CN114716533B - 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 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 of carbohydrate, protein, fat and the like caused by 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 (statistically, approximately 90%). Type 2diabetes (diabetes mellitus type2, T2 DM), 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 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 risk of hypoglycemia, and a weight-loss effect and cardiovascular protection, and are becoming the main 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 the GLP-1 analogue and the glucagon are almost half the same, and the GLP-1 analogue and the glucagon 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 ] Tianjin medicine, 2012, 40 (2): 181-184.); thus, GLP-1 and its analogs and derivatives play an important role in the development and progression of type 1 and 2 diabetes.
The results of studies conducted by Nauck M et al on 10 patients with type 2diabetes with poorly controlled blood glucose, administered to the patients GLP-1 or placebo, respectively, showed that after GLP-1 infusion, the patients had significantly increased insulin and C-peptide levels, significantly decreased glucagon levels, and fasting blood glucose levels 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 further 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 exert the blood sugar reduction effect when the blood sugar rises, inhibit the secretion of glucagon, increase the satiety and reduce the hunger so as to achieve the blood sugar reduction effect (normal-insulin-dependent diabetes mellitus by exogenous glucose-like peptide 1 (7-36 amide) in type2 (non-insulin-dependent) diabetes mellitus (1993, 36)). Lancet et al have shown that GLP-1 produces weight loss via a variety of pathways, including inhibition of gastrointestinal motility and gastric secretion, inhibition of appetite and ingestion, and delay of gastric content emptying. In addition, GLP-1 also acts on the central nervous system (especially the hypothalamus) to suppress appetite, reduce food intake, thereby causing satiety and a decrease in appetite in humans, and reduce calorie intake, thereby achieving weight loss (Effect of 6-week heart of glucose-lipid peptides 1on glucose control, insulin sensitivity, and β -cell function in type 2diabetes, ([ J ]. Lancet, zander, mette, 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, lissamide, exenatide microsphere formulations, albiglutide, polyethylene glycol loxapide and somaglutide (also known as semaglutide). Wherein the Somalide is a representative of GLP-1 derivative medicaments.
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.
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 groups,Acyl, ester, and the like. An example of a GLP-1 (7-37) derivative is N-epsilon 26 - ((4S) -4- (hexadecanoylamino) -carboxy-butyryl) [ Arg 34 Lys 26 ]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 a 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, the 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 present invention provides a GLP-1 (7-37) analog consisting of a polypeptide having an amino acid sequence represented by the formula:
YX 8 EGTFTSDVSSYLEX 22 QAAX 26 EFIX 30 WLVX 34 X 35 X 36 X 37 ；
wherein:
X 8 selected from Aib, V, I, T, L, G or S;
X 22 selected from G or E;
X 26 is selected from K or R;
X 30 selected from A, K, E or R;
X 34 is selected from R or G;
X 35 is selected from R or G;
X 36 is selected from R or G;
X 37 is selected from R or G;
and X 26 And X 30 Only one of which is K.
Preferably, said X 8 Selected from V, X 22 Selected from E, X 26 Selected from R, X 30 Selected from K, X 34 Selected from R, X 35 Is selected from G.
More preferably, (1) said X 36 Selected from R, X 37 Is selected from G; or, (2) X 36 Selected from G, X 37 Is selected from R.
When the GLP-1 (7-37) analogue is [ Tyr 7 Val 8 Glu 22 Arg 26 Lys 30 Arg 34 ]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 [ Tyr 7 Val 8 Glu 22 Arg 26 Lys 30 Arg 34 Gly 36 Arg 37 ]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) analogue, preferably via the epsilon amino group on the K residue.
As a preferred embodiment of the present invention, the long-acting GLP-1 (7-37) derivative of the present invention isThe fatty acid side chain structure used 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 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 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, 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 ) 16 CO-, and 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][Tyr 7 Val 8 Glu 22 Arg 26 Lys 30 Arg 34 ]GLP-1 (7-37) (abbreviated as HS-Y1), [ Tyr 7 Val 8 Glu 22 Arg 26 Lys 30 Arg 34 ]The amino acid sequence of GLP-1 (7-37) 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][Tyr 7 Val 8 Glu 22 Arg 26 Lys 30 Arg 34 Gly 36 Arg 37 ]GLP-1 (7-37) (abbreviated as HS-Y2), [ Tyr 7 Val 8 Glu 22 Arg 26 Lys 30 Arg 34 Gly 36 Arg 37 ]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 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 bacterium 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 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 somagluteptide, 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 developed 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 somagluteptide, the long-acting GLP-1 derivative provided by the invention has more excellent and remarkable weight-losing capacity and more excellent weight-losing 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 mouse models;
each group of data corresponding to each time point in the figure is a blank control group, a model control group, a Somali peptide group, an HS-Y1 group and an HS-Y2 group from left to right in sequence;
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, 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 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 (taking HS-Y1 as an example):
(1) Construction of the code [ Tyr 7 Val 8 Glu 22 Arg 26 Lys 30 Arg 34 ]Expression plasmid for GLP-1 (7-37)
Through a great deal of research and experiments in the early stage, the inventionObviously selecting FKFEFKFE as an inclusion body promoting sequence, DDDDK as an EK enzyme digestion sequence, and sequentially fusing the inclusion body promoting sequence, the EK enzyme digestion sequence and a GLP-1 analogue coding gene sequence in series to obtain a coding gene fragment shown as SEQ ID NO. 3; the fragment is inserted into a prokaryotic expression plasmid pET-30a (+) through NdeI and XhoI sites and sequenced and verified to obtain an expression plasmid which is called pET-30a (+) - [ Tyr (+) 7 Val 8 Glu 22 Arg 26 Lys 30 Arg 34 ]-GLP-1(7-37)。
(2) Construction of expression of [ Tyr 7 Val 8 Glu 22 Arg 26 Lys 30 Arg 34 ]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, 30min on an ice bath, heat shock in a water bath at 42 ℃ for 30s, and then the centrifuge tubes were quickly transferred to an ice bath for 2min without shaking the centrifuge tubes.
Adding 500 mu L of sterile LB culture medium without antibiotics into a centrifuge tube, uniformly mixing, and then placing at 37 ℃ for culturing for 1h at 180rpm so as to enable bacteria to recover; 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 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 the first-level seed culture solution cultured overnight, inoculating into 200mL of 2YT culture medium according to the proportion of 1.
Taking 60mL of secondary seed liquid, inoculating the secondary seed liquid into an FDM culture medium (600 mL) according to the proportion of 1.
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 10g/L.
(4) Recombinant [ Tyr 7 Val 8 Glu 22 Arg 26 Lys 30 Arg 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 (50 mM Tris-HCl, 50mM NaCl, pH 8.0), 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.
Concentrating the sample before enzyme digestion by UniPS30-300 (purchased from Suzhou Naichi Microscience, inc.) which is balanced by equilibrium liquid 3 (10 mM ammonium acetate and 20% acetonitrile), eluting by the equilibrium liquid 3, then eluting by a gradient of 0-100% eluent (10 mM ammonium acetate and 80% acetonitrile), analyzing by RP-HPLC, and obtaining the GLP-1 intermediate product with the purity higher than 70% by the purification process.
The tag sequence was cleaved off using EK enzyme: the intermediate product was diluted three times by adding 20mM PB buffer solution of pH7.4, and after adding EK enzyme at 20 ℃ according to EK enzyme: intermediate product = 1.
[Tyr 7 Val 8 Glu 22 Arg 26 Lys 30 Arg 34 ]-fine purity of GLP-1 (7-37): concentrating UniPS30-300 (from Suzhou Nami science and technology Co., ltd.) equilibrated with equilibration solution 3 (10 mM ammonium acetate, 20% acetonitrile), washing with equilibration solution 3, and washing with water at a ratio of 0-100% eluent (10 mM ammonium acetate, 80% acetonitrile) was eluted in a gradient with an analytical purity of about 90% by RP-HPLC.
0.2M Na was added to the eluted sample 2 HPO 4 Adjusting pH to 4.8-5.0 with 1M citric acid to 20mM, acid precipitating at 4 deg.C overnight, with RP-HPLC detection yield above 90%, centrifuging at 13000g for 30min at 4 deg.C, collecting precipitate, and storing at-20 deg.C to obtain [ Tyr 7 Val 8 Glu 22 Arg 26 Lys 30 Arg 34 ]-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: tyr obtained in step (4) 7 Val 8 Glu 22 Arg 26 Lys 30 Arg 34 ]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 protein, and quantifying the polypeptide concentration by HPLC; weighing fatty acid powder according to a molar ratio of 1.
Deprotection and purification of fatty acid: to the resulting precipitate was added TFA up to a final peptide concentration of about 10mg/mL, the precipitate was dissolved by shaking, left to stand at room temperature for deprotection for 30min, and then the pH was adjusted to 7.5 to 8.5 by dropping 4M NaOH to terminate the reaction.
Pumping the reaction solution after the reaction termination into UniPS10-300 (purchased from Suzhou Naichi technology Co., ltd.) equilibrated in advance by using a protein purification chromatography system (Seiko SDL 100) at a flow rate of 4mL/min, concentrating, eluting by using equilibration solution 3 (10 mM ammonium acetate, 20% acetonitrile), eluting by using a gradient of 0-100% eluent (10 mM ammonium acetate, 80% acetonitrile), and collecting an elution peak with a purity of about 90% by RP-HPLC.
Diluting the elution peak with water by 3 times, adjusting pH to 4.80 by acid precipitation, acid precipitating at 4 deg.C30min, centrifuging, adding PBST buffer (pH7.0) into the precipitate for redissolving, 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][Tyr 7 Val 8 Glu 22 Arg 26 Lys 30 Arg 34 ]GLP-1 (7-37) (abbreviated as 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 code [ Tyr 7 Val 8 Glu 22 Arg 26 Lys 30 Arg 34 Gly 36 Arg 37 ]-a gene segment 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) 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, 0.004096nM. The test results showed that the test results showed positive control group (Somalu peptide), experimental group 1 (HS-Y1) and experimental group 2 (HS-Y2).
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 digestion for 3 minutes, adding DMEM basal medium to terminate digestion, and centrifuging to collect cells. The cell density was adjusted to 8.0X 10 with DMEM blank medium 5 cell/mL, 50. Mu.L/well in 96 well cell culture plates, at 37 ℃ 5% 2 Incubated overnight under conditions.
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.
Taking out the cultured cell culture plate from the incubator, diluting50. Mu.L/well of the assay medium (2) was added to the cell plate, and the cell plate was incubated at 37 ℃ and 5% CO 2 Incubate 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 adopting a four-parameter regression calculation method, and the EC50 value of the sample to be detected 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 |
As can be seen from the results in the table, the HS-Y1 and HS-Y2 of the invention have lower EC50 values than the somaglutide, and HS-Y1 and HS-Y2 are about 1/2 and 1/4 of the somaglutide respectively, which shows that the invention has about 2-fold and 4-fold better binding affinity with human insulin receptor than the somaglutide respectively.
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/mLNa 2 HPO 4 ，5.5mg/mLPhenol, 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: comprises 10 mice, glucose (4 g/kg) is perfused, and the abdomen is injected with a blank subcutaneously;
positive control group: comprises 10 mice, glucose (4 g/kg) is perfused into stomach, and the abdomen is injected with medicine subcutaneously;
example 1 group: comprises 10 mice, glucose (4 g/kg) is used for gastric lavage, and the abdomen is injected with medicine subcutaneously;
example 2 group: the mice contained 10 mice, were gavaged with glucose (4 g/kg), and were injected with the drug subcutaneously in the abdomen.
(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 test, measuring blood glucose for 0h before oral glucose after administration, measuring blood glucose for 0.5h, 1h, 2h after oral glucose, then measuring blood glucose for 23h after oral glucose, and measuring blood glucose for 1h after oral glucose, 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 are superior to the somaglutide in controlling the blood sugar of mice in the whole experiment, and have better blood sugar control capability.
Example 4: study of weight loss Effect in mice
(1) Experimental Material
a. Experimental formulation:
GLP-1 derivative injection (HS-Y1 and HS-Y2) 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 0.045mg/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 body weight of the HFD group, screening out unmolded mice, randomly grouping the rest mice according to the body weight, and dividing each group into a model control group (blank solvent), a positive control group (Somalutide, 0.45 mg/kg), an experimental group 1 (HS-Y1, 0.45 mg/kg), an experimental group 2 (HS-Y1, 0.75 mg/kg), an experimental group 3 (HS-Y2, 0.45 mg/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 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 can be seen from 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 still maintain good GLP-1 activity, have good weight loss ability, and the effect is superior to that of somaglutide.
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 phrases "comprising one of 8230; \8230;" 8230; "does not exclude the presence of additional like elements in a process, method, article, or apparatus that comprises the element.
The above description is merely illustrative of particular embodiments of the invention that enable those skilled in the art to understand or practice the 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
<130> KP2211659.2
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Gln Ala Ala Arg Glu Phe Ile Lys Trp Leu Val Arg Gly Arg Gly
20 25 30
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Tyr Val Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
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cgcggcggcc gc 132
Claims (3)
1. An acylated long-acting GLP-1 derivative, or a pharmaceutically acceptable salt thereof, 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][Tyr 7 Val 8 Glu 22 Arg 26 Lys 30 Arg 34 ]-GLP-1(7-37)，[Tyr 7 Val 8 Glu 22 Arg 26 Lys 30 Arg 34 ]The amino acid sequence of GLP-1 (7-37) 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][Tyr 7 Val 8 Glu 22 Arg 26 Lys 30 Arg 34 Gly 36 Arg 37 ]-GLP-1(7-37)，[Tyr 7 Val 8 Glu 22 Arg 26 Lys 30 Arg 34 Gly 36 Arg 37 ]The amino acid sequence of GLP-1 (7-37) is shown in SEQ ID NO. 2.
2. Recombinant engineering bacteria for expressing GLP-1 (7-37) analogues are characterized in that the GLP-1 (7-37) analogues have sequences shown as SEQ ID NO.1 or SEQ ID NO. 2;
the recombinant engineering bacterium is a recombinant escherichia coli engineering bacterium, and the construction method of the recombinant engineering bacterium comprises the following steps:
(1) Serially connecting and fusing the inclusion body promoting sequence, the EK enzyme digestion sequence and the GLP-1 (7-37) analogue coding gene sequence in 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 recombinant engineering bacteria for expressing the GLP-1 (7-37) analogue.
3. Use of an acylated long-acting GLP-1 derivative according to claim 1 or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of diabetes or weight loss.
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