CN114621340B - Acylated long-acting GLP-1 derivative - Google Patents
Acylated long-acting GLP-1 derivative Download PDFInfo
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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
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 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 2 diabetes, with the majority of patients being type 2 diabetes patients (statistically, about 90%). Type 2 diabetes (diabetes mellitus type 2, T2DM), old-called non-insulin dependent diabetes mellitus (NIDDM) or adult-onset diabetes (adult-offset diabetes), patients are characterized by hyperglycemia, relative lack of insulin, insulin resistance, etc. At present, clinically used medicaments for treating type 2 diabetes 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 type 2 diabetes.
GLP-1 derivatives belong to GLP-1 receptor agonists, and are a class of secretin drugs, secretin is a hormone which is secreted into blood by intestinal cells after food stimulation and can stimulate insulin secretion, the insulin secretion capacity caused by the secretin accounts for 50-70% of the total insulin secretion amount, and the function of stimulating insulin secretion has the characteristic of glucose concentration dependence.
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 analogues also have multiple functions of glucose-dependent insulinotropic secretion and biosynthesis, glucagon secretion inhibition, gastric emptying inhibition and the like (Dugang, Long, Xuhuman glucagon-like peptide 1 and receptor agonist research progress [ J ] Tianjin medicine, 2012, 40(2):181- > 184.).
The research results of 10 patients with type 2 diabetes 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 patient does not increase the insulin level and the blood sugar level is stable and does not further decrease although GLP-1 is continuously infused, which indicates that GLP-1 is an enterogenic hormone which is released into the blood under the stimulation of nutrient substances (especially carbohydrate), the insulin secretion promotion effect of the enterogenic hormone is glucose concentration dependent, and the insulin secretion promotion effect can play a role of reducing the blood sugar when the blood sugar level is increased, inhibiting the secretion of glucagon, increasing the satiety and reducing the hunger (Normalization of the stimulating hyperglycemic by exogenous glucose-like peptide 1(7-36amide) in type 2(non-insulin-dependent) diabetes polypeptides ([ 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 2 diabetes: 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.
At present, GLP-1 derivatives are marketed mainly as exenatide, liraglutide, dulaglutide, lissamide, exenatide microsphere formulations, albiglutide, macrogol loxapide and somaglutide (also named semaglutide). Wherein the Somalide is a representative of GLP-1 derivative medicaments.
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 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.
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. Through long-term research in the laboratory, a series of GLP-1 derivatives with the potential of reducing blood sugar and weight better than the somaglutide are developed. Accordingly, an object of the present invention is to provide a GPL-1 derivative 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 loss and blood sugar reduction capability, 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, [ Val8] GLP-1(7-37) refers to GLP-1(7-37) analogs in which the naturally occurring histidine at position 8 has been substituted with valiVal.
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 ε26 - ((4S) -4- (Hexadecanoylamino) -carboxy-butyryl) [ Arg34, Lys26]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 nucleotide sequence fragment 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 invention provides a GLP-1(7-37) analog consisting of a polypeptide having an amino acid sequence represented by the formula:
HX 8 EGTFTSDVSSYLEEQAAREFIKWLVRRGG;
wherein X 8 Selected from V, I, T, L, G or S; preferably, X 8 Selected from V, I, T.
In another aspect, the invention provides a long-acting GLP-1(7-37) derivative comprising fatty acid side chains attached to the K residues of said GLP-1(7-37) analogs, respectively, preferably via the epsilon amino group on the K residue.
When the GLP-1(7-37) analog is Val 8 Glu 22 Arg 26 Lys 30 Arg 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. 1.
When the GLP-1(7-37) analogue is Ile 8 Glu 22 Arg 26 Lys 30 Arg 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. 2.
When the GLP-1(7-37) analogue is Thr 8 Glu 22 Arg 26 Lys 30 Arg 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.
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 chains 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 amino acid residue through a linker.
As a preferred embodiment of the present invention, said fatty acid side chain is linked to the epsilon amino group of Lys 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 and n is 1 or 2.
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 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 a specific embodiment of the present invention, said 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, 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 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-ε 30 - [2- (2- [2- (2- [2- (2- [4- (17-carboxyheptadecanoylamino) -4(S) -carboxybutanoylamino)]Ethoxy) ethoxy]Acetylamino) ethoxy]Ethoxy) acetyl group][Val 8 Glu 22 Arg 26 Lys 30 Arg 34 Arg 35 Gly 36 ]GLP-1(7-37) (abbreviated as HS-G5), the amino acid sequence of which is shown in SEQ ID NO. 1; or
N-ε 30 - [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 30 Arg 34 Arg 35 Gly 36 ]GLP-1(7-37) (abbreviated as HS-G6), the amino acid sequence of which is shown in SEQ ID NO. 2; or
N-ε 30 - [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 30 Arg 34 Arg 35 Gly 36 ]GLP-1(7-37) (abbreviated as HS-G7), the amino acid sequence of which is shown in SEQ ID NO. 3.
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 connecting fatty diacid side chains through acylation at specific positions has more excellent blood sugar-reducing and weight-losing activity through partial site change.
On the other hand, 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 another aspect, the invention provides a construction method of the recombinant engineering bacteria of the GLP-1 analogue, which comprises the following steps: (1) sequentially serially fusing the inclusion body promoting sequence, the EK enzyme digestion sequence and the GLP-1 analogue coding gene sequence 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.
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.4 to 6.
The in vitro binding activity shows that compared with the somaglutide, the long-acting GLP-1 derivative provided by the invention has better GLP-1R binding affinity. The blood sugar reduction experiment in a diabetes animal model also shows that the long-acting GLP-1 derivative has the blood sugar reduction effect which is obviously 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. 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.
In addition, the invention provides a pharmaceutical composition, which comprises the long-acting GLP-1 derivative or the pharmaceutically acceptable salt thereof and pharmaceutically acceptable auxiliary materials.
In another aspect, the invention provides the use of said 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 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 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 or technical solutions in the prior art of the present invention, 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 to obtain other drawings without inventive labor.
FIG. 1 is a bar graph of the effect of long-acting GLP-1 derivatives on blood glucose changes in diabetic mouse models: in the figure, for each group of data, a model control group, a somalutide group, an HS-G5 group, an HS-G6 group and an HS-G7 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 weight change in diabetic mouse models; in the figure, a model control group, a somarutide group, an HS-G5 group, an HS-G6 group and an HS-G7 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 feeding inhibition in a mouse model of diabetes; in the figure, the sequence from left to right is a somalutide group, an HS-G5 group, an HS-G6 group and an HS-G7 group.
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 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-G5):
(1) construction of coded Val 8 Glu 22 Arg 26 Lys 30 Arg 34 Arg 35 Gly 36 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 gene segment shown as SEQ ID NO. 4; the fragment is inserted into a prokaryotic expression plasmid pET-30a (+) through NdeI and XhoI sites and is subjected to sequencing verification to obtain an expression plasmid called pET-30a (+) -Val 8 Glu 22 Arg 26 Lys 30 Arg 34 Arg 35 Gly 36 -Glp-1(7-37)。
(2) Construction of expression Val 8 Glu 22 Arg 26 Lys 30 Arg 34 Arg 35 Gly 36 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 the 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 left at 37 ℃ until the liquid was absorbed, the plate was inverted, cultured overnight at 37 ℃, and a single colony in the transformed plate was picked up using an inoculating loop and inoculated in 15mL of a sterile LB medium containing kanamycin antibiotic and cultured overnight at 37 ℃. Adding 500 μ L of overnight culture bacteria solution into 1.5ml sterile 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 μ L glycerol frozen bacteria solution into 50mL 2YT culture medium, adding 50 μ L kanamycin, mixing, placing in constant temperature oscillator, culturing at 37 deg.C and 200rpm overnight, and measuring OD600 > 5.0 to obtain first-grade seed culture solution.
Taking 40mL of first-level seed culture solution cultured overnight, inoculating 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, 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 bacterial liquid for 30min at 8000g to obtain thallus cytoplasm, wherein the thallus cytoplasm is not less than 290g of thallus/L fermentation liquid, and measuring the expression amount of the target protein by using the thallus obtained by centrifugation, wherein the expression amount is not less than 10 g/L.
(4) Recombinant Val 8 Glu 22 Arg 26 Lys 30 Arg 34 Arg 35 Gly 36 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%.
Val 8 Glu 22 Arg 26 Lys 30 Arg 34 Arg 35 Gly 36 -purification of GLP-1 (7-37): using equilibration solution 3(10mM ammonium acetate, 2)0% acetonitrile), concentrated, and the equilibrium solution 3 washed, then eluted with a gradient of 0-100% eluent (10mM ammonium acetate, 80% acetonitrile) with an RP-HPLC purity of about 90%.
0.2M Na was added to the eluted sample 2 HPO 4 Adjusting pH to 4.8-5.0 with 1M citric acid to give final concentration of 20mM, acid precipitating at 4 deg.C overnight, detecting by RP-HPLC with yield of above 90%, centrifuging at 13000g for 30min at 4 deg.C, collecting precipitate, storing at-20 deg.C to obtain Val 8 Glu 22 Arg 26 Lys 30 Arg 34 Arg 35 Gly 36 -GLP-1 (7-37); and (3) sequencing verification, wherein 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: val obtained in step (4) 8 Glu 22 Arg 26 Lys 30 Arg 34 Arg 35 Gly 36 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 a molar ratio of the polypeptide to the 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 of the sample to 4.8 by using 1M citric acid (or 10% acetic acid) to terminate the reaction, standing the sample at 4 ℃ for acid precipitation for 10min, centrifuging the sample at 4 ℃ for 30min by 13000g after the acid precipitation, and then storing the precipitate at-80 ℃.
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 for 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, dissolving, and freezing at-80 deg.C to obtain N-epsilon 30 - [2- (2- [2- (2- [2- (2- [4- (17-carboxyheptadecanoylamino) -4(S) -carboxybutanoylamino group)]Ethoxy) ethoxy]Acetylamino) ethoxy]Ethoxy) acetyl group][Val 8 Glu 22 Arg 26 Lys 30 Arg 34 Arg 35 Gly 36 ]GLP-1(7-37) (abbreviated as HS-G5).
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 respectively obtain the coding Ile 8 Glu 22 Arg 26 Lys 30 Arg 34 Arg 35 Gly 36 GLP-1(7-37) and Thr 8 Glu 22 Arg 26 Lys 30 Arg 34 Arg 35 Gly 36 -GLP-1(7-37) gene segments, the nucleotide sequences of which are shown in SEQ ID No.5-6, respectively; the remaining steps were the same, producing HS-G6 and HS-G7.
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: DMEM blank medium, GLP-1 derivatives: 320nM, 64nM, 12.8nM, 2.56nM, 0.512nM, 0.1024nM, 0.02048nM, 0.004096 nM. The test results comprise a positive control group (Somalutide), an experimental group 1(HS-G5), an experimental group 2(HS-G6) and an experimental group 3 (HS-G7).
Selecting HEK293/Luc/GLP1R cells in good culture state, discarding culture solution in bottle, washing with PBS buffer solution for 1 time, adding 0.05% Trypsin digestive juice, and digesting for 3 minAfter a while, the digestion was terminated by adding DMEM basal medium, and the cells were collected by centrifugation. The cell density was adjusted to 8.0X 10 with DMEM blank medium 5 50. mu.L/well of each of the cells were plated in 96-well cell culture plates at 37 ℃ with 5% CO 2 Incubated 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 by steps by a DMEM blank culture medium, wherein the single dilution multiple does not exceed 10 times; then, 5-fold serial dilutions were performed in 96-well plates for 8 gradients, with 2 duplicate wells for each dilution.
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% CO 2 Incubate under conditions for 6 hr. The sample plate was removed from the incubator and allowed to stand at room temperature. Adding 100ul 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 2:
TABLE 2
Sample (I) | HS-G5 | HS-G6 | HS-G7 | Somalide |
EC50(nM) | 0.405 | 0.399 | 0.464 | 0.734 |
As can be seen from the results in the table, the HS-G5, HS-G6 and HS-G7 of the present invention have lower EC50 values than the thaumareuptade, indicating that they have better binding affinity to human insulin receptor than thaumareuptade.
Example 3: study of hypoglycemic Effect in db/db mice
(1) Experimental materials:
the experimental animals are: BKS-Lepr REM A number of 25/Gpt mice, 6 weeks old, 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 6-week BKS-Lepr male SPF grade REM 25 mice/Gpt were divided into 5 groups at random according to body weight and blood glucose, each group was provided with 5 test animals, which were respectively a model control group (solvent), a positive control group (somaglutide), an experimental group 1(HS-G5), an experimental group 2(HS-G6), and an experimental group 3 (HS-G7); 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: the blood glucose levels before and 2h after the first administration were measured, and the blood glucose levels 0h before and 2h after each administration were measured for the following three administrations. Pre-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 last dose and is also 0h before the current dose.
(3) Results of the experiment
Statistics of experimental data are shown in Table 4, which correspond to the histogram shown in FIG. 1, where each set of data is, from left to right, a model control, Somalutide, HS-G5, HS-G6, and HS-G7.
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; the blood sugar can be controlled within the ideal range in the whole administration period.
Example 4: study of weight loss Effect in db/db mice
(1) Experimental materials:
the product is BKS-Lepr REM Gpt mice, 25 in number, 6 weeks old, 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 method
a. Modeling and grouping:
selecting 25 SPF male BKS-LeprRem/Gpt mice at 6 weeks, randomly grouping according to weight and blood sugar, dividing into 5 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-G5), an experimental group 2(HS-G6) and an experimental group 3 (HS-G7). 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.
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 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 was recorded, and the results are shown in table 5, and fig. 2 is a graph of the weight change rate of the mice for 9 days after administration.
TABLE 5
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.
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
Sample (I) | Model control group | Positive control group | Experimental group 1 | Experimental group 2 | Experimental group 3 |
DAY1 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 |
DAY3 | 65.3 | 25.0 | 12.2 | 11.4 | 18.6 |
DAY5 | 65.7 | 35.9 | 25.6 | 22.1 | 19.9 |
DAY7 | 63.8 | 37.3 | 31.1 | 25.7 | 27.6 |
DAY9 | 54.0 | 41.1 | 40.0 | 32.8 | 32.2 |
Cumulative food intake/g | 248.8 | 139.3 | 108.9 | 92.0 | 98.3 |
Inhibition of food intake/%) | 0.0 | 44.0 | 56.2 | 63.0 | 60.5 |
As can be seen from tables 5-6 and figures 2-3 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 the somaglutide, and the difference is obvious.
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
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<120> an acylated long-acting GLP-1 derivative
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<213> GLP-1 analog (GLP-1)
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cgtcgtggtg gt 132
Claims (6)
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 EGTFTSDVSSYLEEQAAREFIKWLVRRGG;
wherein, X 8 Selected from V, I, T;
the derivatives are linked to the fatty acid side chain via a linker through the epsilon amino group on the amino acid K residue on a GLP-1(7-37) analog thereof;
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 or a pharmaceutically acceptable salt thereof according to claim 2, wherein said long-acting GLP-1 derivative is:
N-ε 30 - [2- (2- [2- (2- [2- (2- [4- (17-carboxyheptadecanoylamino) -4(S) -carboxybutanoylamino)]Ethoxy) ethoxy]Acetylamino) ethoxy]Ethoxy) acetyl group][Val 8 Glu 22 Arg 26 Lys 30 Arg 34 Arg 35 Gly 36 ]GLP-1(7-37), the amino acid sequence of which is shown in SEQ ID No. 1; or
N-ε 30 - [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 30 Arg 34 Arg 35 Gly 36 ]GLP-1(7-37), the amino acid sequence of which is shown in SEQ ID No. 2; or
N-ε 30 - [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 30 Arg 34 Arg 35 Gly 36 ]GLP-1(7-37), the amino acid sequence of which is shown in SEQ ID NO. 3.
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.
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