CN113493504B - Molecular modification of GIP-Exendin-4 chimeric peptide and application of dimer thereof in treatment of diabetes - Google Patents

Molecular modification of GIP-Exendin-4 chimeric peptide and application of dimer thereof in treatment of diabetes Download PDF

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CN113493504B
CN113493504B CN202110322498.3A CN202110322498A CN113493504B CN 113493504 B CN113493504 B CN 113493504B CN 202110322498 A CN202110322498 A CN 202110322498A CN 113493504 B CN113493504 B CN 113493504B
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dimer
αaib
peptide
glu
glp
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CN113493504A (en
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唐松山
张旭东
罗群
杨莉
谭宏梅
唐婧晅
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Tang Lin
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Shenzhen Nafu Biomedical Co ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/605—Glucagons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Abstract

The present invention provides the use of a glucagon-like peptide-1 receptor activator (GLP-1R) -based glucose-dependent insulin nutritional polypeptide (GIP) -Exendin 4 chimeric polypeptide molecule allosteric and dimers thereof for lowering blood glucose and treating metabolic syndrome. The dimer is formed by connecting two identical GIP-Exendin-4 chimeric peptide monomers containing single cysteine allosteric through disulfide bonds formed by cysteine. The H-type dimer (single Ser-Cys replacement in the molecule) of the invention obviously increases the duration of blood glucose reduction of the dimer under the condition of not reducing the activity, and the dimer of the invention has continuous activity in vivo for 22 days, which is obviously prolonged compared with a positive control drug Lixisenatide (2 days). Compared with Lixisenatide, the dimer 2G21 with the same molar concentration has the effects of similar hypoglycemic activity and weight reduction, induction of doubled insulin secretion and improvement of organ toxicity, and expands the medical application of the dimer, such as application in the treatment of metabolic syndrome, including diabetes and obesity, and reduction of excessive food intake and the like.

Description

Molecular modification of GIP-Exendin-4 chimeric peptide and application of dimer thereof in treatment of diabetes
Technical Field
The invention belongs to the field of medical biology, and particularly relates to molecular modification of GLP-1R activator similar peptide and application of homodimer thereof in treating metabolic diseases.
Background
Exendin-4 is an incretin analog isolated from Heloderma suspectum saliva, has 39 amino acids, and has 53% sequence homology with GLP-1. GIP is a 42 amino acid gastrointestinal regulatory peptide that has the functions of regulating body glucose metabolism, promoting insulin release from islet beta cells, and reducing body weight. GLP-1 is an incretin analog peptide of 30 amino acid residues that is released by intestinal L cells upon nutrient ingestion. Exendin-4 and GLP-1 are two GLP-1R activators currently found. Based on these three active polypeptide amino acid sequences that regulate carbohydrate metabolism, through significant structural changes of recent decades, each allosteric birth has obtained a glucose-lowering GLP-1R activator marketed or clinically approved by the us FDA or chinese SFDA, such as once daily administration of Liraglutide (marketed in 2011) and Lixisenatide (marketed in 2016), twice daily administration of Exenatide (marketed in 2010) and once weekly administration of Polyethylene Clycol Loxenatide (marketed in 2019), albiglutide (marketed in 2014), dulaglutide (marketed in 2014), semaglutide (marketed in 2017), tasnoglutide (clinical failure in 2012015) and Tirzepatide (clinical in 2018). Exenatide, polyethylene Clycol Loxenatide and Lixisenatide are allosteric molecules based on the amino acid sequence of the active polypeptide Exendin-4, which have been brought into the market clinically. Liraglutide, albiglutide, dulaglutide, semaglutide and Taspoglutide are amino acid sequence allosteric analogues based on the active polypeptide GLP-1, which are produced by chemical synthesis or by a combination of gene recombination and chemical synthesis. Tirzepatide is a synthetic molecule based on GIP-Exendin-4 bifunctional receptor activator, which was developed by Lily and has been completed in phase II clinic.
Nausea and vomiting occur in most GLP-1R activator treatments. Since The hypothalamic-pituitary-adrenal axis (HPA or HTPA) is part of The physiological stress response, GLP-1R activators stimulate The HPA axis resulting in an increase in corticosterone leading to The occurrence of partial heart rate abnormalities. Thus, there remains a need for: (1) antagonizing the activation of both glucagon and GLP-2 receptors upon activation of the GIP receptor and/or GLP-1 receptor; (2) there is a need to provide weight loss, antagonism of DPP-4 and other forms of degradation mechanisms by activating GIP receptor and/or GLP-1 receptor effects, while maintaining lower immunogenicity; (3) GLP-1R activators still need to be optimized because current long-acting activators have proven to be less effective than natural GLP-1 or Exendin-4 molecules in terms of specific activity (hypoglycemic effect per unit mass), dosing, weight loss and side effects, while technological advances are needed to provide longer-acting hypoglycemic activators and overcome toxicity.
Disclosure of Invention
The invention aims to provide a GLP-1R activator analogue peptide. The GLP-1R activator analogue peptide disclosed by the invention is prepared by respectively carrying out molecular allosteric on Exendin-4, GIP-Exendin-4 and GLP-1. On the one hand, substitution of single Cys.fwdarw.Ser inside the monomeric peptide, the position of cysteine in the peptide chain was studied. On the other hand, modification of the fatty acid or fatty acid substituent on the epsilon-amino group of lysine (K) side chain in the peptide chain is allosteric, with different fatty acids or fatty acid substituents having different effects on the activity of GLP-1R activator-like peptides. The invention also provides homodimers formed by the GLP-1R activator similar peptide, and the homodimers with H-type structures formed by different cysteine positions (single Cys-Ser substitution in the molecule) are found to generate different activities, and the continuous blood glucose reduction time can reach 22 days at most, so that the increase is obvious compared with the clinical medicines of which the time is 1-7 days at present.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows: a glucagon-like peptide-1 receptor (GLP-1R) activator-like peptide, which is an Exendin-4, GIP-Exendin-4 chimeric peptide or GLP-1 sequence modified with a lysine epsilon-amino fatty acid chain. The GLP-1R activator similar peptide takes the amino acid sequence of Exendin-4, GIP-Exendin-4 or GLP-1 peptide as a main chain, replaces Ser on the main chain with Cys, and only contains one Cys in the main chain amino acid sequence; the side chain is the fatty acid chain of one of the epsilon-amino groups of the lysine of the main chain.
Preferably, the specific sequence of the GLP-1R activator-like peptide is any one of the following:
(1)(HN 2 )H-X 2 -EGTFTCDLS-X 12 -QMEEEAV-X 20 -LFIEWL-X 27 -NGGPSSGAPP-X 38 or;
(2)(HN 2 )H-X 2 -EGTFTSDLC-X 12 -QMEEEAV-X 20 -LFIEWL-X 27 -NGGPSSGAPP-X 38 or;
(3)(HN 2 )H-X 2 -EGTFTSDLS-X 12 -QMEEEAV-X 20 -LFIEWL-X 27 -NGGPCSGAPP-X 38 or;
(4)(HN 2 )H-X 2 -EGTFTSDLS-X 12 -QMEEEAV-X 20 -LFIEWL-X 27 -NGGPSCGAPP-X 38 or;
(5)(HN 2 )Y-X 2 -EGTFTCDYSI-X 13 -LDKIAQ-X 20 -AFVQWLIAGGPSSGAPP-X 38 or;
(6)(HN 2 )Y-X 2 -EGTFTSDYCI-X 13 -LDKIAQ-X 20 -AFVQWLIAGGPSSGAPP-X 38 or;
(7)(HN 2 )Y-X 2 -EGTFTSDYSI-X 13 -LDKIAQ-X 20 -AFVQWLIAGGPCSGAPP-X 38 or;
(8)(HN 2 )Y-X 2 -EGTFTSDYSI-X 13 -LDKIAQ-X 20 -AFVQWLIAGGPSCGAPP-X 38 or;
(9)(HN 2 )H-X 2 -EGTFTSDVSCYLEGQAA-X 20 -EFIAWLV-X 28 -GRG(NH 2 )；
wherein X is 2 Or X 13 Is L-alpha-glycine or L-alpha-alanine or alpha-amino isobutyric acid (alpha Aib); x is X 12 Or X 20 Or X 27 Or X 28 Is lysine, arginine, or glutamyl fatty acid [ gamma-Glu (N-alpha-fatty acid) on the side chain epsilon-amino group]Or glutamyl fatty diacid [ gamma-Glu (N-alpha-fatty diacid)]Modified lysine, or side chain epsilon-amino [2 XAEEAC-gamma-Glu- (N-alpha-fatty diacid)]Modified lysine; x is X 38 Is PS (HN) 2 ) Or SKKKKKK (HN 2 ). The capital letter is abbreviation of L-alpha-amino acid or ammoniaSubstitution of the symbols with basic acids, arabic numerals in the order of amino acid residues, NH 2 Representing an N-terminal or C-terminal amide structure.
Preferably, the GLP-1R activator resembles a peptide when X 12 Or X 20 Or X 27 Or X 28 Is glutamyl fatty acid [ gamma-Glu (N-alpha-fatty acid) on the side chain epsilon-amino group]Or glutamyl fatty diacid [ gamma-Glu (N-alpha-fatty diacid)]When modified, the structure of the modified lysine is shown in a chemical formula 1; when X is 12 Or X 20 Or X 27 Or X 28 For side-chain epsilon-amino [2 XAEEAC-gamma-Glu- (N-alpha-fatty diacid)]When modified, the structure of the lysine is shown in chemical formula 2.
The invention also provides a hypoglycemic peptide-like homodimer, which is formed by connecting identical monomers according to any one of claims 1-3 through disulfide bonds formed by cysteine, and is formed into the H-type GLP-1R activator peptide-like homodimer.
Preferably, the amino acid sequence of the dimer is any one of the following:
wherein X is 2 Or X 13 Is L-alpha-glycine or L-alpha-alanine or alpha-amino isobutyric acid (alpha Aib); x is X 12 Or X 20 Or X 27 Or X 28 Is lysine, arginine, or glutamyl fatty acid [ gamma-Glu (N-alpha-fatty acid) on the side chain epsilon-amino group]Or glutamyl fatty diacid [ gamma-Glu (N-alpha-fatty diacid)]Modified lysine, or side chain epsilon-amino [2 XAEEAC-gamma-Glu- (N-alpha-fatty diacid)]Modified lysine; x is X 38 Is PS (HN) 2 ) Or SKKKKKK (HN 2 ) The method comprises the steps of carrying out a first treatment on the surface of the "|" indicates the disulfide bond formed between two cysteines.
Preferably, when X 12 Or X 20 Or X 27 Or X 28 Is glutamyl fatty acid [ gamma-Glu (N-alpha-fatty acid) on the side chain epsilon-amino group]Or glutamyl fatty diacid [ gamma-Glu (N-alpha-fatty diacid)]When modified, the structure of the modified lysine is shown in a chemical formula 1; when X is 12 Or X 20 Or X 27 Or X 28 Is [2 XAEEAC-gamma-Glu- (N-alpha-fatty diacid) on side chain epsilon-amino group]When modified, the structure of the lysine is shown in chemical formula 2.
The invention also provides application of the GLP-1R activator similar peptide or the homodimer in preparing medicines for treating metabolic syndrome symptoms. Preferably, the metabolic syndrome disorder includes hyperglycemia, diabetes, and obesity.
The present invention also provides a medicament for treating a disorder of metabolic syndrome, which comprises as an active ingredient a GLP-1R activator like peptide or homodimer as described above and pharmaceutically acceptable salts thereof.
The invention has the beneficial effects that: under the condition that the glucose-lowering strength of the homodimer of the H-type GLP-1R activator analogue is not lower than that of the corresponding monomeric peptide, the glucose-lowering action time of the corresponding monomeric activator or a GLP-1R activator clinical drug approved by FDA or SFDA is obviously prolonged by about 2-3 times, and the activity maintenance time of the homodimer of the GLP-1R activator analogue in the human body is prolonged by 22 days, which is obviously prolonged compared with that of a positive drug Lixinaglutinde (drug effect maintenance for 2 days). Compared with clinical GLP-1R activator, the novel dimer has obvious structural change, and greatly facilitates the clinical application and market popularization.
Drawings
FIG. 1 is a graphical representation of the results of a single OGTT blood glucose test.
Fig. 2 is a graph of body weight statistical analysis of the T2D model of 2G21 treatment.
Fig. 3 is a graph of blood glucose statistical analysis in a 2G21 treatment T2D model.
Fig. 4 is a graph of a statistical analysis of glycosylated hemoglobin in a T2D model of 2G21 treatment.
Fig. 5 is a graph of statistical analysis of insulin in a 2G21 treatment T2D model.
FIG. 6 is a graph showing the statistical analysis of glutamic pyruvic transaminase in the T2D model of 2G21 treatment.
FIG. 7 is a graph showing the statistical analysis of amylopsin in the T2D model of 2G21 treatment.
Detailed Description
In order to more clearly demonstrate the technical scheme, objects and advantages of the present invention, the present invention is described in further detail below with reference to the specific embodiments and the accompanying drawings.
EXAMPLE 1 preparation of monomeric peptides and dimers
1. The solid phase chemical synthesis process of monomer peptide: manual solid-phase polypeptide synthesis operation steps.
1. Swelling of the resin: amino resin (amino resin for C-terminal amidation sequence) (available from Nankai, tianjin) was placed in a reaction kettle, and 15ml/g of dichloromethane (DCM, dikmaTechnologies Inc.) was added to the resin and the mixture was shaken for 30min. SYMPHONY 12 channel polypeptide synthesizer (SYMPHONY model, software version.201, protein Technologies Inc.).
2. The first amino acid: the solvent was removed by sand core suction filtration, 3-fold moles of the C-terminal first Fmoc-amino acid (all Fmoc-amino acids were supplied by sumac pharmaceutical group fine chemicals limited), 10-fold molar amounts of 4-Dimethylaminopyridine (DMAP) and N, N' -Dicyclohexylcarbodiimide (DCC) were added, and finally Dimethylformamide (DMF) (from Dikma Technologies inc.) was added for dissolution with shaking for 30min. Blocking with acetic anhydride.
3. Deprotection: DMF was removed, 20% piperidine-DMF solution (15 ml/g) was added, the solvent was removed by filtration for 5min, and 20% piperidine-DMF solution (15 ml/g) was added for 15min. Piperidine is supplied by the national pharmaceutical group Shanghai chemical reagent company.
4. And (3) detection: the solvent was pumped off. Taking more than ten pieces of resin, washing with ethanol for three times, adding ninhydrin, KCN and phenol solution into the resin, heating the mixture at 105-110 ℃ for 5min, and turning deep blue to be positive reaction.
5. Washing resin: the washing was performed twice with DMF (10 ml/g), twice with methanol (10 ml/g) and twice with DMF (10 ml/g).
6. Condensation: depending on the specific synthesis conditions, the following methods may be used alone or in admixture in the synthesis of the polypeptide:
method a: three times the amount of protected amino acid and three times the amount of 2- (7-azobenzotriazole) -tetramethylurea hexafluorophosphate (HBTU, suzhou Tianma pharmaceutical Co., ltd.) were dissolved in DMF as little as possible and added to the reaction vessel. Immediately adding N-methylmorpholine (NMM, suzhou Tianma pharmaceutical Co., ltd.) in ten times amount, reacting for 30min, and detecting to be negative.
Method b: three times the amount of the protected amino acid FMOC-amino acid and three times the amount of 1-hydroxybenzotriazole (HOBt, suzhou Tianma pharmaceutical Co., ltd.) were dissolved in DMF as little as possible, and three times the amount of N, N' -Diisopropylcarbodiimide (DIC) was added immediately to the reaction tube, and the reaction was carried out for 30min.
7. Washing resin: DMF (10 ml/g) was washed once, methanol (10 ml/g) was washed twice, and DMF (10 ml/g) was washed twice.
8. The procedure of steps 2 to 6 was repeated, and corresponding amino acids were sequentially linked from right to left as shown in the GLP-1R-activating peptide having no side chain modification of the amino acids in Table 1, or the GLP-1R-activating peptide having side chain modification. With K 12 Or K 20 Or K 27 Or K 28 Modified, synthesized according to the following 9 method.
9. Synthesis of K { N- ε - [ γ -Glu- (N- α -fatty acid or fatty diacid) ] }: 10ml of 2% hydrazine hydrate is added for reaction for 30min, the protecting group Dde of Fmoc-Lys (Dde) -OH is removed, the side chain amino group is exposed, the reaction mixture is alternately washed with DMF and methanol for six times, and ninhydrin is detected as blue. 550mg of Fmoc-Glu-OTBU, HOBT 250mg, are weighed, dissolved in DMF, 0.3ml of DIC is added, mixed evenly, added into a reactor to react with lysine side chain amino groups for 1h, pumped out, washed 4 times with DMF, and ninhydrin is detected to be colorless. 5ml of 20% piperidine DMF solution was added to the reactor and reacted for 20min, the amino protecting group Fmoc of Fmoc-Glu-OTBU was removed, and the reaction was washed six times with DMF and methanol alternately, and ninhydrin was detected as blue. 300mg of fatty acid or fatty diacid, HOBT 250mg, was weighed, dissolved in DMF, and 0.3ml of DIC was added, mixed well, added to the reactor for reaction for 1h, dried by suction, washed 4 times with DMF, detected as colorless by ninhydrin, and washed 2 times with methanol.
Synthesis of K { N- ε - [2 XAEEAC- γ -Glu- (N- α -fatty diacid) ] }: after Dde-Lys (Fmoc) was removed from Fmoc group, 2mM Fmoc-AEEAC-OH and 2mM benzotriazole-1-oxy-tripyrrolidinylphosphine hexafluorophosphate (PyBOP) were added, 45mM HOBt was dissolved in DMF, 0.375mM N, N' -Diisopropylethylamine (DIPEA) was added under ice water bath to activate for 3min, and the mixture was reacted for 2h with a reaction column to determine the end point of the experiment by ninhydrin method. At the end of the reaction, fmoc was removed from a 20% piperidine-DMF solution (15 ml/g) and washed 6 times with DMF. Fmoc-AEEAC-OH, fmoc-Glu-OtBu and fatty diacid chain groups were again coupled in the same manner. The reaction is carried out for 30min with 2% hydrazine hydrate to remove the protecting group Dde of the sequence lysine, and the protecting group Dde is connected to the epsilon amino group of the lysyl side chain through the step 8.
10. The condensed polypeptide was passed through DMF (10 ml/g) twice, DCM (10 ml/g) twice, DMF (10 ml/g) twice and dried for 10min. Ninhydrin assay was negative.
11. Removing FMOC protecting group of final N-terminal amino acid of peptide chain, detecting positive, and draining solution for standby.
12. The resin was washed twice with DMF (10 ml/g), twice with methanol (10 ml/g), twice with DMF (10 ml/g), twice with DCM (10 ml/g) and dried for 10min.
13. Cleavage of polypeptide from resin: preparing cutting fluid (10 ml/g): TFA94% (J.T. Baker Chemical Company), water 2.5%, ethane dithiol (EDT, sigma-Aldrich Chemistry) 2.5% and triisoopropylsilane (TIS, sigma-Aldrich Chemistry) 1%. Cutting time: 120min.
14. Drying and washing: drying the lysate with nitrogen as much as possible, washing with diethyl ether for six times, and volatilizing at normal temperature.
15. The polypeptides were purified by HPLC, identified and stored at-20℃in the absence of light as follows.
2. The test method is as follows:
1. purification of the polypeptide by HPLC: the crude peptide was dissolved with pure water or a small amount of acetonitrile and purified according to the following conditions: high performance liquid chromatography (analytical; software Class-VP. Seal System; manufacturer, japan SHIMADZU) and Venusi MRC-ODS C18 column (30X 250mm, tianjin Bonna-Agela Technologies). Mobile phase a liquid: 0.1% aqueous trifluoroacetic acid solution, mobile phase B: 0.1% trifluoroacetic acid +99.9% acetonitrile solution (acetonitrile Fisher Scientific company). Flow rate: 1.0ml/min, a loading volume of 30 μl and a detection wavelength of 220nm. Elution procedure: 0 to 5min:90% of solution A and 10% of solution B; 5-30 min:90% of solution A/10% of solution B, 20% of solution A/80% of solution B.
2. And finally, freeze-drying the purified effective solution (Freeze zone Plus 6 model of freeze dryer, LABCONCO manufacturer) to obtain a finished product.
3. And (3) identification: taking a small amount of finished polypeptide, and performing HPLC analysis on the purity of the finished polypeptide: high performance liquid chromatography (manufacturer, SHIMADZU) and Venusi MRC-ODS C18 column (4.6X1150 mm, tianjin Bonna-Agela Technologies). Mobile phase a liquid: 0.1% aqueous trifluoroacetic acid, mobile phase B: 99.9% acetonitrile +0.1% trifluoroacetic acid solution, flow rate: 1.0ml/min, a loading volume of 10 μl and a detection wavelength of 220nm. Elution procedure: 0 to 5min:100% solution a; 5-30 min:100% of solution A, 20% of solution A/80% of solution B. The purity is required to be greater than 95%. For specific methods, see our issued patent (chinese patent ZL 201410612382.3).
Identification of polypeptide molecular weight by MS method: and adding water into the polypeptide with qualified purity for dissolution, and adding 5% acetic acid+8% acetonitrile+87 water for dissolution to test electrospray ionization mass spectrometry to determine the molecular weight, wherein the specific method is described in our patent (Chinese patent ZL 201410612382.3).
4. Sealing and packaging the powdery polypeptide, and storing at-20deg.C in dark place.
3. Formation of dimers: monomeric peptides with single cysteines within peptide chains at a concentration of 1mg/ml were incubated overnight at 37 ℃ in aqueous disodium hydrogen phosphate at ph=9.5 to form homodimeric peptides. For the slightly poorly soluble dimeric peptide, the precipitate was taken as a pure dimeric peptide by centrifugation at 4000 rpm for 20min, and the precipitate was dissolved in NaCl-PB solution (ph=8.0 of physiological saline adjusted with disodium hydrogen phosphate) for use. The dissolved dimer peptide or the above supernatant was separated and identified by Sephadex G-25 chromatography (at 2X 60cm G-25 column and natural flow rate, naCl-PB solution was used as flow term, dimer component was the first peak, residual impurity component was the subsequent peak). The dimeric peptide can be identified by peptide PAGE electrophoresis or mass spectrometry without thiol reducing agent, see our issued patent (Chinese patent ZL 201410612382.3).
4. GLP-1R activators like peptide monomers and dimers thereof were synthesized by the present laboratory or commercial company, and the inventors confirmed their structure by HPLC purity, ESI or laser flight mass spectrometry, and cysteine oxidation. The GLP-1R activator monomers synthesized by the invention are shown in table 1, and the amino acid sequences of the homodimeric peptides are shown in table 2.
Table 1: the GLP-1R activator synthesized by the invention is similar to the amino acid sequence of peptide monomer and has continuous hypoglycemic activity of single injection with the same dosage
Note that: tirzepatide in the table is a chimeric peptide of GIP-Exendin-4 peptide; lixisenatide is also an allosteric for Exendin 4; CFA (carbon fatty acid) or CFDA (carbon fatty diacid) is a carbon fatty acid or a carbon fatty diacid; the K [ N-epsilon- (gamma-Glu-N-alpha-CFA or CFDA) ], K [ N-epsilon- (2 XAEEAC-gamma-Glu-N-alpha-CFDA) ] represents fatty acyl or fatty diacid monoacylglutamyl modification of lysine K side chain epsilon-amino, and the specific structure is shown in formula 1 or 2.
EXAMPLE 2 persistence study of the hypoglycemic Effect of GLP-1R activators of the invention
1. The experimental method comprises the following steps: normal KM mice were purchased at the animal center in guangdong province for glucose tolerance measurement (OGTT): glucose tolerance measurements in normal Kunming mice were used to screen for hypoglycemic activity and persistence of the drug. Male Kunming mice (5 weeks old) were divided into groups (NaCl-PB group, lixisenatide group, monomer G9-G12 series and dimer 2G9-2G12 series) according to indiscriminate fasting blood glucose (n=6). After a conditioning period of 14-10 hour feeding, KM mice were immediately subjected to glucose tolerance measurements after each 10 hour feeding. Drug or monomer (dissolved in physiological saline ph6.5, blank control using physiological saline) or dimer peptide (dissolved in NaCl-PB solution ph8.0, blank control using NaCl-PB solution generally) was injected subcutaneously into the back for 30min, and mice were perfused with 5% glucose solution on time, and the blood glucose level in the rat tail was accurately determined after the lavage. Blood glucose meters and blood glucose test papers are products of Bayer HeathCare LLC company. Taking the average blood glucose value of each group as a judgment standard: when the average blood glucose level of each group OGTT was higher than the average blood glucose level of the same day blank group twice in succession, the measurement was stopped, and the duration of blood glucose lower than the blank group was the duration of efficacy.
2. Experimental results
2.1 oral glucose tolerance test
Blood glucose was measured by taking blood from the tail of the mice 30 minutes after the single administration, on time before (0 min) and after 10, 20, 40, 60, 120min of the single oral administration of glucose, and physiological saline was used for the blank group (fig. 1). The results show that: at 10min, a significant glucose increase (P < 0.05) occurred in group 2G33 compared to the Lixisenatide and G21 groups. At 20min, the Lixisenatide and G21 groups showed a significant decrease (P <0.05 or 0.01) compared to the blank group. Compared to the Lixisenatide and G21 groups, the 2G21 group showed significant glucose increase (P < 0.05). At 40min, a significant decrease (P <0.05 or 0.01) occurred in the Lixisenatide, G and G33 groups compared to the blank group. Compared to the blank, lixisenatide or G33 group, the 2G33 group showed a significant glucose decrease or increase (P < 0.05). At 60min, a significant decrease (P < 0.05) occurred in group G21 compared to the blank; compared to the Lixisenatide and G21 groups, significant glucose increases occurred in groups 2G21 or 2G33 (P < 0.05). The results showed that the dimer blood glucose level was significantly higher than that of the monomeric peptide within the same time period, showing significantly delayed absorption by the dimer.
After a single dose, the OGTT test (mmogtt) was continued multiple times for multiple days: 30min after back subcutaneous injection of drug or monomeric or dimeric peptide, mice were perfused with 5% glucose solution on time, and the rat tail blood glucose value was determined exactly 35min after lavage. The average value of blood sugar is used as a judging standard, and the blood sugar of the Lixisenatide positive medicine is reduced for 2 days, the G9 series is maintained for 2-9 days, the G10 series is maintained for 2-11 days, and the G11 series is maintained for 2-11 and the G12 series is maintained for 7-9 days. Dimer 2G9 series for 4-21 days, 2G10 series for 4-22 days, 2G11 series for 6-21 days, and 2G12 series for 16-20 days. Each monomer set (shown in table 1) was about 1/2 the duration of its corresponding dimer set (shown in table 2). Comparison revealed that dimeric peptides with disulfide bond formation at position 11 or 12 and lysine epsilon-amino side chain fatty acid modification at position 20 (with 20 carbon fatty acids or fatty diacids being preferred) had significant weight loss and significantly increased duration of blood glucose reduction. Comparison revealed that the 11 th peptide in the 2G9 series, the 33 th and 34 th dimeric peptides in the 20 th and 21,2G11 th series in the 2G10 series lasted up to 21-22 days. The 21 st peptide (2G 21) in the 2G10 series is in an Exendin 4 variant form, has high homology with the sequence of Lixisenatide positive control drug and has the same side chain fatty acid modification, so the 2G21 peptide is selected for treating type II diabetes mellitus (T2D) in vivo and subsequent experiments. The mOGTT experiment needs to be accurately implemented, otherwise, the result may have a jump.
TABLE 2 GLP-1R activator dimer sequences and Single subcutaneous injections of the same dose for duration of hypoglycemic Activity
Note that: CFA (carbon fatty acid) or CFDA (carbon fatty diacid) in the table is a carbon fatty acid or a carbon fatty diacid; the K [ N-epsilon- (gamma-Glu-N-alpha-CFA or CFDA) ], K [ N-epsilon- (2 XAEEAC-gamma-Glu-N-alpha-CFDA) ] represents fatty acyl or fatty diacid monoacyl glutamyl modification of K side chain epsilon-amino, and the specific structure is shown in formula 1 or 2. The dimer is connected by disulfide bonds formed between cysteines in the monomeric peptide to form an H-type dimer; wherein "|" indicates the formation of disulfide bonds between two cysteine residues between monomers.
EXAMPLE 3 therapeutic Effect of dimer on type II diabetes model
1. Construction of type II diabetes (T2D) mouse model
C57Bl6/J mice were placed in a standard diet SPF grade environment, free-drinking. All experimental operations are in accordance with the guidelines of ethics and use of experimental animals. After feeding the day on a standard diet, 5 week old C57B16/J male mice were divided into 6 groups: naCl-PB group, placebo group (model control group), lixisenatide group, low, medium and high dose dimeric peptide 2G21 group. NaCl-PB was blank control and Placebo was T2D model control, which were injected with NaCl-PB solution. All T2D model groups were fed a 60kcal% high fat diet (D12492, rat-mouse two biotechnology limited, everstate, china) until the end of the experiment, and the blank group remained on the standard diet until the end of the experiment. The method for establishing the diabetes model comprises the following steps: mice were fed with high fat for 4 weeks, intraperitoneally injected with 75mg/kg streptozotocin (STZ, sigma chemical Co., USA), intraperitoneally injected with 50mg/kg dose of STZ after 3 days, and mice with blood glucose equal to or greater than 11mM after 3 weeks were considered diabetic mice. These diabetic groups were further studied for 35 days on a high-fat diet.
2. Therapeutic effect on type II diabetes
Solubility of peptide: for the monomeric peptide which contains fatty acid modified structure on the Lys side chain and does not contain Aib amino acid composition, the monomeric peptide shows a suspension state in water, and the corresponding homodimeric peptide is completely dissolved in water; for those containing fatty acid modified structures in the Lys side chains, monomeric peptides containing Aib amino acids or/and having C-terminal amidated structures show complete dissolution in water, while their corresponding homodimeric peptides are poorly soluble in water. For fatty acid modifications without Lys side chains, both monomeric peptide and dimer were completely dissolved in water. All dimer peptides were separately injected with NaCl-PB (pH 8.0) and low, medium and high different doses of homodimer 2G21 were separately dissolved in NaCl-PB solution [ Na ] 2 HPO 4 Buffered saline (pH 8.0)]The animals were injected. The monomeric peptide was dissolved in physiological saline solution for injection (pH 6.5 or so).
Drug administration concentration setting: we have shown from preliminary experiments that a single subcutaneous injection of 0.624 nmol/100. Mu.l Lixisenatide peptide, the aging relationship of the multiple OGTT's over multiple days is readily observed. Therefore, in all glucose tolerance experiments, normal Kunming mice were subcutaneously injected at the buttocks with a single dose of 0.624nmol/100 μl of Lixisenatide or monomeric or dimeric peptide, and blood glucose was measured and weighed by 9-point tail-cutting blood sampling every day. For OGTT, the administration, lavage and blood glucose taking times for each animal need to be accurate to seconds. T2D animal experiments Lixisenatide was chosen as positive control and mode of administration (subcutaneous administration).
Lixisenatide at 0.624 nmol/100. Mu.l induced postprandial blood glucose levels of 8-11mM in the T2D diabetes model (postprandial blood glucose up to 20 mM). At this threshold, the effect-dose relationship of the positive drug Lixisenatide to GLP-1R dimer is readily observed. In the T2D treatment study, T2D model mice were subcutaneously injected into the buttocks at a dose of 100 μl each for 30min, blood glucose values were measured every five days, and the whole test was completed within 40 min. The dimer 2G21 peptide has high, medium and low doses of 1.873, 0.624 and 0.208nmol/100 μl respectively, and the positive drug Lixisenatide has a dose of 0.624nmol/100 μl (the bulk drug is synthesized by commercial company), and is injected once a day until the experiment is completed for 35 days.
1. Changes in body weight after T2D treatment: there was no significant difference in body weight of the T2D model group prior to dosing. At the end of the experiment, placebo, lixisenatide, L-2G21 (low dose), M-2G21 (medium dose) showed significant weight gain (P <0.05 or 0.001) compared to NaCl-PB, but H-2G21 (high dose) was not different from normal, indicating effective treatment. The body weight loss was significant in the Lixisenatide and H-2G21 groups compared to Placebo groups (P < 0.05). The body weight of each group 2G21 decreased in a dose-dependent manner, and the M-2G21 group was similarly changed to the Lixisenatide group (FIG. 2).
2. Hypoglycemic effects in T2D treatment: compared with NaCl-PB group, placebo, lixisenatide, L-and M-2G21 groups had significantly higher fasting blood glucose values (FIG. 3) (P<0.05 or 0.001), or Placebo, lixisenatide, L-and M-, H-2G21 group with significant hyperglycosylated hemoglobin (HbA) 1c ) (FIG. 4) (P<0.001 The T2D model was shown to be successful. The Lixisenatide and H-2G21 groups showed significantly reduced fasting glucose (P <0.05 or 0.01) compared to Placebo groups, or the Lixisenatide, M-2G21 and H-2G21 groups HbA 1c The significance was reduced (P < 0.05). The fasting blood glucose was significantly elevated (P < 0.05) in the L-2G21 group compared to the Lixisenatide group. After peptide injection, blood glucose levels decreased in a dose-dependent manner, with greater number of doses being used, the better the effect. The blood glucose changes in the M-2G21 group were similar to those in the Lixisenatide group. HbA 1c And blood glucose levels produce similar changes in T2D therapy.
3. Blood biochemical index detection in T2D treatment: following T2D treatment experiments, the Placebo, lixisenatide or L-2G21 groups showed significantly lower fasting insulin levels than the NaCl-PB group (P <0.01 or 0.001). Each group 2G21 had a dose-dependent increase in fasting insulin, with a 2-3 fold increase in insulin content (P <0.05 or 0.01) in the M-2G21 group compared to the Lixisenatide group and the L-2G21 group. The H-2G21 group insulin content was increased 2-4 fold (P <0.05 or 0.001) compared to Placebo, lixisenatide or L-2G21 groups (FIG. 5), showing that equimolar concentrations of dimeric peptide induced 2-3 fold insulin secretion. Group 2G21 has a dose-dependent decrease in glutamic pyruvic transaminase (ALT), with the ALT level of H-2G21 being lower than that of NaCl-PB or Placebo, group L-2G21 (P < 0.05) (FIG. 6). Each 2G21 group serum amylase decreased dose-dependently, but was not statistically different (P > 0.05) from either the blank or Placebo, lixisenatide (fig. 7).
From the above embodiments, the following conclusions can be drawn: the homodimer series we developed can significantly increase the duration of the drug effect. Studies have shown that dimer sequences show the most promising application prospects for rodent T2D models, such as the longest duration hypoglycemic effect and weight loss effects.
The structure-activity relationship shows that the dimer without fatty acid modification has the best solubility in water, and the dimer with fatty acid modification peptide contains Aib amino acid structure, even has a C-terminal amidation structure, and the solubility in water is slightly poor, which is important data of drug preparation research, and is the root cause of different duration of hypoglycemic activity of GLP-1R activators with different structures in the research, namely different spatial conformations, form different physicochemical properties and generate different duration of hypoglycemic activity.
The results of a single OGTT experiment show that a longer hypoglycemic effect is produced due to slow dimer absorption. The results of multiple OGTT experiments indicate that longer duration effects involve allosteric correlations such as dimer amino acid 2, disulfide bond position, symmetrical fatty acid or fatty diacid modified Lys and C-terminal amidation. Table 2 shows that the longest active dimer structure contains 2 αAib、 11 Or (b) 12 Cys-Cys disulfide bond, symmetrical 20 fatty acyl or fatty diacid monoacyl-L-gamma-glutamyl- 20 Lysine or 20-carbon fatty diacid monoacyl-gamma-Glu-2 XAEEAC- 20 Lys and C-terminal amidation, and the like. These modifications are characterized as follows: (1) Alpha Aib → 2 Ala substitution results in longer activity; (2) With other fatsFatty acid modification comparison, lys [2 XAEEAC-gamma-Glu- (N-alpha-20 carbon fatty diacid)]Modification achieves the best results; (3) C-terminal amidation significantly prolonged activity; (4) The disulfide structure at position 12 or 11 in the dimer molecule shows the best activity. Monomeric peptide activity was only 1/2 of that of the corresponding dimer.
In T2D treatment experiments, group 2G21 model HbA of diabetes 1c Or the fasting blood glucose (FPG) value is obviously reduced, the effect of obviously reducing blood glucose is obvious, and the same molar concentration of 2G21 peptide and Lixisenatide is used for PPG (postprandial blood glucose) or FPG and HbA 1c Has similar reduction effect.
The body weight of group 2G21 decreased in a dose-dependent manner, and the M-2G21 group was consistent with the Lixisenatide group in terms of weight loss. Better weight loss dimeric peptide structure relates to 11 Or (b) 12 Cys-Cys disulfide bonds, symmetry 20 Lys [2 XAEEAC-gamma-Glu-20 carbon fatty acid]Modification, C-terminal amidation, and the like.
2G21 reduces glutamic pyruvic transaminase (ALT) in a dose-dependent manner, which shows that the medicine has a strong protective effect on liver.
In T2D treatment experiments, the insulin in group 2G21 increased in a dose-dependent manner, and the M-or H-2G21 induced insulin levels 2-4 times higher than Placebo, lixisenatide and L-2G21, thus 2G21 had better hypoglycemic effects.
In conclusion, the dimeric peptide of the present invention can induce more insulin release, thereby producing better hypoglycemic effect.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Claims (4)

1. A glucagon-like peptide-1 receptor (GLP-1R) activator-like peptide, characterized in that the specific sequence of said analog peptide is any one of the following:
①(HN 2 )Y-αAib-EGTFTCDYSI-αAib-LDKIAQK[N-ε-(2×AEEAC-γ-Glu-N-α-20CFDA)]AFVQWLIAGGPSSGAPPPS(NH 2 ) Or;
②(HN 2 )Y-αAib-EGTFTCDYSI-αAib-LDK[N-ε-(2×AEEAC-γ-Glu-N-α-20CFDA)]IAQKAFVQWLIAGGPSSGAPPPS(NH 2 ) Or;
③(HN 2 )Y-αAib-EGTFTCDYSI-αAib-LDKIAQK[N-ε-(2×AEEAC-γ-Glu-N-α-20CFDA)]AFVQWLIAGGPSSGAPPSKKKKKK(NH 2 ) Or;
④(HN 2 )Y-αAib-EGTFTCDYSI-αAib-LDKIAQK[N-ε-(L-γ-Glu-N-α-20CFDA)]AFVQWLIAGGPSSGAPPPS(NH 2 ) Or;
⑤(HN 2 )Y-αAib-EGTFTSDYCI-αAib-LDKIAQK[N-ε-(2×AEEAC-γ-Glu-N-α-20CFDA)]AFVQWLIAGGPSSGAPPPS(NH 2 ) Or;
⑥(HN 2 )Y-αAib-EGTFTSDYCIALDKIAQK[N-ε-(2×AEEAC-γ-Glu-N-α-20CFDA)]AFVQWLIAGGPSSGAPPPS(NH 2 ) Or;
⑦(HN 2 )Y-αAib-EGTFTSDYCI-αAib-LDKIAQK[N-ε-(L-γ-Glu-N-α-20CFDA)]AFVQWLIAGGPSSGAPPPS(NH 2 ) Or;
⑧(HN 2 )Y-αAib-EGTFTSDYCI-αAib-LDKIAQK[N-ε-(2×AEEAC-γ-Glu-N-α-20CFDA)]AFVQWLIAGGPSSGAPPSKKKKKK(NH 2 ) Or;
⑨(HN 2 )Y-αAib-EGTFTSDYCI-αAib-LDKIAQK[N-ε-(L-γ-Glu-N-α-18CFA)]AFVQWLIAGGPSSGAPPPS(NH 2 ) Or;
⑩(HN 2 )Y-αAib-EGTFTSDYSI-αAib-LDKIAQK[N-ε-(2×AEEAC-γ-Glu-N-α-20CFDA)]AFVQWLIAGGPCSGAPPPS(NH 2 ) Or;
(HN 2 )Y-αAib-EGTFTSDYSI-αAib-LDKIAQK[N-ε-(2×AEEAC-γ-Glu-N-α-20CFDA)]AFVQWLIAGGPSCGAPPSKKKKKK(NH 2 )。
2. a hypoglycemic peptide-like homodimer, characterized in that the dimer is formed by connecting identical monomers through disulfide bonds formed by cysteines, thus forming an H-type GLP-1R activator peptide-like homodimer, wherein the cysteines used for connection are positioned at the 8 th position or 11 th position or 32 rd position or 33 rd position on the dimer monomers; wherein the dimer of cysteine 11 is formed by the activator-like peptide monomer of claim 1, and the specific sequence is any one of the structures shown in the following:
wherein, the specific structure of the dimer of the 11 th cysteine is one of the following two types:
wherein, the specific structure of the dimer of the 32 nd cysteine is one of the following two types:
wherein, the specific structure of the dimer of the 33 rd cysteine is one of the following two types:
wherein \' represents the disulfide bond formed between the two cysteines.
3. Use of a GLP-1R activator like peptide according to claim 1, or a homodimer according to claim 2, for the preparation of a medicament for the treatment of hyperglycemia, diabetes and obesity.
4. A medicament for treating hyperglycemia, diabetes and obesity, characterized in that the medicament comprises the GLP-1R activator-like peptide or/and pharmaceutically acceptable salt thereof as an active ingredient; or the homodimer or/and pharmaceutically acceptable salt thereof as claimed in claim 2 as active ingredient.
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