon secretion, inhibiting gastric emptying and the like (Dugang, Long, Xuren, glucagon-like peptide 1 and receptor agonist research progress [ J ] Tianjin medicine, 2012, 40(2): 181-) 184.).

In the literature, the authors studied 10 patients with type 2diabetes with poor glycemic control and administered GLP-1 or placebo in the fasting state, and the results showed that after GLP-1 infusion, the patient had significantly increased insulin and C peptide levels, significantly decreased glucagon levels, and the fasting blood levels became normal after 4 hours; after the blood sugar level is normal, the insulin level of a patient can not rise any more although GLP-1 is continuously infused, the blood sugar level is also kept stable and does not further drop, which shows that GLP-1 is used as an enterogenous hormone which is released into blood under the stimulation of nutrient substances (particularly carbohydrate), the insulin secretion promotion effect of the GLP-1 is glucose concentration dependent, and the GLP-1 can play a role in reducing blood sugar when the blood sugar rises, inhibiting the secretion of glucagon, increasing satiety and reducing hunger, thereby achieving the effect of reducing blood sugar. In the literature, however, Effect of 6-seek core of glucose-like peptide 1on glycolic acid control, insulin sensitivity, and β -cell function in type 2diabetes, a parallel-group study, ([ J ]. Lancet, Zander, Mette, Madsbad, et al., 2002), researchers have shown that GLP-1 produces weight-reducing effects through a variety of pathways, including inhibition of gastrointestinal motility and gastric secretion, inhibition of appetite and ingestion, and delay of gastric emptying. In addition, GLP-1 can also act on central nervous system (especially hypothalamus) to suppress appetite, reduce food intake, thereby causing satiety and appetite reduction in human body, and reducing calorie intake, thereby achieving the purpose of reducing weight.

Currently, among the GLP-1 derivatives that are marketed are exenatide, liraglutide, dulaglutide, risanatide, exenatide microsphere formulations, albiglutide, polyethylene glycol loxapide and somaglutide (also known as semaglutide). Wherein the Somalutide is a representative of GLP-1 derivative medicines.

Somaglutide is a long-acting GLP-1 derivative developed by Novoglide, which requires only once weekly subcutaneous administration and is currently marketed in many countries. Moreover, by formulation technology, norshanodond developed an oral formulation of somaglutide. Structurally, the somaglutide is obtained by connecting the 26 th Lys position on a GLP-1(7-37) chain to AEEA, glutamic acid and octadecane fatty diacid side chains, and replacing the 8 th amino acid with the unnatural amino acid aminoisobutyric acid (Aib) to obtain the somaglutide. Compared with liraglutide, the fat chain of the soraglutide is longer, the hydrophobicity is increased, but the hydrophilicity of the soraglutide is greatly enhanced through short-chain AEEA modification. After AEEA modification, the modified polypeptide can be tightly combined with albumin to cover DPP-4 enzyme hydrolysis sites, and can also reduce renal excretion, prolong the biological half-life and achieve the effect of long circulation. The soxhlet peptide is proved to be capable of effectively controlling blood sugar by combining different oral hypoglycemic drugs in a plurality of clinical trial researches, and can reduce the weight of a patient, reduce systolic pressure and improve the function of islet beta cells.

Accordingly, it is an object of the present invention to provide a GPL-1 derivative and a preparation thereof having excellent weight-loss and glucose-lowering ability.

Disclosure of Invention

To solve the above technical problem or to at least partly solve the above technical problem, the present invention provides a GLP-1(7-37) polypeptide analogue and a long-acting derivative thereof. The long-acting GPL-1(7-37) derivative provided by the invention has excellent weight-losing and sugar-reducing capabilities, the weight-losing effect of the derivative can even reach nearly twice that of the somaglutide, and the sugar-reducing effect of the derivative is also obviously superior to that of the somaglutide.

In a first aspect, the present invention provides a GLP-1(7-37) analog, said GLP-1(7-37) analog consisting of a polypeptide having an amino acid sequence represented by the formula:

HX ₈ EGTFTSDVSSYLE _E QAAX ₂₆ EFIAWLVX ₃₄ X ₃₅ X ₃₆ G；

wherein, X ₈ Selected from V, I, T, L, G or S, X ₂₆ Is R or K, X ₃₄ Is R or K, X ₃₅ Is G or R, X ₃₆ Is G or R, wherein X ₂₆ And X ₃₄ Only one of which isK；

In a second aspect, the present invention provides a long-acting GLP-1(7-37) derivative comprising fatty acid side chains attached to the K residues of said GLP-1(3-37) analogues, respectively, preferably via the epsilon amino group on the K residue.

When the GLP-1(7-37) analogue is Ile ⁸ Glu ²² Arg ²⁶ Lys ³⁴ When GLP-1(7-37) is adopted, the amino acid sequence of the GLP-1(7-37) analogue is shown as SEQ ID NO. 1.

When the GLP-1(7-37) analogue is Thr ⁸ Glu ²² Arg ²⁶ Lys ³⁴ When GLP-1(7-37) is adopted, the amino acid sequence of the GLP-1(7-37) analogue is shown as SEQ ID NO. 2.

When the GLP-1(7-37) analogue is Ile ⁸ Glu ²² Arg ²⁶ Lys ³⁴ Arg ³⁵ Gly ³⁶ When GLP-1(7-37) is adopted, the amino acid sequence of the GLP-1(7-37) analogue is shown as SEQ ID NO. 3.

When the GLP-1(7-37) analogue is Thr ⁸ Glu ²² Arg ²⁶ Lys ³⁴ Arg ³⁵ Gly ³⁶ When GLP-1(7-37) is adopted, the amino acid sequence of the GLP-1(7-37) analogue is shown as SEQ ID NO. 4.

As a preferred embodiment of the present invention, the side chain structure of the fatty acid used in the long-acting GLP-1(7-37) derivative of the present invention is HOOC (CH) ₂ ) _n CO-where n is an integer selected from 10 to 24, more preferably 16 to 20. In particular, the fatty acid side chain may be selected from HOOC (CH) ₂ ) ₁₄ CO-、HOOC(CH ₂ ) ₁₅ CO-、HOOC(CH ₂ ) ₁₆ CO-、HOOC(CH ₂ ) ₁₇ CO-、HOOC(CH ₂ ) ₁₈ CO-、HOOC(CH ₂ ) ₁₉ CO-、HOOC(CH ₂ ) ₂₀ CO-、HOOC(CH ₂ ) ₂₁ CO-or HOOC (CH) ₂ ) ₂₂ CO-, preferably the fatty acid side chain structure is HOOC (CH) ₂ ) ₁₆ CO-。

In a preferred embodiment of the present invention, the fatty acid side chain is linked to the residue via a linker.

As a preferred embodiment of the present invention, the fatty acid side chain is linked to position X on the GLP-1(3-37) analog via a linker ₂₆ Or X ₃₄ The epsilon amino group of Lys is linked.

As a preferred embodiment of the present invention, the linker is selected from the group consisting of:

wherein m is an integer from 0 to 6, such as 0, 1, 2, 3, 4, 5, 6, etc., n is an integer from 1 to 3, such as 1, 2, 3, etc., s is an integer from 0 to 3, such as 0, 1, 2, 3, etc., t is an integer from 0 to 4, such as 0, 1, 2, 3, 4, etc., p is an integer from 1 to 23, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, etc.

As a specific embodiment of the present invention, the joint is:

wherein s is 1 and n is 1 or 2.

As for the above-mentioned preferred linker moiety (when n is 1), it may be represented by γ -Glu-OEG-OEG wherein OEG represents a group-NH (CH) according to IUPAC nomenclature ₂ ) ₂ O(CH ₂ ) ₂ CO-i.e. "2- [2- (2-aminoethoxy) ethoxy]Abbreviation for acetyl ". When HOOC (CH) is selected ₂ ) ₁₆ When CO-is used as a side chain, the combination of the above side chain and linker (acyl group) can be referred to as "[ 2- (2- [2- (2- [2- (2-)4- (17-carboxyheptadecanoylamino) -4(S) -carboxybutyrylamino group" according to IUPAC nomenclature]Ethoxy) ethoxy]Acetylamino) ethoxy]Ethoxy) acetyl group]”。

As the inventionIn a specific embodiment, the derivative of the invention comprises a fatty acid side chain attached to the epsilon amino group of the lysine at position 34 of the GLP-1(7-37) analog, said fatty acid side chain being HOOC (CH) ₂ ) ₁₆ CO-, the fatty acid side chain is linked to the epsilon amino group of lysine at position 34 via-gamma-Glu-OEG-OEG-.

Thus, preferably, the long-acting GLP-1 derivative according to the invention is selected from the group consisting of:

N-ε ³⁴ - [2- (2- [2- (2- [2- (2- [4- (17-carboxyheptadecanoylamino) -4(S) -carboxybutanoylamino)]Ethoxy) ethoxy]Acetylamino) ethoxy]Ethoxy) acetyl group][Ile ⁸ Glu ²² Arg ²⁶ Lys ³⁴ ]GLP-1(7-37) (abbreviated as HS-G1); or

N-ε ³⁴ - [2- (2- [2- (2- [2- (2- [4- (17-carboxyheptadecanoylamino) -4(S) -carboxybutanoylamino group)]Ethoxy) ethoxy]Acetylamino) ethoxy]Ethoxy) acetyl group][Thr ⁸ Glu ²² Arg ²⁶ Lys ³⁴ ]GLP-1(7-37) (abbreviated HS-G2); or

N-ε ³⁴ - [2- (2- [2- (2- [2- (2- [4- (17-carboxyheptadecanoylamino) -4(S) -carboxybutanoylamino group)]Ethoxy) ethoxy]Acetylamino) ethoxy]Ethoxy) acetyl group][Ile ⁸ Glu ²² Arg ²⁶ Lys ³⁴ Arg ³⁵ Gly ³⁶ ]GLP-1(7-37) (abbreviated HS-G3); or

N-ε ³⁴ - [2- (2- [2- (2- [2- (2- [4- (17-carboxyheptadecanoylamino) -4(S) -carboxybutanoylamino group)]Ethoxy) ethoxy]Acetylamino) ethoxy]Ethoxy) acetyl group][Thr ⁸ Glu ²² Arg ²⁶ Lys ³⁴ Arg ³⁵ Gly ³⁶ ]GLP-1(7-37) (abbreviated as HS-G4).

In the invention, the inventor finds that the GLP-1 derivative obtained by modifying amino acids at 35 th position and 36 th position of GLP-1(7-37) analogue polypeptide into amino acids Arg and Gly respectively and then acylating specific positions has more excellent blood sugar reducing and weight reducing activities through partial site change.

In a third aspect, the invention provides a recombinant engineering bacterium for highly expressing the GLP-1 analogue, wherein the engineering bacterium is preferably a recombinant Escherichia coli engineering bacterium, and more preferably a recombinant Escherichia coli BL21 engineering bacterium.

In a fourth aspect, the invention provides a construction method of the recombinant engineering bacteria of the GLP-1 analogue, and the method comprises the following steps: (1) sequentially fusing the inclusion body promoting sequence, the EK enzyme digestion sequence and the GLP-1 analogue coding gene sequence in series to prepare a gene expression fragment of the GLP-1 analogue; (2) inserting the gene expression fragment into a prokaryotic expression plasmid to obtain an expression plasmid of the GLP-1 analogue; (3) and transferring the expression plasmid into escherichia coli to prepare the recombinant engineering bacteria for expressing the GLP-1 analogue.

Preferably, the prokaryotic expression plasmid is pET-30a (+) and the gene expression fragment is inserted into the plasmid through NdeI and XhoI sites, or the prokaryotic expression plasmid is pET-28a (+) and the gene expression fragment is inserted into the plasmid through NcoI and XhoI sites.

Preferably, the amino acid sequence of the inclusion body promoting sequence is FKFEFKFE, and the EK enzyme digestion sequence is DDDDK.

More preferably, the nucleic acid sequence of the gene expression fragment according to the invention is selected from one of SEQ ID NO.5 to 8.

The in vitro binding activity shows that compared with the somaglutide, the long-acting GLP-1 derivative provided by the invention maintains GLP-1R binding affinity equivalent to that of the somaglutide. However, the blood sugar reduction experiment in a diabetes animal model shows that the long-acting GLP-1 derivative has the blood sugar reduction effect which is obviously better than that of the somataltide, and particularly has the blood sugar reduction effect of HS-G3 and HS-G4. Another important role of GLP-1 derivatives is their weight loss effect, which can be exploited as weight loss indication drugs, and somaglutide has been approved by the FDA in the united states as a weight loss indication drug. Research results in the obese animal model show that compared with the somaglutide, the GLP-1 derivative (especially HS-G3 and HS-G4) has approximately two times or more weight loss effect in a normal animal model and a diabetic animal model, and does not have hypoglycemia risk, so the long-acting GLP-1 derivative has wider commercial development value.

In a fifth aspect, the invention provides a pharmaceutical composition comprising said long-acting GLP-1 derivative or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

In a sixth aspect, the invention provides the application of the long-acting GLP-1 derivative or the pharmaceutically acceptable salt thereof or the pharmaceutical composition thereof in preparing a medicament for treating diabetes.

In a seventh aspect, the invention provides the use of the long-acting GLP-1 derivative or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, for the preparation of a weight loss formulation.

Compared with the prior art, the long-acting GLP-1 derivative provided by the invention has the following advantages:

(1) the GLP-1 derivative provided by the invention has more excellent blood sugar reducing capability in diabetics;

(2) compared with the somaglutide, the long-acting GLP-1 derivative provided by the invention has more excellent and remarkable weight loss capability for normal people and diabetic patients, and has more excellent weight loss application potential.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the invention and together with the description, serve to explain the principles of the invention.

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without inventive exercise.

FIG. 1 is a bar graph of the effect of long-acting GLP-1 derivatives on blood glucose changes in db/db mice:

in the figure, for each group of data, a model control group, a somalvidin group, an HS-G1 group, an HS-G2 group, an HS-G3 group and an HS-G4 group are arranged from left to right;

FIG. 2 is a bar graph of the effect of long acting GLP-1 derivatives on the rate of change of body weight in db/db mice;

in the figure, a model control group, a somarutide group, an HS-G1 group, an HS-G2 group, an HS-G3 group and an HS-G4 group are arranged from left to right in sequence;

FIG. 3 is a bar graph of the effect of long-acting GLP-1 derivatives on the rate of change of body weight in normal mice;

in the figure, from left to right, the model control group, the Somalitin group, the HS-G3 group and the HS-G4 group are arranged in sequence.

Detailed Description

In order that the above objects, features and advantages of the present invention may be more clearly understood, a solution of the present invention will be further described below. It should be noted that the embodiments of the present invention and features of the embodiments may be combined with each other without conflict.

In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways than those described herein; it is to be understood that the embodiments described in this specification are only some embodiments of the invention, and not all embodiments.

EXAMPLE 1 preparation of Long-acting GLP-1 derivatives

This example provides various long-acting GLP-1(7-37) derivatives and methods for their preparation, and in particular, a recombinant engineered bacterium capable of efficiently expressing the derivatives of the present invention is constructed by the following methods (for example, HS-G1):

(1) construction of the code Ile ⁸ Glu ²² Arg ²⁶ Lys ³⁴ Expression plasmid for GLP-1(7-37)

Through a large number of researches and experiments, FKFEFKFE is finally selected as an inclusion body promoting sequence, DDDDK is selected as an EK enzyme digestion sequence, and the inclusion body promoting sequence, the EK enzyme digestion sequence and a GLP-1 analogue coding gene sequence are sequentially fused in series to obtain a gene segment shown as SEQ ID NO. 5; inserting the fragment into prokaryotic expression plasmid pET-28a (+) through NcoI and XhoI sites, and sequencing to verify to obtain expression plasmid named pET-28a (+) -Ile ⁸ Glu ²² Arg ²⁶ Lys ³⁴ -Glp-1(7-37)。

(2) Construction of expression Ile ⁸ Glu ²² Arg ²⁶ Lys ³⁴ Recombinant engineering bacterium of (7-37) GLP-1

BL21 competent cells (TransGenBiotech) were thawed (50. mu.L) on an ice bath, the expression plasmid constructed in step (1) was added, shaken gently, and placed in the ice bath for 30 min. Then carrying out water bath heat shock for 30s at 42 ℃, and then quickly transferring the centrifugal tube into an ice bath for placing for 2min without shaking the centrifugal tube;

adding 500. mu.L of sterile LB medium (containing no antibiotic) into a centrifuge tube, mixing uniformly, culturing at 37 ℃ for 1h at 180rpm to resuscitate the bacteria, then sucking 200. mu.L of transformed competent cells, adding the cells to a plate of LB agar medium containing kanamycin resistance, spreading the cells uniformly, placing the plate at 37 ℃ until the liquid is absorbed, inverting the plate, culturing overnight at 37 ℃, the next day, picking up a monoclonal colony in the transformation plate by using an inoculating loop, inoculating into 15mL of sterile LB medium (containing antibiotic), and culturing overnight at 30 ℃. Adding 500 μ L of overnight culture liquid into a sterile 1.5ml centrifuge tube, adding 500 μ L of 50% sterile glycerol, and mixing to obtain glycerol cryopreserved bacteria, and storing at-80 deg.C.

(3) Fermentation expression of recombinant engineering bacteria

Adding 50 mu L of glycerol frozen bacteria liquid into 50mL of 2YT culture medium, simultaneously adding 50 mu L of kanamycin, uniformly mixing, placing in a constant temperature oscillator, culturing at 37 ℃ and 200rpm overnight to obtain a first-grade seed culture solution, and measuring OD600 to be more than 5.0;

taking 40mL of first-level seed culture solution cultured overnight, inoculating the first-level seed culture solution into 200mL of 2YT culture medium according to the proportion of 1:5, simultaneously adding 200 mu L of kanamycin, uniformly mixing, putting into a constant temperature oscillator, culturing at 37 ℃ and 200rpm for 3h, wherein OD600 is more than 3.0, and obtaining second-level seed culture solution;

taking 60mL of secondary seed liquid, inoculating the secondary seed liquid into an FDM culture medium (600mL) according to the proportion of 1:10, culturing in a 2L fermentation tank, starting to insert IPTG (isopropyl-beta-D-thiogalactoside) when detecting that the OD600 value of the culture bacterial liquid reaches about 160, ending the culture after the induction culture at 30 ℃ for 24 hours, and putting the culture tank for centrifugation;

the expressed bacteria liquid is centrifuged for 30min at 8000g to obtain thallus cytoplasm, the thallus yield is about 300g of thallus/L of fermentation liquid, and the thallus obtained by centrifugation is subjected to target protein expression amount measurement, and the expression amount is not less than 10 g/L.

(4) Recombinant Ile ⁸ Glu ²² Arg ²⁶ Lys ³⁴ Purification of GLP-1(7-37)

Weighing 100g of the cell paste obtained in the step (3), suspending the cell paste in 500mL of solution (50mM Tris-HCl, 50mM NaCl, pH8.0), carrying out ultrasonic treatment for 30min by using an ultrasonic cell crusher to crush the cells, centrifuging the obtained homogenate for 30min at 13000g at 4 ℃, collecting precipitates after the centrifugation is finished, and dissolving the precipitates by using 8M urea to obtain a sample before enzyme digestion;

concentrating a sample before enzyme digestion by UniPS30-300 (purchased from Suzhou Naichi Microscience, Inc.) which is balanced by equilibrium liquid 3(10mM ammonium acetate, 20% acetonitrile) in advance, eluting by the equilibrium liquid 3, then carrying out gradient elution by 0-100% eluent (10mM ammonium acetate, 80% acetonitrile), and analyzing by SDS-PAGE, wherein the purity of the GLP-1 intermediate product generated by the purification process is higher than 70%;

the tag sequence was cleaved using EK enzyme: adding 20mM PB buffer solution with the pH value of 7.4 into the intermediate product to dilute the intermediate product by three times, adding EK enzyme according to the ratio of EK enzyme to intermediate product of 1:15 at the temperature of 20 ℃, uniformly mixing, then carrying out enzyme digestion overnight, and analyzing the enzyme cleavage rate by SDS-PAGE to be close to 80%;

Ile ⁸ Glu ²² Arg ²⁶ Lys ³⁴ -purification of GLP-1 (7-37): concentrating UniPS30-300 (purchased from Suzhou Naichi Microscience, Inc.) equilibrated with equilibration solution 3(10mM ammonium acetate, 20% acetonitrile), eluting with equilibration solution 3, and gradient-eluting with 0-100% eluent (10mM ammonium acetate, 80% acetonitrile), wherein the purity of the eluate is about 90% by SDS-PAGE;

0.2M Na was added to the eluted sample ₂ HPO ₄ Adjusting pH to 4.8-5.0 with 1M citric acid to final concentration of 20mM, acid precipitating at 4 deg.C overnight, centrifuging at 13000g for 30min at 4 deg.C with SDS-PAGE detection yield of above 90%, collecting precipitate, and storing at-20 deg.C to obtain Ile ⁸ Glu ²² Arg ²⁶ Lys5 ³⁴ -GLP-1(7-37)。

(5) Preparation of long-acting GLP-1 derivatives

Fatty acid modification: ile obtained in step (4) ⁸ Glu ²² Arg ²⁶ Lys ³⁴ -GLP-1(7-37) Adding water to prepare a dissolving solution of 4-6 mg/mL, adding 1M sodium hydroxide to adjust the pH value to 11.0-11.5, shaking up to completely dissolve the protein, and quantifying the polypeptide concentration by HPLC; weighing fatty acid powder according to a molar ratio of polypeptide to octadecanedioic acid mono-tert-butyl ester-glutamic acid (1-tert-butyl ester) -AEEA-AEEA-OSU (sucrose synthase inhibitor) of 1:4, dissolving the fatty acid powder in acetonitrile, mixing a polypeptide sample with the fatty acid solution, standing the mixed solution for one hour at 4 ℃, then adding water to dilute the sample by 5 times, adjusting the pH value to 4.8 by using 1M citric acid (or 10% acetic acid) to terminate the reaction, standing the mixture at 4 ℃ for acid precipitation for 10min, centrifuging the mixture at 4 ℃ for 30min by 13000g after the acid precipitation, and then storing the precipitate at-80 ℃;

and (3) deprotection and purification of fatty acid: adding TFA to the obtained precipitate until the final concentration of the polypeptide is about 10mg/mL, shaking to dissolve the precipitate, standing at room temperature for deprotection for 30min, and then dropping 4M NaOH to adjust the pH value to 7.5-8.5 to terminate the reaction;

pumping the reaction solution after the reaction termination into UniPS10-300 (purchased from Suzhou Naichi technology Co., Ltd.) which is balanced by equilibrium liquid 3(10mM ammonium acetate, 20% acetonitrile) at the flow rate of 4mL/min by using a protein purification chromatography system (SecureSDL 100) for concentration, after the equilibrium liquid 3 is subjected to leaching, performing gradient elution by using eluent (10mM ammonium acetate, 80% acetonitrile) of 0-100%, and collecting an elution peak, wherein the purity of the elution peak is about 90% through RP-HPLC detection;

diluting the elution peak with water by 3 times, adjusting pH to 4.80 by acid precipitation, precipitating at 4 deg.C for 30min, adding PBST buffer solution with pH of 7.0 into the precipitate after centrifugation, redissolving, and freezing at-80 deg.C to obtain N-epsilon ³⁴ - [2- (2- [2- (2- [2- (2- [4- (17-carboxyheptadecanoylamino) -4(S) -carboxybutanoylamino)]Ethoxy) ethoxy]Acetylamino) ethoxy]Ethoxy) acetyl group][Ile ⁸ Glu ²² Arg ²⁶ Lys ³⁴ ]GLP-1(7-37) (abbreviated as HS-G1).

According to the step (1) in the embodiment, the inclusion body promoting sequence, the EK enzyme digestion sequence and the GLP-1 analogue coding gene sequence are sequentially fused in series to obtain the polypeptide containing the coding Thr ⁸ Glu ²² Arg ²⁶ Lys ³⁴ -GLP-1(7-37)、Ile ⁸ Glu ²² Arg ²⁶ Lys ³⁴ Arg ³⁵ Gly ³⁶ GLP-1(7-37) and Thr ⁸ Glu ²² Arg ²⁶ Lys ³⁴ Arg ³⁵ Gly ³⁶ -GLP-1(7-37) gene segments, the nucleotide sequences of which are shown in SEQ ID No.6-8, respectively; wherein, the first and the second end of the pipe are connected with each other,

Thr ⁸ Glu ²² Arg ²⁶ Lys ³⁴ GLP-1(7-37) was prepared in exactly the same manner as in steps (1) to (5) ³⁴ - [2- (2- [2- (2- [2- (2- [4- (17-carboxyheptadecanoylamino) -4(S) -carboxybutanoylamino)]Ethoxy) ethoxy]Acetylamino) ethoxy]Ethoxy) acetyl group][Thr ⁸ Glu ²² Arg ²⁶ Lys ³⁴ ]GLP-1(7-37) (abbreviated HS-G2);

Ile ⁸ Glu ²² Arg ²⁶ Lys ³⁴ Arg ³⁵ Gly ³⁶ GLP-1(7-37) and Thr ⁸ Glu ²² Arg ²⁶ Lys ³⁴ Arg ³⁵ Gly ³⁶ GLP-1(7-37), which is obtained by inserting the above fragment into a prokaryotic expression plasmid pET-30a (+) and verifying the sequencing according to the substantially same method as that of the steps (1) to (5) except that NdeI and XhoI sites are selected, and finally obtaining expression plasmids (the other steps are the same) by: n-epsilon ³⁴ - [2- (2- [2- (2- [2- (2- [4- (17-carboxyheptadecanoylamino) -4(S) -carboxybutanoylamino)]Ethoxy) ethoxy]Acetylamino) ethoxy]Ethoxy) acetyl group][Ile ⁸ Glu ²² Arg ²⁶ Lys ³⁴ Arg ³⁵ Gly ³⁶ ]GLP-1(7-37) (abbreviated HS-G3), and, N- ε ³⁴ - [2- (2- [2- (2- [2- (2- [4- (17-carboxyheptadecanoylamino) -4(S) -carboxybutanoylamino)]Ethoxy) ethoxy]Acetylamino) ethoxy]Ethoxy) acetyl group][Thr ⁸ Glu ²² Arg ²⁶ Lys ³⁴ Arg ³⁵ Gly ³⁶ ]GLP-1(7-37) (abbreviated as HS-G4).

In the examples, the expression of the recombinant engineered bacteria constructed in steps (1) to (4) is shown in table 1:

TABLE 1

Example 2: in vitro cell affinity activity assay

(1) Preparing different long-acting GLP-1 derivative injection

The specific formula is as follows: 1.133mg/mL Na ₂ HPO ₄ 5.5mg/mL phenol, 14.0mg/mL propylene glycol, GLP-1 derivatives: 1200ng/mL, 300ng/mL, 75ng/mL, 18.75ng/mL, 4.6875ng/mL, 1.172ng/mL, 0.293ng/mL, 0.073 ng/mL.

Selecting RIN-m5F cells in good culture state, discarding culture solution in bottle, washing with Versene solution (ethylenediamine tetraacetic acid solution) for 1 time, adding 0.05% TRYPSIN digestive juice for digestion, adding RPMI1640 basic culture solution for termination of digestion, centrifuging to collect cells, adjusting cell density to 6.7 × 10 with RPMI1640 basic culture solution ⁵ one/mL, 100. mu.L/well of the culture medium was inoculated into a 96-well cell culture plate at 37 ℃ in 5% CO ₂ Incubated overnight under conditions.

In vitro activity of derivatives of GLP-1 analogs using cAMP detection kit (R & D): preparing a determination culture solution, diluting a sample to 1200ng/mL step by using the determination culture solution, wherein the single dilution multiple does not exceed 10 times; then, 4-fold serial dilutions were performed in 96-well plates for 8 gradients, with 2 duplicate wells for each dilution.

Taking out the cultured cell culture plate from the incubator, discarding the old culture solution, and blotting the old culture solution on filter paper; adding the diluted solution into cell plate at 100 μ L per well, and standing at 37 deg.C with 5% CO ₂ The drug was allowed to act for 3.5min under conditions, the sample solution on the cell culture plate was decanted, blotted dry on filter paper, and diluted cell lysate (1 × Lysis Buffer) was added to the cell culture plate, 120 μ L per well, frozen at-80 ℃ and thawed by shaking at room temperature.

(2) And (3) cAMP content detection:

the desired strips were lined up in a 96-well plate rack and primary antibodies were incubated: adding 50 microliter of primary antibody solution into each well, sealing the plate by using a sealing plate membrane, and putting the plate into a microplate oscillator to incubate for 1 hour at room temperature.

Washing: discarding the liquid, spin-drying, adding 300 μ L washing buffer solution into each well, placing into a micropore plate oscillator, shaking for 1min, discarding, repeating the steps for 4 times, and patting on filter paper to dry.

Loading and secondary antibody: adding 50 mu L of secondary antibody into each hole, taking 100 mu L of cell lysate from each hole in the cell plate, transferring the cell lysate into the holes of the enzyme label plate, sealing the plate by a sealing plate membrane, uniformly mixing, putting into a micropore plate oscillator, and incubating for 2 hours at room temperature.

Washing: discarding the liquid, spin-drying, adding 300 μ L of washing buffer solution into each well, placing into a microplate oscillator, shaking for 1min, discarding, repeating the above steps for 4 times, and drying on filter paper.

Color development: and (3) uniformly mixing the color development liquid A and the color development liquid B in equal amount, adding 200 mu L of mixed liquid into each hole, putting the mixed liquid into a micropore plate oscillator, and performing light-proof color development for 30 minutes.

And (4) terminating: the reaction was stopped by adding 100. mu.L of stop solution to each well.

And (3) determination: placing the sample in an enzyme-labeling instrument, and detecting the OD value of each hole by using 570nm as reference wavelength and 450nm as detection wavelength; completing detection within 30min, drawing a cAMP analysis curve graph and calculating an EC50 value; the test data were processed using a four parameter regression algorithm and the EC50 value of the test sample was calculated with the results shown in table 2:

TABLE 2

Sample(s)	HS-G1	HS-G2	HS-G3	HS-G4	Somazutide
						EC50(nM)	4.814	9.269	3.842	2.463	1.892

From the results in the table, it can be seen that HS-G3 and HS-G4 according to the present invention have lower EC50 values compared to HS-G1 and HS-G2, which are closer to EC50 values of somaglutide, indicating that they have binding affinity to the receptor comparable to somaglutide.

Example 3: study of hypoglycemic Effect in db/db mice

(1) Experimental materials:

the experimental animals are BKS-LeprREM/Gpt mice, the number of which is 30, the week age of which is 6 weeks old, and the animals are male;

experimental pharmaceutical formulation: 1.133mg/mL Na ₂ HPO ₄ 5.5mg/mL phenol, 14.0mg/mL propylene glycol, 0.045mg/mL GLP-1 derivative.

(2) The experimental method comprises the following steps:

a. modeling and grouping:

selecting 30 SPF male BKS-LeprRem/Gpt mice at 6 weeks, randomly grouping according to weight and blood sugar, dividing into 6 groups, setting 5 test animals in each group, and respectively setting a model control group (solvent), a positive control group (Somalou peptide), an experimental group 1(HS-G1), an experimental group 2(HS-G2), an experimental group 3(HS-G3) and an experimental group 4 (HS-G4); wherein the control group of the model was injected with a blank solvent intraperitoneally and subcutaneously.

b. The administration mode comprises the following steps:

the administration was carried out in the manner shown in Table 3, once every 48h, four times, and by intraperitoneal injection.

TABLE 3

c. Detecting the index

Blood glucose value: measuring the blood glucose value before administration and 2h after administration at the first administration, and measuring the blood glucose value 0h before administration and 2h after administration at the next three administrations;

pre-re-dose 0h is 48h as described in table 4 and in DAY3, DAY5, DAY7 and DAY9 of fig. 1, which refers to 48h after the previous dose and is also pre-re-dose 0 h.

(3) Results of the experiment

The statistics of the experimental data are shown in Table 4, the data correspond to a histogram shown in FIG. 1, and in the graph, each group of data sequentially comprises a model control group, somaglutide, HS-G1, HS-G2, HS-G3 and HS-G4 from left to right;

table 4: mean blood glucose condition

Statistical analysis of all data for this experiment was performed using SPSS software. All values are expressed as means ± standard deviation (mean ± SD). For normal distribution data, differences between groups were compared using one-way ANOVA. For all analyses, P <0.05 was considered statistically significant.

As can be seen from the data results of the experiment (table 4 and fig. 1), the derivatives of the present invention have significantly better hypoglycemic effects than the somaglutide when administered for 2h and 48h almost every time; wherein, the HS-G3 has the most obvious effect and the best blood sugar reducing effect, and is also superior to HS-G1 with similar sequence; and the blood sugar reducing effect of HS-G4 is better than that of the somaglutide and is also better than that of HS-G2. Therefore, the GLP-1 derivative has very excellent hypoglycemic effect.

Example 4: study of weight loss Effect in db/db mice

(1) Experimental materials:

the product is BKS-LeprREM/Gpt mice, the number of which is 30, the age of6 weeks, and males;

experimental pharmaceutical formulation: 1.133mg/ml Na ₂ HPO ₄ 5.5mg/ml phenol, 14.0mg/ml propylene glycol, 0.045mg/ml GLP-1 derivative

(2) Experimental methods

a. Modeling and grouping:

30 SPF male 6-week BKS-LeprRem/Gpt mice are selected, randomly grouped according to weight and blood sugar and divided into 6 groups, and each group is provided with 5 test animals which are respectively a model control group (solvent), a positive control group (Somalulptin), an experimental group 1(HS-G1), an experimental group 2(HS-G2), an experimental group 3(HS-G3) and an experimental group 4 (HS-G4). Wherein the model control group was injected intraperitoneally subcutaneously with a blank solvent.

b. The administration mode comprises the following steps:

the administration was carried out in the same manner as shown in Table 3 in example 3, once every 48 hours, four times, and subcutaneously via intraperitoneal injection.

c. Detection indexes are as follows:

weight: mice were weighed before each dose and 48h after the last dose;

food intake: a fixed food intake (200g) was set per cage of mice before the first dose, and then the food intake of the mice was measured 48h after each (including the first) dose.

(3) The experimental results are as follows:

the weight change data of the mice for 9 days are recorded, and the results are shown in table 5, and fig. 2 is a graph of the weight change rate of the mice after 9 days of administration;

TABLE 5

Statistical analysis of all data from this experiment was performed using SPSS software. All values are expressed as means ± standard deviation (mean ± SD). For normal distribution data, differences between groups were compared using one-way ANOVA. For all analyses, P <0.05 was considered statistically significant.

Meanwhile, the food intake of the mice for 9 days was recorded, wherein the food intake inhibition rate was (average of food intake of model control group-average of food intake of administration group)/average of food intake of model control group, and the results are shown in table 6:

TABLE 6

As can be seen from tables 5-6 and figure 2 for recording weight loss and food intake data of mice, the long-acting GLP-1 derivative provided by the invention has better weight loss effect and better food intake inhibition than thaumatin, and the difference is obvious. Especially HS-G3 and HS-G4, the weight change rate reaches more than 2 times of thaumatin, the weight loss effect is particularly obvious, and even if the HS-G1 and the HS-G2 are compared, the weight loss effect is close to 1.5-2 times.

Example 5: study of weight loss Effect in Normal mice

(1) Experimental Material

a. Experimental formulation:

GLP-1 derivative injection (HS-G3 and HS-G4) is prepared, and the specific formula is 1.133mg/mLNa ₂ HPO ₄ 5.5mg/mL phenol, 14.0mg/mL propylene glycol, and GLP-1 derivative 0.045 mg/mL.

b. Animal experiments:

selecting 50 healthy SPF male KM mice with the body weight of 18-20g and the ages of 6-8 weeks, and dividing the mice into a Normal Diet (ND) group and an HFD (high fat diet) group, wherein the normal diet group comprises 10 mice and is fed with a common maintenance diet, the HFD group comprises 40 mice and is fed with 60% high fat diet, and the feeding period is 5-8 weeks;

detecting the weight of the HFD group, screening out unmolded mice, randomly grouping the remaining mice according to the weight, and dividing the mice into a blank control group (used for judging whether a DIO model succeeds or not and does not participate in a drug test), a positive control group (somaglutide), an experiment group 1, an experiment group 2 and a model control group (blank solvent);

(2) experimental methods

a. The administration mode comprises the following steps:

the medicine is taken once every two days, the administration route is intraperitoneal injection and four times of administration, and the contents of specific administration dosage and the like are shown in a table 7:

TABLE 7

b. Detection indexes are as follows:

weight: detecting body weight at each administration;

(3) results of the experiment

The test results of this example are shown in Table 8, and the rate of change of body weight after 9 days of administration is shown in FIG. 3;

TABLE 8

Statistical analysis of all data in this experiment was performed using SPSS software, and all values are expressed as means ± standard deviation (mean ± SD); for normal distribution data, differences between groups were compared using one-way anova; for non-normal distribution data, differences between groups were analyzed using the Kruskal-WallisH test or Mann-WhitneyU test; the correlation analysis adopts a Spearman test; for all analyses, P <0.05 was considered statistically significant.

FIG. 3 is a graph of the weight change rate of a normal mouse after 9 days of administration, and it can be seen from the results of FIG. 3 and Table 8 that, in the normal mouse, the long-acting GLP-1 derivatives HS-G3 and HS-G4 provided by the invention have better weight-reducing capability than thaumatin, and achieve the weight-reducing effect of approximately 2 times and more than 2 times of that of the thaumatin; meanwhile, the long-acting GLP-1 derivative does not cause excessive lower blood sugar, and has no risk of hypoglycemia. Thus, the long-acting GLP-1 derivatives of the invention are more suitable for the development of weight-reducing indications.

In addition, the results of a group of experiments on HS-G1 and HS-G2 in a normal mouse model show that the two have weight loss effects equivalent to those of the thaumatin, and the weight change rates of the thaumatin are 8.94% and 6.26% corresponding to the weight change rate of 8.19%.

Example 6: pharmacokinetic Studies

(1) Experimental Material

Experimental mice: SD rats, 50, with body weights between 180-200g, male mice 6-8 weeks old.

Experimental formulation: the formulation of the injection of the Somalutide, the HS-G3 and the HS-G4 is 1.133mg/mLNa ₂ HPO ₄ 5.5mg/mL phenol, 14.0mg/mL propylene glycol, and GLP-1 derivative were 0.015mg/mL, 0.045mg/mL, and 0.075mg/mL, respectively.

(2) Experimental methods

a. Grouping condition: experimental rats were grouped according to table 9:

b. the administration mode comprises the following steps: the administration route is intraperitoneal subcutaneous injection, and the contents of specific administration dosage and the like are shown in a table 9:

TABLE 9

(3) Results of the experiment

Half-lives were measured for different doses in different experiments, and the results are shown in table 10:

watch 10

Statistical analysis of all data for this experiment was performed using SPSS software. All values are expressed as means ± standard deviation (mean ± SD). For normal distribution data, differences between groups were compared using one-way ANOVA. For non-normal distribution data, differences between groups were analyzed using the Kruskal-Wallis H test or the Mann-Whitney U test. The correlation analysis was performed using Spearman's test. For all analyses, P <0.05 was considered statistically significant.

From the results of the above experiments, it can be seen that the long-acting GLP-1 derivatives HS-G3 and HS-G4 provided by the present invention have a half-life in rats superior to thaumatin, and both have a long half-life of about 1.5 times or more relative to thaumatin at different doses.

It is noted that, in this document, relational terms such as "first" and "second," and the like, are used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising an … …" does not exclude the presence of other identical elements in a process, method, article, or apparatus that comprises the element.

The foregoing are merely exemplary embodiments of the present invention, which enable those skilled in the art to understand or practice the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Sequence listing

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Claims

1. A long-acting GLP-1 derivative or a pharmaceutically acceptable salt thereof, wherein the amino acid sequence of a GLP-1(7-37) analogue in said long-acting GLP-1 derivative is:

HX ₈ EGTFTSDVSSYLEEQAAREFIAWLVKRGG；

wherein X ₈ Is selected from I or T;

the derivative is linked to a fatty acid side chain via a linker through the epsilon amino group on the amino acid K residue on the GLP-1(7-37) analog;

the fatty acid side chain is selected from HOOC (CH) ₂ ) ₁₄ CO-、HOOC(CH ₂ ) ₁₅ CO-、HOOC(CH ₂ ) ₁₆ CO-、HOOC(CH ₂ ) ₁₇ CO-、HOOC(CH ₂ ) ₁₈ CO-、HOOC(CH ₂ ) ₁₉ CO-、HOOC(CH ₂ ) ₂₀ CO-、HOOC(CH ₂ ) ₂₁ CO-or HOOC (CH) ₂ ) ₂₂ CO-；

The joint is as follows:

wherein s and n are both 1.

2. The long-acting GLP-1 derivative or a pharmaceutically acceptable salt thereof according to claim 1, wherein said fatty acid side chain is selected from the group Consisting of HOOC (CH) ₂ ) ₁₆ CO-。

3. The long-acting GLP-1 derivative according to claim 1, or a pharmaceutically acceptable salt thereof, wherein said long-acting GLP-1 derivative is:

N-ε ³⁴ - [2- (2- [2- (2- [2- (2- [4- (17-carboxyheptadecanoylamino) -4(S) -carboxybutanoylamino)]Ethoxy) ethoxy]Acetylamino) ethoxy]Ethoxy) acetyl group][Ile ⁸ Glu ²² Arg ²⁶ Lys ³⁴ Arg ³⁵ Gly ³⁶ ]GLP-1 (7-37); or

N-ε ³⁴ - [2- (2- [2- (2- [2- (2- [4- (17-carboxyheptadecanoylamino) -4(S) -carboxybutanoylamino)]Ethoxy) ethoxy]Acetylamino) ethoxy]Ethoxy) acetyl group][Thr ⁸ Glu ²² Arg ²⁶ Lys ³⁴ Arg ³⁵ Gly ³⁶ ]GLP-1(7-37)。

4. A pharmaceutical composition comprising a long-acting GLP-1 derivative according to any one of claims 1-3 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.

5. Use of a long-acting GLP-1 derivative according to any one of claims 1-3 or a pharmaceutically acceptable salt thereof, a pharmaceutical composition according to claim 4 for the manufacture of a medicament for the treatment of diabetes.

6. Use of a long-acting GLP-1 derivative according to any one of claims 1-3 or a pharmaceutically acceptable salt thereof, a pharmaceutical composition according to claim 4 for the manufacture of a weight loss medicament.