CN114716533A

CN114716533A - Acylated long-acting GLP-1 derivative

Info

Publication number: CN114716533A
Application number: CN202210381817.2A
Authority: CN
Inventors: 曹海燕; 林兆生; 朱志伟; 王娟; 王含
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: 2022-04-12
Filing date: 2022-04-12
Publication date: 2022-07-08
Anticipated expiration: 2042-04-12
Also published as: CN114716533B; CN114958883B; CN114958883A

Abstract

The invention relates to an acylated long-acting GLP-1 derivative, in particular to a long-acting GLP-1 derivative modified by fatty acid acylation. The long-acting GLP-1 derivative has good binding affinity with a GLP-1 receptor and remarkably prolonged action time, and can be used for treating diseases such as diabetes, impaired glucose tolerance, obesity, hypertension, metabolic syndrome, dyslipidemia and the like.

Description

Acylated long-acting GLP-1 derivative

Technical Field

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

Background

Diabetes is a metabolic disorder disease such as carbohydrate, protein, fat and the like caused by the absolute or relative insulin secretion deficiency and/or insulin utilization disorder, 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, the 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, almost no hypoglycemic risk, and a weight-reducing effect and a cardiovascular protection function, and thus are becoming major therapeutic drugs and research hotspots for type2 diabetes.

GLP-1 (glucagon-like peptide 1) is a secretive insulinotropic hormone which is secreted into blood by intestinal tract cells after food stimulation and can stimulate insulin secretion, the insulin secretion capacity caused by the GLP-1 is about 50-70% of the total insulin secretion, and the function of stimulating insulin secretion has the characteristic of glucose concentration dependence, and has the functions of promoting insulin secretion, inhibiting glucagon release, stimulating insulin beta cell proliferation, inducing insulin beta cell regeneration, preventing insulin beta cell apoptosis, improving insulin sensitivity, increasing glucose utilization and the like. GLP-1 derivatives are a class of secretin drugs, and belong to GLP-1 receptor agonists; the amino acid sequences of GLP-1 analogues and glucagon are nearly half the same, and the analogues also have multiple functions of glucose-dependent insulin secretion promotion and biosynthesis, glucagon secretion inhibition, gastric emptying inhibition and the like (Dugang, Long, Xuren. glucagon-like peptide 1 and receptor agonist research progress [ J. Thus, GLP-1 and its analogs and derivatives play an important role in the development and progression of type 1 and 2 diabetes.

The research results of 10 patients with type 2diabetes with poor blood sugar control conducted by Nauck M, etc. and the patients were respectively given GLP-1 or placebo in a fasting state show that after the patients were infused with GLP-1, the insulin and C peptide levels were significantly increased, the glucagon level was significantly decreased, and the fasting blood sugar level became normal after 4 hours; after the blood sugar level is normal, the insulin level of the patient does not rise any more and the blood sugar level is kept stable and does not drop any more although GLP-1 is continuously infused, which indicates that GLP-1 is released into the blood under the stimulation of nutrient substances (particularly carbohydrate), the insulin secretion promotion effect of GLP-1 is glucose concentration dependent, and the GLP-1 can play the role of reducing the blood sugar when the blood sugar rises, inhibiting the secretion of glucagon, increasing satiety and reducing hunger so as to achieve the effect of reducing the blood sugar (normal-insulin-dependent) diabetes by way of glucose excretion-like peptide 1(7-36amide) in type2(non-insulin-dependent) diabetes peptides ([ J ]. Diabetologia, Nauck M., 1993, 36)). Lancet et al, however, have shown that GLP-1 produces weight loss through a variety of pathways, including inhibition of gastrointestinal motility and gastric secretion, inhibition of appetite and ingestion, delay of emptying of gastric contents, etc. In addition, GLP-1 can also act on the central nervous system (especially hypothalamus) to suppress appetite, reduce food intake, thus causing satiety and appetite reduction in human body, and reduce calorie intake, thereby achieving the purpose of reducing weight (Effect of 6-week heart of glucose-lipid 1on glucose control, insulin sensitivity, and β -cell function in type 2diabetes: a parallel-group study. ([ J ]. Lancet, Zander, method, Madsbad, et al, 2002)).

GLP-1 is rapidly degraded by dipeptidyl peptidase 4(DPP-4) in vivo, so that the action time of the GLP-1 is greatly limited, and the GLP-1 is difficult to be directly used as a medicament. Therefore, the existing method mainly for improving blood sugar control by GLP-1 mainly takes GLP-1 derivatives which simulate GLP-1 function exogenously and prolong the activity of endogenous GLP-1 as main components. 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 Novonide, which requires only once weekly subcutaneous administration and is currently approved for sale 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 somalutide has been proved in a plurality of clinical trial studies to be capable of effectively controlling blood sugar by combining different oral hypoglycemic drugs, and reducing the weight of a patient, reducing systolic pressure and improving the function of islet beta cells.

Due to the large number of patients with diabetes and obesity, the market is huge and the market demand for related GLP-1 derivatives is still huge. Thus, there is still a need to provide new GLP-1 derivatives with excellent glucose and weight reduction potential.

Disclosure of Invention

In order to solve the technical problems, the invention provides a GLP-1(7-37) polypeptide analogue and a long-acting derivative thereof. The long-acting GLP-1(7-37) derivative provided by the invention has excellent weight loss and blood sugar reduction capabilities, has a weight loss effect and a blood sugar reduction effect which are obviously superior to those of the somaglutide, and has more excellent and wide clinical and market application prospects.

The term GLP-1(7-37) analog in the present invention refers to a polypeptide obtained by modifying a human native GLP-1(7-37) amino acid, said modification comprising the removal and/or substitution (substitution) and/or addition (elongation) of one or more amino acid residues, which may be naturally occurring amino acids or artificially synthesized amino acids. The present invention describes the analogs with a simple nomenclature: for example [ Val ]⁸]GLP-1(7-37) refers to GLP-1(7-37) analogs in which the histidine naturally occurring at position 8 has been substituted with valine Val.

In the present invention, the term "derivative" with respect to a peptide (e.g., GLP-1 or insulin) means a chemically modified (e.g., covalently modified, etc.) peptide or an analog thereof. Typical modifications are amides, sugars, alkyl, acyl, esters, and the like. An example of a GLP-1(7-37) derivative is N-epsilon²⁶- ((4S) -4- (Hexadecanoylamino) -carboxy-butyryl) [ Arg³⁴Lys²⁶]GLP-1-(7-37)。

In the present invention, the term "aliphatic diacid" includes straight or branched chain aliphatic dicarboxylic acids. Non-limiting examples of aliphatic diacids are succinic acid, adipic acid, suberic acid, sebacic acid, dodecanedioic acid, tetradecanedioic acid, hexadecanedioic acid, heptadecanedioic acid, octadecanedioic acid, and eicosanedioic acid.

In the present invention, the term "pharmaceutically acceptable salt" refers to a salt of a polypeptide or protein that retains the biological activity of the parent.

The term "vector" refers to a vehicle into which nucleotide fragments encoding a protein or polypeptide can be operably inserted to cause expression of the protein or polypeptide. The vector may be used to transform, transduce or transfect a host cell so that it expresses the carried genetic element in the host cell. Examples of vectors include plasmids, artificial chromosomes, bacteriophages, viral particles, and the like. The vector may contain a variety of elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. The vector may also include materials that facilitate its entry into the cell, including but not limited to viral particles, liposomes, or protein envelopes.

The term "recombinant expression vector" in the present invention is a nucleic acid molecule encoding a gene, which is expressed in a host cell and contains the necessary elements to control the expression of the gene. Typically, an expression vector comprises a transcription promoter, a gene of interest, and a transcription terminator.

The host cell in the present invention refers to a cell into which a vector comprising a segment of a nucleotide sequence encoding a protein or polypeptide of interest can be introduced for cloning or gene expression. Suitable host cells for cloning or expressing the DNA in the vectors herein are prokaryotes, yeast or higher eukaryote cells.

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

YX₈EGTFTSDVSSYLEX₂₂QAAX₂₆EFIX₃₀WLVX₃₄X₃₅X₃₆X₃₇；

wherein:

X₈selected from Aib, V, I, T, L, G or S;

X₂₂selected from G or E;

X₂₆is selected from K or R;

X₃₀selected from A, K, E or R;

X₃₄is selected from R or G;

X₃₅is selected from R or G;

X₃₆is selected from R or G;

X₃₇is selected from R or G;

and X₂₆And X₃₀Only one of which is K.

Preferably, said X₈Selected from V, X₂₂Selected from E, X₂₆Selected from R, X₃₀Selected from K, X₃₄Selected from R, X₃₅Is selected from G.

More preferably, (1) said X₃₆Selected from R, X₃₇Is selected from G; or, (2) X₃₆Selected from G, X₃₇Is selected from R.

When the GLP-1(7-37) analogue is [ Tyr⁷Val⁸Glu²²Arg²⁶Lys³⁰Arg³⁴]When GLP-1(7-37) is used, the amino acid sequence is shown in SEQ ID NO. 1.

When the GLP-1(7-37) analogue is [ Tyr⁷Val⁸Glu²²Arg²⁶Lys³⁰Arg³⁴Gly³⁶Arg³⁷]When GLP-1(7-37) is used, the amino acid sequence is shown in SEQ ID NO. 2.

In another aspect, the invention provides an acylated long acting GLP-1(7-37) derivative comprising a fatty acid side chain attached to the K residue of said GLP-1(7-37) analog, preferably via the epsilon amino group on the K residue.

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)_nCO-wherein 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)₁₄CO-、HOOC(CH₂)₁₅CO-、HOOC(CH₂)₁₆CO-、HOOC(CH₂)₁₇CO-、HOOC(CH₂)₁₈CO-、HOOC(CH₂)₁₉CO-、HOOC(CH₂)₂₀CO-、HOOC(CH₂)₂₁CO-or HOOC (CH)₂)₂₂CO-, preferably the fatty acid side chain structure is HOOC (CH)₂)₁₆CO-。

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

As a preferred embodiment of the present invention, said fatty acid side chain is linked to the epsilon amino group of amino acid K at position 30 on said GLP-1(7-37) analog via a linker.

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, n is 1 or 2, preferably n is 1.

The above-mentioned preferred linker moiety (when n is 1) may be represented by γ -Glu-OEG-OEG according to IUPAC nomenclature; wherein OEG is "2- [2- (2-aminoethoxy) ethoxy]Abbreviation for acetyl ". When HOOC (CH) is selected₂)₁₆When 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 a specific embodiment of the present invention, the derivative of the present invention comprises a fatty acid side chain attached to the epsilon amino group of lysine at position 30 of said GLP-1(7-37) analog, preferably said fatty acid side chain is HOOC (CH)₂)₁₆CO-, the fatty acid side chain is linked to the epsilon amino group of lysine at position 30 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-ε³⁰- [2- (2- [2- (2- [2- (2- [4- (17-carboxyheptadecanoylamino) -4(S) -carboxybutanoylamino)]Ethoxy) ethoxy]Acetylamino) ethoxy]Ethoxy) acetyl group][Tyr⁷Val⁸Glu²²Arg²⁶Lys³⁰Arg³⁴]GLP-1(7-37) (abbreviated as HS-Y1), [ Tyr⁷Val⁸Glu²²Arg²⁶Lys³⁰Arg³⁴]The amino acid sequence of GLP-1(7-37) is shown in SEQ ID NO. 1; or

N-ε³⁰- [2- (2- [2- (2- [2- (2- [4- (17-carboxyheptadecanoylamino) -4(S) -carboxybutanoylamino)]Ethoxy) ethoxy]Acetylamino) ethoxy]Ethoxy) acetyl group][Tyr⁷Val⁸Glu²²Arg²⁶Lys³⁰Arg³⁴Gly³⁶Arg³⁷]GLP-1(7-37) (abbreviated as HS-Y2), [ Tyr⁷Val⁸Glu²²Arg²⁶Lys³⁰Arg³⁴Gly³⁶Arg³⁷]The amino acid sequence of GLP-1(7-37) is shown in SEQ ID NO. 2.

In general, histidine H at position 7 in the amino acid sequence of GLP-1(7-37) is believed to be critical for maintaining its GLP-1 activity, and thus there were no changes to this site in numerous GLP-1(7-37) -based studies. However, the inventor of the application unexpectedly finds that the site is mutated into tyrosine Tyr in research, so that the activity of GLP-1 is retained, and the activity is enhanced in aspects of weight loss and blood sugar reduction. Perhaps under the influence of changes in the amino acid sequence at other positions, the changes at that position instead synergistically potentiate its GLP-1 activity. Therefore, based on the discovery, the invention obtains a group of drugs which are expected to be more effective in reducing blood sugar and weight.

On the other hand, the invention provides a recombinant engineering bacterium for highly expressing the GLP-1(7-37) analogue, wherein the recombinant engineering bacterium is preferably a recombinant Escherichia coli engineering bacterium, and more preferably a recombinant Escherichia coli BL21 engineering bacterium.

In another aspect, the invention provides a construction method of the recombinant engineering bacteria of the GLP-1(7-37) analogue, and the method comprises the following steps: (1) sequentially and serially fusing the inclusion body promoting sequence, the EK enzyme digestion sequence and the GLP-1(7-37) analogue coding gene sequence to prepare a gene expression fragment of the GLP-1(7-37) analogue; (2) inserting the gene expression fragment into a prokaryotic expression plasmid to obtain an expression plasmid of the GLP-1(7-37) analogue; (3) and transferring the expression plasmid into escherichia coli to prepare the recombinant engineering bacteria for expressing the GLP-1(7-37) analogue.

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

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

In vitro binding activity indicates:

(1) compared with the somaglutide, the acylated long-acting GLP-1 derivative provided by the invention has better GLP-1R binding affinity. A hypoglycemic experiment in a diabetes animal model also shows that the acylated long-acting GLP-1 derivative has a hypoglycemic effect similar to or remarkably superior to that of the somaglutide. The selection of the mutation site of the invention is proved to bring significant beneficial hypoglycemic activity to GLP-1.

(2) In addition, 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 also show that compared with the somaglutide, the GLP-1 derivative has obvious and beneficial weight loss effect in the diabetic animal model, and does not have the risk of hypoglycemia. Therefore, the long-acting GLP-1 derivative has wider commercial development value compared with the somaglutide.

Furthermore, the present invention provides a pharmaceutical composition comprising said acylated long-acting GLP-1 derivative or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

In another aspect, the invention provides the use of the acylated long-acting GLP-1 derivative, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, for the preparation of a medicament for the treatment of diabetes.

In another aspect, the invention provides the use of said acylated 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 equivalent or excellent blood sugar reducing capability in a diabetic patient, and the 7 th amino acid is modified into Y, so that the GLP-1 derivative has excellent enzymolysis resistance, excellent stability and half-life potential;

(2) compared with the somaglutide, the long-acting GLP-1 derivative provided by the invention has more excellent and obvious weight loss capability and better 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 mouse models;

each group of data corresponding to each time point in the figure is a blank control group, a model control group, a somarutide group, an HS-Y1 group and an HS-Y2 group from left to right;

FIG. 2 is a bar graph of the effect of long-acting GLP-1 derivatives on the rate of body weight change in a mouse model.

Detailed Description

In order that the above objects, features and advantages of the present invention may be more clearly understood, aspects 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 acylated 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-Y1):

(1) construction of the code [ Tyr⁷Val⁸Glu²²Arg²⁶Lys³⁰Arg³⁴]Expression plasmid for GLP-1(7-37)

Through a large number of previous researches and experiments, FKFEFKFE is 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 coding gene segment shown as SEQ ID NO. 3; the fragment is inserted into a prokaryotic expression plasmid pET-30a (+) through NdeI and XhoI sites and is sequenced and verified to obtain an expression plasmid which is called pET-30a (+) - [ Tyr +⁷Val⁸Glu²²Arg²⁶Lys³⁰Arg³⁴]-GLP-1(7-37)。

(2) Construction of expression [ Tyr⁷Val⁸Glu²²Arg²⁶Lys³⁰Arg³⁴]Recombinant engineering bacterium of (7-37) GLP-1

50 μ L of BL21 competent cells (TransGenBiotech) were thawed on an ice bath, the expression plasmid constructed in step (1) was added and shaken up, placed in an ice bath for 30min, heat-shocked in a water bath at 42 ℃ for 30s, and then the centrifuge tubes were quickly transferred into an ice bath for 2min without shaking the centrifuge tubes.

Adding 500 mu L of antibiotic-free sterile LB culture medium into a centrifuge tube, uniformly mixing, and culturing at 37 ℃ and 180rpm for 1h to recover bacteria; then, 200. mu.L of transformed competent cells were pipetted and applied to a plate of LB agar medium containing kanamycin resistance, the cells were spread out uniformly, the plate was placed at 37 ℃ until the liquid was absorbed, the plate was inverted, cultured overnight at 37 ℃, a single colony in the transformation plate was picked up using an inoculating loop and inoculated in 15mL of a sterile LB medium containing kanamycin antibiotic, cultured overnight at 37 ℃, 500. mu.L of overnight culture broth was added to a 1.5mL sterile centrifuge tube, and 500. mu.L of 50% sterile glycerol was added and mixed uniformly to obtain glycerol cryopreserved cells, which were stored at-80 ℃.

(3) Fermentation expression of recombinant engineering bacteria

Adding 50 μ L glycerol into 50mL 2YT culture medium, freezing to obtain bacterial liquid, adding 50 μ L kanamycin, mixing, placing in a constant temperature oscillator, culturing at 37 deg.C and 200rpm overnight, and measuring OD600 > 5.0 to obtain first-grade seed culture solution.

And (3) taking 40mL of the 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 a 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, leading the final concentration to be 1mmol/L, carrying out induced culture at 30 ℃ for 24h, finishing the culture, and putting the tank for centrifugation.

Centrifuging the collected bacteria liquid for 30min at 8000g to obtain thallus cytoplasm to obtain not less than 290g thallus/L fermentation liquid, and measuring the expression amount of target protein in the thallus obtained by centrifugation, wherein the expression amount is not less than 10 g/L.

(4) Recombinant [ Tyr⁷Val⁸Glu²²Arg²⁶Lys³⁰Arg³⁴]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 in an ultrasonic cell crusher for 30min to crush the cells, centrifuging the obtained homogenate at 13000g for 30min at 4 ℃, collecting a precipitate after the centrifugation is finished, and dissolving the precipitate by using 8M urea to obtain a sample before enzyme digestion.

The GLP-1 intermediate product prepared by the purification process is analyzed by RP-HPLC, and the purity of the GLP-1 intermediate product is higher than 70 percent by the steps of concentrating a sample before enzyme digestion by UniPS30-300 (purchased from Suzhou Nami micro technology Co., Ltd.) which is balanced by equilibrium liquid 3(10mM ammonium acetate and 20 percent acetonitrile), eluting by equilibrium liquid 3, eluting by a gradient of 0-100 percent eluent (10mM ammonium acetate and 80 percent acetonitrile), and purifying by the purification process.

The tag sequence was cleaved using EK enzyme: adding 20mM PB buffer solution with pH7.4 into the intermediate product to dilute the intermediate product three times, adding EK enzyme according to EK enzyme: intermediate product: 1:15 at 20 ℃, uniformly mixing, performing enzyme digestion overnight, and analyzing the enzyme digestion rate by RP-HPLC to be approximately 80%.

[Tyr⁷Val⁸Glu²²Arg²⁶Lys³⁰Arg³⁴]-purification of GLP-1 (7-37): UniPS30-300 (from Sozhou, Nami, Inc.) equilibrated with equilibration solution 3(10mM ammonium acetate, 20% acetonitrile) was concentrated, and after elution with equilibration solution 3, the product was eluted with a gradient of 0-100% eluent (10mM ammonium acetate, 80% acetonitrile) and analyzed for purity of about 90% by RP-HPLC.

0.2M Na was added to the eluted sample₂HPO₄Adjusting pH to 4.8-5.0 with 1M citric acid to obtain final concentration of 20mM, acid precipitating at 4 deg.C overnight, detecting with RP-HPLC with yield over 90%, centrifuging at 13000g for 30min at 4 deg.C, collecting precipitate, storing at-20 deg.C to obtain [ Tyr ℃⁷Val⁸Glu²²Arg²⁶Lys³⁰Arg³⁴]-GLP-1 (7-37); and sequencing verification shows that the amino acid sequence of the polypeptide is shown as SEQ ID NO. 1.

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

Fatty acid modification: tyr obtained in step (4)⁷Val⁸Glu²²Arg²⁶Lys³⁰Arg³⁴]Adding water into GLP-1(7-37) to prepare a 4-6 mg/mL solution, 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 the 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 in acetonitrile, mixing the polypeptide sample with the fatty acid solution, and heating the mixture at 4 deg.CStanding for one hour, diluting the sample with water 5 times, adjusting pH to 4.8 with 1M citric acid (or 10% acetic acid) to terminate the reaction, standing at 4 deg.C for acid precipitation for 10min, centrifuging at 13000g at 4 deg.C for 30min, and storing the precipitate at-80 deg.C.

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

The reaction solution after the reaction was terminated was pumped into UniPS10-300 (purchased from Suzhou Naichi Microscience Co., Ltd.) equilibrated with equilibration solution 3(10mM ammonium acetate, 20% acetonitrile) at a flow rate of 4mL/min by using a protein purification chromatography system (Seikagaku SDL100) to concentrate, after elution with equilibration solution 3, the eluate was eluted at a gradient of 0-100% (10mM ammonium acetate, 80% acetonitrile) and the eluate was collected and the purity was about 90% by RP-HPLC.

Diluting the peak with water by 3 times, adjusting pH to 4.80 by acid precipitation, precipitating at 4 deg.C for 30min, centrifuging, adding PBST buffer (pH7.0) into the precipitate for redissolving, and freezing at-80 deg.C to obtain N-epsilon³⁰- [2- (2- [2- (2- [2- (2- [4- (17-carboxyheptadecanoylamino) -4(S) -carboxybutanoylamino)]Ethoxy) ethoxy]Acetylamino) ethoxy]Ethoxy) acetyl group][Tyr⁷Val⁸Glu²²Arg²⁶Lys³⁰Arg³⁴]GLP-1(7-37) (abbreviated HS-Y1).

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 gene containing the coding [ Tyr⁷Val⁸Glu²²Arg²⁶Lys³⁰Arg³⁴Gly³⁶Arg³⁷]-a gene fragment of GLP-1(7-37) having the sequence shown in SEQ ID No. 4; the remaining steps were carried out as in this example to prepare HS-Y2.

Example 2: in vitro cell affinity activity assay

(1) Different long-acting GLP-1 derivative injection prepared

The specific formula is as follows: DMEM blank medium, GLP-1 derivatives: 320nM, 64nM, 12.8nM, 2.56nM, 0.512nM, 0.1024nM, 0.02048nM, 0.004096 nM. The positive control group (Somalutide), the experimental group 1(HS-Y1) and the experimental group 2(HS-Y2) are included.

Selecting HEK293/Luc/GLP1R cells with good culture state, discarding culture solution in the bottle, washing with PBS buffer solution for 1 time, adding 0.05% Trypsin digestive juice for digesting for 3 minutes, adding DMEM basal medium to terminate the digestion, and centrifuging to collect the cells. DMEM blank medium was used to adjust the cell density to 8.0X 10⁵50. mu.L/well of cells were seeded in 96-well cell culture plates at 37 ℃ in 5% CO₂Incubated under conditions overnight.

In vitro activity of derivatives of GLP-1 analogs was tested using Fire-Lumi luciferase assay kit: preparing a determination culture solution, diluting a sample to 320nM step by using a DMEM blank medium, wherein the single dilution multiple does not exceed 10 times, and then performing 5-time serial dilution in a 96-well plate to obtain 8 gradients, wherein each dilution is performed by 2 duplicate wells.

The cultured cell culture plate was taken out from the incubator, and 50. mu.L of diluted assay medium was added to the cell plate at 37 ℃ with 5% CO₂Incubate for 6h under conditions. The sample plate was removed from the incubator and allowed to stand at room temperature. Adding 100 mu L Fire-Lumi detection solution, reacting for 5min, shaking for 10s, and detecting fluorescence intensity.

The test data is processed by a four-parameter regression calculation method, and the EC50 value of the sample to be tested can be calculated. The results are shown in table 1:

TABLE 1

Sample (I)	HS-Y1	HS-Y2	Somazutide
				EC50(nM)	0.336	0.1746	0.6611

From the results in the table, it can be seen that HS-Y1 and HS-Y2 of the present invention have lower EC50 values than thaumalu peptide, and HS-Y1 and HS-Y2 are about 1/2 and 1/4 of thaumalu peptide, respectively, indicating that they have about 2-fold and 4-fold better binding affinity to human insulin receptor than thaumalu peptide.

Example 3: study of hypoglycemic Effect in mice

(1) Experimental materials:

a. experimental pharmaceutical formulation:

respectively preparing injections of different GLP-1 derivatives, wherein the specific formula is 1.133mg/mLNa₂HPO₄5.5mg/mL phenol, 14.0mg/mL propylene glycol, and GLP-1 derivatives;

b. animal experiments:

selecting 50 healthy SPF male KM mice with the age of 6-8 weeks, weighing 18-20g, and dividing into a blank control group, a model control group, a positive control group (a somatid group), an example 1 group and an example 2 group, wherein:

blank control group: comprises 10 mice, and is prepared by intragastric administration of pure water and subcutaneous injection of a blank solvent into abdomen;

model control group: containing 10 mice, glucose (4g/kg) was gavaged, and a blank was injected subcutaneously into the abdomen;

positive control group: comprises 10 mice, glucose (4g/kg) is used for gastric lavage, and the abdomen is injected with medicine subcutaneously;

example 1 group: comprises 10 mice, glucose (4g/kg) is perfused into stomach, and the abdomen is injected with medicine subcutaneously;

example 2 group: 10 mice were included, and the stomach was perfused with glucose (4g/kg) and the drug was injected ventrally, subcutaneously.

(2) The experimental method comprises the following steps:

a. the administration method comprises the following steps:

the mice of each experimental group identified above were dosed according to the specific dosing schedule shown in table 2:

TABLE 2

b. Blood sugar value detection:

fasting for 16h before blood glucose testing, blood glucose for 0h before oral glucose after administration, and blood glucose for 0.5h, 1h, 2h after oral glucose, followed by oral glucose for 23h and blood glucose for 1h after oral glucose, were performed, and the results are shown in table 3 and fig. 1:

TABLE 3

Group of	0h	0.5h	1h	2h	24h
						Blank control group	4.51±0.55	7.01±1.24**	6.01±1.14**	4.73±0.69**	10.83±1.13*
Model control group	4.49±0.54	18.76±3.44	15.98±3.15	9.21±1.12	12.26±1.84
						Positive control group	4.32±0.45	10.94±2.88**	6.21±1.39**	4.32±0.79**	8.95±1.94**
EXAMPLE 1 group	4.4±0.74	8.4±4.03**	4.91±1.5**	3.69±0.49**	8.42±1.43**
						EXAMPLE 2 group	4.23±0.54	10.24±4.53**	5.66±1.67**	4.52±0.75**	9.07±2.46**

Note: "" means, p < 0.05 relative to model control; ". indicates, p < 0.01 relative to model control.

FIG. 1 is a graph showing the trend of blood glucose change of experimental mice of this example, wherein each time point is a blank control group, a model control group, a positive control group (Somalutide), an example 1 group (HS-Y1) and an example 2 group (HS-Y2) from left to right. As can be seen from figure 1, the HS-Y1 and HS-Y2 of the invention have better blood sugar control capability than thaumalutide in the whole experiment.

Example 4: study of weight loss Effect in mice

(1) Experimental materials

a. Experimental formulation:

GLP-1 derivative injection (HS-Y1 and HS-Y2) is prepared, and the specific formula is 1.133mg/mLNa₂HPO₄5.5mg/mL phenol, 14.0mg/mL propylene glycol, and GLP-1 derivative 0.045 mg/mL.

b. Animal experiments:

selecting healthy SPF male KM mice with the age of 6-8 weeks, weighing 18-20g, and dividing into normal diet group (ND group) and high fat diet group (HFD group), wherein the ND group is fed with common maintenance diet, the HFD group is fed with 60% high fat diet, and the feeding period is 12-18 weeks;

detecting the weight of an HFD group, screening unmolded mice, randomly grouping the remaining mice according to the weight, and dividing each group into 5 mice, namely a model control group (blank solvent), a positive control group (Somalutide, 0.45mg/kg), an experimental group 1(HS-Y1, 0.45mg/kg), an experimental group 2(HS-Y1, 0.75mg/kg), an experimental group 3(HS-Y2, 0.45mg/kg) and an experimental group 4(HS-Y2, 0.75 mg/kg);

(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 4:

TABLE 4

b. Detection indexes are as follows:

weight: detecting the body weight at each administration;

(3) results of the experiment

The test results of this example are shown in table 5, corresponding to the bar chart shown in fig. 2:

TABLE 5

As shown in Table 5 and FIG. 2, HS-Y1 and HS-Y2 of the present invention change H at position 7 in the amino acid sequence to Y, but unexpectedly maintain good GLP-1 activity, have good weight loss ability, and have better effect than thaumalutide.

It is noted that, in this document, relational terms such as "first" and "second," and the like, may be 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

<110> Jilin Hui Sheng biopharmaceutical Co., Ltd

<120> an acylated long-acting GLP-1 derivative

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Gln Ala Ala Arg Glu Phe Ile Lys Trp Leu Val Arg Gly Arg Gly

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Thr Thr Cys Ala Ala Ala Thr Thr Thr Gly Ala Ala Thr Thr Thr Ala

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Ala Ala Thr Thr Thr Gly Ala Ala Gly Ala Thr Gly Ala Cys Gly Ala

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Thr Gly Ala Thr Ala Ala Ala Thr Ala Thr Gly Thr Gly Gly Ala Ala

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Gly Gly Cys Ala Cys Cys Thr Thr Cys Ala Cys Gly Ala Gly Cys Gly

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Ala Cys Gly Thr Gly Ala Gly Thr Ala Gly Cys Thr Ala Thr Cys Thr

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Gly Gly Ala Gly Gly Ala Ala Cys Ala Ala Gly Cys Gly Gly Cys Gly

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Cys Gly Cys Gly Ala Ala Thr Thr Thr Ala Thr Thr Ala Ala Ala Thr

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Gly Gly Thr Thr Ala Gly Thr Gly Cys Gly Cys Gly Gly Cys Cys Gly

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Thr Thr Cys Ala Ala Ala Thr Thr Thr Gly Ala Ala Thr Thr Thr Ala

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Cys Gly Ala Thr Ala Ala Ala Thr Ala Thr Gly Thr Gly Gly Ala Ala

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Ala Thr Gly Thr Gly Ala Gly Cys Ala Gly Cys Thr Ala Thr Cys Thr

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130

Claims

1. An acylated 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:

YX₈EGTFTSDVSSYLEX₂₂QAAX₂₆EFIX₃₀WLVX₃₄X₃₅X₃₆X₃₇；

wherein:

X₈selected from Aib, V, I, T, L, G or S;

X₂₂selected from G or E;

X₂₆is selected from K or R;

X₃₀selected from A, K, E or R;

X₃₄is selected from R or G;

X₃₅is selected from R or G;

X₃₆is selected from R or G;

X₃₇is selected fromR or G;

and X₂₆And X₃₀Is K, said long-acting GLP-1 derivative comprising a fatty acid side chain attached to the amino acid K in position 26 or position 30 of said GLP-1(7-37) analogue.

2. The acylated long-acting GLP-1 derivative or a pharmaceutically acceptable salt thereof according to claim 1, wherein said X₈Selected from V, X₂₂Selected from E, X₂₆Selected from R, X₃₀Selected from K, X₃₄Selected from R, X₃₅Is selected from G.

3. An acylated long-acting GLP-1 derivative according to claim 2, or a pharmaceutically acceptable salt thereof, characterized in that X₃₆Selected from R, X₃₇Is selected from G; or, X₃₆Selected from G, X₃₇Is selected from R.

4. An acylated long-acting GLP-1 derivative or a pharmaceutically acceptable salt thereof, according to any one of claims 1-3, wherein said derivative is attached to the fatty acid side chain via the epsilon amino group on the amino acid K residue.

5. The acylated long-acting GLP-1 derivative or a pharmaceutically acceptable salt thereof according to claim 4, wherein said fatty acid side chain is selected from HOOC (CH)₂)₁₄CO-、HOOC(CH₂)₁₅CO-、HOOC(CH₂)₁₆CO-、HOOC(CH₂)₁₇CO-、HOOC(CH₂)₁₈CO-、HOOC(CH₂)₁₉CO-、HOOC(CH₂)₂₀CO-、HOOC(CH₂)₂₁CO-or HOOC (CH)₂)₂₂CO-。

6. The acylated long-acting GLP-1 derivative or a pharmaceutically acceptable salt thereof, according to claim 5, wherein said fatty acid side chain is attached to amino acid K through a linker.

7. The acylated long-acting GLP-1 derivative or a pharmaceutically acceptable salt thereof, according to claim 6, wherein said linker is:

wherein s and n are both 1.

8. An acylated long-acting GLP-1 derivative, or a pharmaceutically acceptable salt thereof, according to claim 7, wherein said long-acting GLP-1 derivative is:

N-ε³⁰- [2- (2- [2- (2- [2- (2- [4- (17-carboxyheptadecanoylamino) -4(S) -carboxybutanoylamino)]Ethoxy) ethoxy]Acetylamino) ethoxy]Ethoxy) acetyl group][Tyr⁷Val⁸Glu²²Arg²⁶Lys³⁰Arg³⁴]-GLP-1(7-37)，[Tyr⁷Val⁸Glu²²Arg²⁶Lys³⁰Arg³⁴]The amino acid sequence of GLP-1(7-37) is shown in SEQ ID NO. 1; or

N-ε³⁰- [2- (2- [2- (2- [2- (2- [4- (17-carboxyheptadecanoylamino) -4(S) -carboxybutanoylamino)]Ethoxy) ethoxy]Acetylamino) ethoxy]Ethoxy) acetyl group][Tyr⁷Val⁸Glu²²Arg²⁶Lys³⁰Arg³⁴Gly³⁶Arg³⁷]-GLP-1(7-37)，[Tyr⁷Val⁸Glu²²Arg²⁶Lys³⁰Arg³⁴Gly³⁶Arg³⁷]The amino acid sequence of GLP-1(7-37) is shown in SEQ ID NO. 2.

9. A recombinant engineering bacterium for expressing GLP-1(7-37) analogues, which is characterized in that the sequence of the GLP-1(7-37) analogues is as follows:

YX₈EGTFTSDVSSYLEX₂₂QAAX₂₆EFIX₃₀WLVX₃₄X₃₅X₃₆X₃₇；

wherein:

X₈selected from Aib, V, I, T, L, G or S;

X₂₂is selected from G or E;

X₂₆is selected from K or R;

X₃₀selected from A, K, E or R;

X₃₄is selected from R or G;

X₃₅is selected from R or G;

X₃₆is selected from R or G;

X₃₇is selected from R or G, and X₂₆And X₃₀Only one of them is K;

the recombinant engineering bacteria are recombinant escherichia coli engineering bacteria, and the construction method of the recombinant engineering bacteria comprises the following steps:

(1) sequentially and serially fusing the inclusion body promoting sequence, the EK enzyme digestion sequence and the GLP-1(7-37) analogue coding gene sequence to obtain a gene expression fragment of the GLP-1(7-37) analogue;

(2) inserting the gene expression fragment into a prokaryotic expression plasmid to obtain an expression plasmid of the GLP-1(7-37) analogue;

(3) and transferring the expression plasmid into escherichia coli to obtain the recombinant engineering bacteria for expressing the GLP-1(7-37) analogue.

10. Use of an acylated long-acting GLP-1 derivative of any one of claims 1 to 8, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of diabetes or weight loss.