CN116970062B - Ultra-long acting GLP-1 polypeptide derivative and preparation method and application thereof - Google Patents

Ultra-long acting GLP-1 polypeptide derivative and preparation method and application thereof Download PDF

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CN116970062B
CN116970062B CN202211480825.9A CN202211480825A CN116970062B CN 116970062 B CN116970062 B CN 116970062B CN 202211480825 A CN202211480825 A CN 202211480825A CN 116970062 B CN116970062 B CN 116970062B
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acid
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acting glp
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CN116970062A (en
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丁伟
张哲峰
侯雯
潘楚炎
侯吉昊
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Nanjing Zhihe Medical Technology 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
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
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    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P3/00—Drugs for disorders of the metabolism
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    • 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
    • A61P3/06—Antihyperlipidemics
    • 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
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/48—Drugs for disorders of the endocrine system of the pancreatic hormones
    • A61P5/50—Drugs for disorders of the endocrine system of the pancreatic hormones for increasing or potentiating the activity of insulin
    • A—HUMAN NECESSITIES
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives
    • 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 invention discloses an ultra-long acting GLP-1 polypeptide derivative or a salt thereof, wherein the ultra-long acting GLP-1 polypeptide derivative is shown as a formula (I), and only one X is arranged in a main chain 2 The amino group of the side chain of the compound is connected with the branched acyl group shown in the formula (II) to form amide, and the ultra-long-acting GLP-1 polypeptide derivative or salt thereof and a pharmaceutical preparation thereof are applied to the treatment and/or prevention of type II diabetes, impaired glucose tolerance, type I diabetes, obesity-related diseases, metabolic syndrome, nonalcoholic steatohepatitis, nonalcoholic fatty liver disease, neurodegenerative diseases and the like. 7 H‑Aib‑X 1 ‑ 10 G‑T‑F‑T‑S‑D‑V‑S‑S‑Y‑ 20 L‑E‑G‑Q‑A‑ 25 A‑X 2 ‑E‑F‑I‑ 30 A‑W‑L‑V‑X 3 ‑ 35 G‑R‑Z(I)

Description

Ultra-long acting GLP-1 polypeptide derivative and preparation method and application thereof
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to an ultra-long-acting GLP-1 polypeptide derivative, a preparation method and application thereof.
Background
Glucagon-like peptide-1 (GLP-1) is a peptide hormone secreted by human intestinal L cells, and has effects of promoting insulin secretion, inhibiting glucagon secretion, and lowering blood sugar concentration, and can be used for treating type II diabetes and obesity. However, natural GLP-1 is unstable in vivo, is easily rapidly degraded by dipeptidyl peptidase-IV (DPP-IV), and has a half-life of less than 2 minutes. GLP-1 function is mediated by the GLP-1 receptor (GLP-1R). GLP-1R is widely distributed in a plurality of tissues and organs of the whole body such as pancreas, liver, kidney and brain. The physiological action and mechanism of GLP-1 receptor agonist or medicine for reducing blood glucose can regulate the secretion function of islet alpha cells and beta cells bidirectionally, promote the differentiation and proliferation of islet beta cells, inhibit the apoptosis of islet beta cells, reduce the infiltration of inflammatory cells and other modes to improve the functions of islet beta cells, increase the number of beta cells, increase the sensitivity of islet beta cells to glucose, promote the secretion of glucose-dependent insulin and inhibit the release of glucagon. The GLP-1 receptor agonist or medicine has beneficial physiological effects and mechanisms of reducing oxidative stress, promoting NO release, improving endothelial function, inhibiting inflammatory response, reducing left ventricular remodeling, delaying AS progression, reducing systolic pressure and pulmonary capillary wedge pressure, increasing myocardial rescue rate after myocardial infarction, increasing autophagy, reducing myocardial injury, regulating calcium ion concentration in myocardial cells, avoiding myocardial apoptosis, improving metabolic factors and age-induced myocardial fibrosis, thereby improving cardiovascular system function and reducing incidence of cardiovascular death, non-lethal myocardial infarction or non-lethal stroke. The physiological actions of GLP-1 receptor agonists or drugs for weight loss and the mechanisms thereof are as follows: GLP-1 can be synthesized by the central nervous system, and GLP-1 receptors are widely distributed in the brain, namely GLP-1 is not only a gastrointestinal hormone, but also a brain neuropeptide, and the dual purposes of reducing blood sugar and weight are achieved through the effects of inhibiting secretion and movement of the gastrointestinal tract (particularly delaying gastric emptying), increasing satiety, affecting food intake and the like through the mediation of the central nervous system. The physiological action of GLP-1 receptor agonist or medicine for protecting liver and its mechanism are that GLP-1 receptor exists in liver cell, GLP-1 receptor agonist or medicine can activate GLP-1R of liver, directly regulate metabolism of liver lipid, inflammation reaction caused by oxidative stress and endoplasmic reticulum stress, reduce visceral fat content of liver so as to protect liver cell, further prevent and delay generation and development of nonalcoholic fatty liver. The GLP-1 receptor agonist or the medicine has the physiological action and the mechanism of strengthening brain, and can activate GLP-1R of the brain, strengthen nerve growth factor mediated nerve cell differentiation, stimulate neurite growth, reduce the transportation of blood brain barrier to glucose, promote the normalization of dopamine metabolism in the brain and increase dopamine neurons.
The Somatlutide, chinese name is Semaglutide, is a novel long-acting glucagon-like peptide-1 (GLP-1) analogue developed and produced by Daneno and Norde company, has clinical effects of reducing blood sugar, losing weight, protecting cardiovascular and the like, and possibly has therapeutic significance on NASH (non-alcoholic fatty liver) and AD (Alzheimer's disease). The hypoglycemic and cardiovascular risk reducing injectable cord Ma Lutai Ozempic, hypoglycemic oral cord Ma Lutai Rybelsus, and slimming injectable cord Ma Lutai Wegovy are marketed in batches in different countries around the world. After modification of a Lys side chain of the somalupeptide by AEEA (Chinese name 2- (2- (2-aminoethoxy) ethoxy) acetic acid), glu and octadecadicarboxylic acid, the hydrophilicity is greatly improved, and the binding force with albumin is enhanced; meanwhile, after Ala at the 2 nd position of the N end is mutated into Aib, the inactivation caused by DPP-IV enzymolysis is effectively avoided, the average half life of the injectable cable Ma Lutai Ozempic reaches 6.3 days, and patients only need to inject once a week.
Jesper Lau et al (Discovery of the Once-Weekly glucose-Like Peptide-1 (GLP-1) Analogue Semaglutide, J.Med. Chem,2015, 58:7370-7380) and Lotte Bj erre Knudsen details the research process of differently engineering branched modifications of polypeptides to achieve half-life prolongation while maintaining polypeptide affinity, with cable Ma Lutai and other GLP-1 derivative content being presented in China patent CN101133082B and world patent WO2006/097537 A2, respectively. Jinhua Zhang et al (Design, synthesis and biological evaluation of double fatty chain-modified glucagon-like peptide-1conjugates, biorg. Med. Chem.,2021, 44:116291) and jingha et al (Novel fatty chain-modified glucagon-like peptide-1conjugates with enhanced stability and prolonged in vivo activity,Biochem.Pharmacol, 2013, 86:297-308), etc., have attempted new branched modifications. The effect of Glu9 substitution to Asp9 on affinity and activity of (unbranched) GLP-1 with the receptor was studied by Xao et al (Biological Activities of Glucagon-Like Peptide-1Analogues in Vitro and in Vivo,Biochem, 2001, 40:2860-2869); cyril Sarrauste de Menthi re et al (Structural requirements of the N-terminal region of GLP-1- [ 7-37)]NH2 for receptor interaction and cAMP production, eur.j.med.chem.,2004, 39: 473-438) studied the substitution of Glu9 for Asp9 for GLP-1- [7-37 without branching]-NH 2 Influence on receptor affinity and activity.
The invention aims to research GLP-1 polypeptide derivatives with Glu9 replaced by Asp9 and long-acting branched chain modification, and aims to develop potential medicaments for prolonging half-life and simultaneously maintaining polypeptide affinity and activity, thereby meeting the huge clinical demands of GLP-1 medicaments applied to metabolic diseases and more fields.
Disclosure of Invention
The invention provides an ultra-long acting GLP-1 polypeptide derivative or a salt thereof, wherein the ultra-long acting GLP-1 polypeptide derivative is shown as a formula (I), and X is in a main chain 2 An amino group having one and only one side chain thereof is linked to a branched acyl group represented by formula (II) and forms an amide:
7 H-Aib-X 1 - 10 G-T-F-T-S-D-V-S-S-Y- 20 L-E-G-Q-A- 25 A-X 2 -E-F-I- 30 A-W-L-V-X 3 - 35 G-R-G
(I)
wherein Aib is amino isobutyric acid, X 1 Selected from D aspartic acid, X 2 Selected from K lysine, X 3 Selected from R arginine or K lysine, X 2 Amino groups having one and only one side chain thereof are linked to the branched acyl group represented by formula (II) and form an amide; b is octadecanedioic acid or eicosanedioic acid, one end of which forms an amide structure with the alpha amino group of glutamic acid, and the other end of which is a carboxylic acid structure.
For ease of identification and presentation, the amino acid sequence is provided by labeling the amino acid position in the upper left hand corner of the partial amino acid, which position number is derived from and corresponds to the human GLP-1 (7-37) amino acid sequence position.
In one embodiment of the invention, the ultralong acting GLP-1 polypeptide derivative is of the following formula (III):
wherein formula (III) corresponds to polypeptide compound (1): x is X 1 Is D, X 2 K is that epsilon amino group of amino acid side chain is connected with branched structure in form of amide bond, X 3 K and B are octadecanedioic acid, and one end of the octadecanedioic acid forms amide with glutamic acid alpha amino group.
In one embodiment of the invention, the ultra-long acting GLP-1 polypeptide derivative is of formula (IV)
Wherein formula (IV) corresponds to polypeptide compound (2): x is X 1 Is D, X 2 K is that epsilon amino group of amino acid side chain is connected with branched structure in form of amide bond, X 3 R and B are octadecanedioic acid, and one end of the octadecanedioic acid forms amide with glutamic acid alpha amino group.
In one embodiment of the invention, the ultra-long acting GLP-1 polypeptide derivative is of the formula (V)
Wherein formula (V) corresponds to polypeptide compound (3): x is X 1 Is D, X 2 K is that epsilon amino group of amino acid side chain is connected with branched structure in form of amide bond, X 3 And K and B are eicosanedioic acid, and one end of the eicosanedioic acid forms amide with glutamic acid alpha amino.
In one embodiment of the invention, the very long acting GLP-1 polypeptide derivative is of the formula (VI)
Wherein formula (VI) corresponds to polypeptide compound (4): x is X 1 Is D, X 2 K is that epsilon amino group of amino acid side chain is connected with branched structure in form of amide bond, X 3 R, B is eicosanedioic acid, and one end of the eicosanedioic acid forms amide with glutamic acid alpha amino.
The compounds of the invention may exist in specific geometric or stereoisomeric forms. The present invention contemplates all such compounds, including cis and trans isomers, (-) -and (+) -pairs of enantiomers, (R) -and (S) -enantiomers, diastereomers, (D) -isomers, (L) -isomers, and racemic mixtures and other mixtures thereof, such as enantiomerically or diastereomerically enriched mixtures, all of which are within the scope of the invention. All such isomers and mixtures thereof are included within the scope of the present invention. In an embodiment of the present invention, as a preferred embodiment of the present invention, the amino acids in the polypeptide compound are all preferably L-type amino acids.
The amino acids expressed herein are all international standard single or three letter abbreviations.
In another aspect, the invention provides a method for preparing the ultra-long acting GLP-1 polypeptide derivative, which comprises the following step A:
1) Synthesizing branched chain fragments by a solid phase or liquid phase method;
2) Synthesizing peptide chains by a solid phase method;
3) Coupling the branched fragment to a peptide resin;
4) And (3) splitting off and removing the protecting group and mounting the protecting group on resin to obtain the ultra-long-acting GLP-1 polypeptide derivative.
The invention provides a preparation method of the ultra-long acting GLP-1 polypeptide derivative, which comprises the following step B:
1) Synthesizing peptide chains by a solid phase method;
2) Coupling each module of the branched chain with peptide resin successively by a solid phase method;
3) And (3) splitting off and removing the protecting group and mounting the protecting group on resin to obtain the ultra-long-acting GLP-1 polypeptide derivative.
In some embodiments, the protocol for synthesizing branched fragments in step a is as follows:
according to the invention, the branched chain fragments are connected in sequence or in reverse order, and finally the protection of active groups which are required to be combined with peptide chains is removed. The step can be carried out by adopting a solid phase synthesis mode of polypeptide or a liquid phase synthesis mode.
Condensing and coupling benzyl ester mono-protected 3, 6-dioxa-suberic acid (structure see below) and Boc (tert-butoxycarbonyl) mono-protected 3, 6-dioxa-1, 8-subelamine (structure see below) under weak organic base conditions, and removing Boc protection by TFA; condensing and coupling Fmoc-Glu-OtBu with the product of the previous step, and removing Fmoc protection from a Pip/DMF solution or other alkaline solution; the tBu group is singly protected, and octadecanedioic acid (or eicosanedioic acid) is condensed and coupled to the amino position of E; finally, hydrogen is hydrolyzed to remove benzyl ester under the catalysis of palladium carbon, and acyl which is required to be combined with peptide chain is exposed.
In some embodiments, the scheme for synthesizing the backbone of step a is as follows:
sequentially coupling Fmoc-protected amino acids to solid-phase synthetic resin according to the amino acid sequence of the molecular structure of the invention, deprotecting Fmoc by Pip/DMF solution or other alkaline solution, and circulating until the main chain amino acid is completely finished; wherein the alpha amino of the last amino acid His is protected by a Boc group; wherein Lys of the branched fragment to be linked is protected with Alloc (optionally, fmoc-Lys (Alloc) -OH as raw material); the invention adopts CTC resin, wang resin, HMPA-MBHA resin, HMBA-AM resin, hydroxymethyl resin, rink Acid resin and other resins capable of constructing carboxylic Acid type amino Acid for solid phase synthesis.
In some embodiments, the protocol for coupling the branched chain to the peptide chain of step a is as follows:
protecting side chains of the depeptiding resin; coupling the branched fragment to a peptide resin; (removing the Fmoc protection of the first amino acid; finally, splitting, cutting and removing the protecting group and mounting the resin to obtain the ultra-long-acting GLP-1 polypeptide derivative.
Using tetrakis (triphenylphosphine) palladium and PhSiH 3 Take off X 2 Alloc protection of (Lys); condensation coupling of branched fragments to a peptide resin; removing Fmoc protection from the Pip/DMF solution or other alkaline solution; in proportion (TFA: EDT: TIS: H) 2 O=95:2:2:1), the fully protected peptide resin was added to the lysate, cleaved from the resin and the side chains were removed. TFA was removed by rotary evaporation in vacuo and MTBE (methyl tert-butyl ether) was added to the concentrate to precipitate the desired product as a white solid.
In some embodiments, the scheme for synthesizing the backbone of step B is the same as the scheme for synthesizing the backbone of step a.
In some embodiments, the procedure for coupling each module of the branched chain to the peptide resin sequentially in the step B solid phase method is as follows:
the step is carried out by adopting a polypeptide solid-phase synthesis mode, and the sequence of each module structure of the branched chain is sequentially and reversely connected to a polypeptide main chain on the resin according to the invention. Removing X first 2 And (3) side chain protection of (Lys), connecting mono-protected 3, 6-dioxa-suberic acid, connecting 3, 6-dioxa-1, 8-subelamine, connecting Glu, and finally connecting mono-tert-butyl octadecanedioic acid (or eicosanedioic acid) to finish uploading and synthesis of the whole branched chain.
Removal of the main peptide chain X using tetrakis (triphenylphosphine) palladium and PhSiH3 2 Alloc protection of (Lys); the condensation reagent pre-activates 3, 6-dioxa-suberic acid, and then the 3, 6-dioxa-suberic acid is coupled with the main peptide chain reaction for uploading; then Fmoc mono-protected 3, 6-dioxa-1, 8-octanediamine is added for condensation coupling; fmoc protection by Pip/DMF solution or other alkaline solution removal; the Fmoc-Glu-OtBu is pre-activated by a condensing reagent, and then condensation coupling is carried out; removing Fmoc protection by Pip/DMF solution or other alkaline solution; the tBu group is singly protected by octadecanedioic acid (or eicosanedioic acid) condensation coupled to the alpha amino position of Glu; thus, the uploading and the synthesis of the whole branched chain are completed.
In some embodiments, step a and step B may be performed stepwise to construct the polypeptide compounds of the present invention.
A part of fragments of the branched chain are synthesized according to the step A, and then each module of the branched chain is coupled with the peptide resin successively according to the solid phase method of the step B.
Synthesizing 3, 6-dioxa-suberic acid and Fmoc-3, 6-dioxa-1, 8-subelamine into fragments; removal of the main peptide chain X using tetrakis (triphenylphosphine) palladium and PhSiH3 2 Alloc protection of (Lys); coupling and uploading the fragment and the main peptide chain reaction; fmoc protection by Pip/DMF solution or other alkaline solution removal; the Fmoc-Glu-OtBu is pre-activated by a condensing reagent, and then condensation coupling is carried out; removing Fmoc protection by Pip/DMF solution or other alkaline solution; the tBu group is singly protected by octadecanedioic acid (or eicosanedioic acid) condensation coupled to the alpha amino position of Glu; thus, the uploading and the synthesis of the whole branched chain are completed.
Optionally, the method further comprises:
and (3) chromatographic purification: performing multi-step reversed phase chromatography or ion chromatography, and finally converting salt into an aqueous solution containing active ingredients; and lyophilizing.
The preparation method or scheme of the ultra-long acting G LP-1 polypeptide derivative disclosed by the invention adopts a chemical synthesis mode, but according to the prior art, the compound can adopt a semi-synthesis mode of gene expression and recombination and combining chemical synthesis to construct the ultra-long acting GLP-1 polypeptide derivative. All such embodiments are intended to be included within the scope of the present invention.
Abbreviations for some of the chemical groups or chemical agents or formulas used in the context of the present invention are common and well known in the art, and their specific meanings are not individually recited, but do not affect their specific orientation.
The invention discloses a pharmaceutical composition, which comprises the compound or pharmaceutically acceptable salt thereof as an active ingredient or a main active ingredient and a pharmaceutically acceptable carrier.
A pharmaceutically acceptable salt forms part of the present invention and suitable "pharmaceutically acceptable salts" include the conventional non-toxic salts of the compounds of the invention formed by reaction of the free compounds of the invention with an inorganic or organic acid, and the conventional non-toxic salts of the compounds of the invention formed by reaction of an inorganic or organic base. For example, salts derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like are included, as well as salts derived from organic acids such as acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, sulfanilic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, isethionic acid, trifluoroacetic acid and the like. Also for example, salts derived from inorganic bases such as sodium, potassium, calcium, magnesium, zinc, iron, and the like are included, as are salts derived from organic bases such as ammonia, arginine, lysine, citrulline, histidine, and the like.
One of the objects of the present invention is the research and development of the ultra-long acting GLP-1 polypeptide derivatives according to the invention into clinically usable pharmaceutical preparations. The formulation may further comprise buffers, preservatives, isotonic agents, cosolvents, tonicity agents, chelating agents, stabilizers, antioxidants, surfactants, acid-base modifiers and the like. The concentration is typically 0.01mg/ml to 50mg/ml, wherein the formulation has a pH of 3.0 to 9.0.
In one embodiment of the invention, the pharmaceutical formulation is an aqueous formulation, i.e. an aqueous solution, typically a solution, emulsion or suspension.
In another embodiment, the pharmaceutical formulation is a lyophilized formulation, and the solvent and/or diluent is added prior to use until it is sufficiently dissolved for use.
In another embodiment, the pharmaceutical formulation is a ready-to-use dry formulation that does not require pre-dissolution, such as a spray-on lyophilized powder or the like.
In another embodiment of the invention, the pH range of the pharmaceutical formulation is critical, which affects the solubility and stability of the GLP-1 polypeptide derivative and, under certain specific conditions, results in physical aggregation or adsorption of the polypeptide. In one embodiment of the invention, the pH of the pharmaceutical formulation is 3.0 to 4.5. In one embodiment of the invention, the pH of the pharmaceutical formulation is 7.0 to 8.0. In one embodiment of the invention, the pH of the pharmaceutical formulation is between 7.5 and 8.5. In another embodiment of the invention, the pH of the pharmaceutical formulation is between 6.0 and 7.5.
In a further embodiment of the invention, the buffering agent is selected from disodium hydrogen phosphate, sodium acetate and the like, the preservative is selected from phenol, o-cresol, m-cresol, p-cresol and the like, the isotonic agent is selected from sodium chloride salts, sugar or sugar alcohols, amino acids, propylene glycol, mannitol and the like, the cosolvent is selected from mannitol, propylene glycol, PEG, glycerol, tween, ethanol and the like, the tonicity agent is selected from sodium chloride salts, propylene glycol, glycerol, mannitol and the like, the chelating agent is selected from EDTA, citrate and the like, the stabilizer is selected from creatinine, glycine, nicotinamide, PEG and the like, the antioxidant is selected from sodium bisulfite, sodium sulfite, cysteine, methionine and the like, the surfactant is selected from polysorbate, acid base, glycerol, mannitol and the like, and the regulator is selected from hydrochloric acid, phosphoric acid, sulfuric acid, sodium hydroxide, potassium hydroxide and the like.
Other ingredients may be present in the pharmaceutical formulation of the ultra-long acting GLP-1 polypeptide derivatives of the invention depending on the requirements of the pharmaceutical formulation (e.g., long term stability), including emulsifiers, metal ions, oleaginous carriers, proteins (e.g., human serum albumin, gelatin or proteins, etc.), zwitterionic (e.g., arginine, glycine, lysine, histidine, betaine, taurine, etc.), and the like, and other pharmaceutical formulation additives.
The term "pharmaceutically acceptable carrier" refers to any formulation carrier or medium capable of delivering an effective amount of the active agent of the present invention, which does not interfere with the biological activity of the active agent and which does not have toxic or side effects to the host or patient, representative carriers include water, oils, liposomes, and the like.
For a drug or pharmacologically active agent, the term "effective amount" or "therapeutically effective amount" refers to a sufficient amount of the drug or agent that is non-toxic but achieves the desired effect.
The term "active ingredient", "therapeutic agent", "active substance" or "active agent" refers to a chemical entity that is effective in treating a disorder, disease or condition of interest.
"optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.
In a third aspect, the present invention provides the use of the above-described ultra-long acting GLP-1 polypeptide derivative as GLP-1 receptor agonist, said clinical use including, but not limited to, use in the manufacture of a medicament for the treatment or prevention of at least one disease including type II diabetes, impaired glucose tolerance, type I diabetes, obesity, metabolic syndrome, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), neurodegenerative diseases such as Alzheimer's Disease (AD), parkinson's Disease (PD) and the like.
The ultralong-acting GLP-1 polypeptide derivative can be used for free compounds or salts which are acceptable in pharmacy, and can be used for diabetes, obesity-related diseases and diabetes-related diseases and metabolic syndrome. Diabetes mellitus includes a group of metabolic diseases characterized by hyperglycemia due to defects in insulin secretion, insulin action, or both. Diabetes mellitus is classified into type I diabetes mellitus, type II diabetes mellitus and gestational diabetes mellitus according to the mechanism of the disorder.
Preferably, also included is the use in the manufacture of a medicament for the treatment of delayed potency and/or prevention of exacerbation of type II diabetes, and a method of improving glycemic control in an adult with type II diabetes comprising administering to a patient in need thereof an effective amount of a polypeptide derivative as described above as a dietary and exercise supplement.
Preferably, the preparation method also comprises the application in preparing medicines for treating drug effect delay of type II diabetes and/or preventing type II diabetes from worsening.
Preferably, the use of reducing food intake, reducing beta cell apoptosis, increasing islet beta cell function, increasing beta cell mass and/or restoring glucose sensitivity to beta cells is also included.
The ultra-long acting GLP-1 polypeptide derivative disclosed by the invention is a pharmaceutically acceptable free compound or salt, and can be used for treating obesity, insulin resistance, impaired glucose tolerance, prediabetes, elevated fasting blood glucose, type II diabetes, hypertension, dyslipidemia (or a combination of metabolic risk factors), atherosclerosis, arteriosclerosis, coronary heart disease, peripheral arterial disease and stroke. These are all conditions that may be associated with obesity. However, the effect of the compounds used according to the invention on these conditions may be mediated in whole or in part by the effect on body weight or may be independent of said effect.
In certain embodiments, the pharmaceutically acceptable free compounds or salts of the GLP-1 receptor agonist polypeptide derivatives of the invention may be useful in the treatment of obesity-related disorders such as obesity-related inflammation, obesity-related gallbladder disease, and obesity-induced sleep apnea.
The pharmaceutically acceptable free compound or salt of the ultra-long acting GLP-1 receptor agonist polypeptide derivative has positive preventive and therapeutic significance for NAFLD and NASH with complex pathogenesis and related factors of multiple metabolism. Insulin resistance and fat metabolism disorders constitute early damage to the liver, leading to the formation of fat accumulation (NAFLD) in liver cells. With the development of diseases, the formation of immune regulation of the organism, the generation of inflammatory reaction by liver cells, the promotion of fibrosis and the final stage liver disease symptoms such as liver cirrhosis are finally caused. Whereas the GLP-1 receptor agonist polypeptide derivatives of the invention are effective in regulating blood glucose levels to participate in metabolism, they may be fully or partially mediated for NASH, or they may be independent of the effect.
The free compound or salt which is acceptable in pharmacy of the GLP-1 receptor agonist polypeptide derivative has positive preventive and therapeutic significance for neurodegenerative diseases with complex pathogenesis, such as Alzheimer Disease (AD), parkinson Disease (PD) and the like, which are related to a plurality of metabolic related factors.
Preferably, the method further comprises the steps of reducing beta amyloid plaque precipitation in the brain, reducing nerve cell damage caused by oxidative stress, regulating transmission of nerve synapses, increasing synaptic plasticity, stimulating axon lines, affecting long-term enhancement, thereby improving memory and improving cognitive level.
Preferably, the method also comprises activating GLP-1 receptor of brain, enhancing dopamine connection function, exerting anti-inflammatory effect, improving energy generation and opening cell survival signal, thereby improving exercise performance.
In a fourth aspect, although the mutual substitution between conservative amino acids under the conditions of the prior art is considered as no difference, the present inventors have unexpectedly found that the polypeptide derivative exhibits a very significant in vivo long-acting property better than the prior art due to the difference in the branched modification and the conservative substitution of amino acids. The technical advantage is beneficial to reducing the administration frequency and improving the medication compliance of clinical patients.
In a fifth aspect, the present inventors have found that the polypeptide derivatives have quite good GLP-1 receptor agonism due to differences in branching modification and amino acid substitution, achieving GLP-1 receptor agonism similar to or better than that of cable Ma Lutai and other therapeutic benefits. While the prior art has studied the effect of substitution of Glu9 with Asp9 on affinity and activity of non-branched GLP-1 or GLP-1- [7-37] -NH2 with the receptor, the results of various research teams on substitution of Glu9 with Asp9 are not the same, and as the research process of rope Ma Lutai described in the literature, the effect of the branched modification on activity is unknown, and some changes may lead to poor activity or poor drug generation results, and it is surprising that the research of the invention achieves GLP-1 receptor agonism similar to or better than that of rope Ma Lutai and other therapeutic benefits.
The term "conservative amino acid substitutions" refers to the mutual substitution between aromatic amino acids Phe, trp, tyr under known conditions; mutual substitution between hydrophobic amino acids Leu, ile, val; mutual substitution between polar amino acids Gln, asn; mutual substitution between basic amino acids Lys, arg, his; mutual replacement between the acidic amino acids Asp and Glu; mutual replacement between amino acids Ser and Thr of hydroxyl.
Drawings
FIG. 1 shows the plasma concentration versus time profile for a single administration of SD rats.
Detailed Description
Example 1: preparation of polypeptide Compound (2)
ESI-MS：[M+3H]1367.55，[M+4H]1025.96，[M+5H]821.01。
1) Solid phase synthesis of branched fragments
3, 6-dioxa-suberic acid condensation was mounted onto 2-CTC resin, and Fmoc protected 3, 6-dioxa-1, 8-subelamine was then coupled to the resin using HATU/DIEA condensation; removing Fmoc protection by 15% pip/DMF solution; fmoc-Glu-OtBu was coupled to the resin using a HATU/DIEA condensation; removing Fmoc protection by 15% pip/DMF solution; the octadecanedioic acid with the single protection of the tBu group is coupled to the resin by HATU/DIEA condensation; the branched fragment was cleaved with 30% TFE/DCM solution to give a branched fragment intermediate.
2) Solid phase method for synthesizing peptide chain
Sequentially coupling Fmoc-protected amino acids to solid-phase synthetic resin according to the amino acid sequence of a molecular structure, wherein a Rink Amide-AM resin is used as a solid-phase synthetic carrier, fmoc is deprotected by 15% pip/DMF solution, HATU/DIEA is used as a condensation reagent, and the reaction is circulated until all peptide chain amino acids are completed; wherein the protecting amino acid of the branched chain fragment to be connected is Fmoc-Lys (OAlloc) -OH, and the alpha amino of the last amino acid His is protected by adopting a Boc group;
3) Coupling branched fragments to peptide resins
Using tetrakis (triphenylphosphine) palladium and PhSiH 3 Removing the Alloc protection of Lys on the peptide chain; coupling the branched fragment to a peptide resin using HATU/DIEA condensation; removing Fmoc protection by 15% pip/DMF solution;
4) Cleavage to remove protecting group and resin mounting
In proportion (TFA: TIS: H) 2 O=96:3:1), the fully protected peptide resin was added to the lysate, cleaved from the resin and the side chains were removed. TFA is removed by rotary evaporation in vacuum, isopropyl ether is added into concentrated solution to separate out white solid, namely target product;
5) Chromatographic purification and lyophilization
Performing multi-step reversed phase chromatographic purification by adopting a C8 or C18 column, and finally desalting to obtain a free aqueous solution; and (5) freeze-drying.
Example 2: preparation of polypeptide Compound (1)
ESI-MS：[M+3H]1358.23，[M+4H]1018.96，[M+5H]815.38
The preparation process is similar to that of example 1, wang resin is adopted as the solid phase synthesis peptide chain carrier, fmoc protection is removed by 20% pip/DMF solution, and PyBop/DIEA is adopted as the condensation reagent. The branched chain adopts octadecanedioic acid with single protection of tBu group. MTBE crystallization is adopted during cracking. The purification adopts a mode of combining ion chromatography and reverse phase chromatography, and finally, salt conversion is carried out to obtain aqueous acetic acid solution for freeze-drying.
Example 3: preparation of polypeptide Compound (3)
ESI-MS：[M+3H]1367.54，[M+4H]1025.91。
1) Synthesis of branched chain fragment by liquid phase method
Coupling benzyl ester mono-protected 3, 6-dioxa-suberic acid with Boc (t-butoxycarbonyl) mono-protected 3, 6-dioxa-1, 8-octanediamine under N-methylmorpholine conditions using EDC+HOBt condensation, removing Boc protection with 33% TFA/DCM solution; fmoc-Glu-OtBu and the product of the previous step are condensed and coupled by DCC+HOSu, and Fmoc protection is removed by 50% ethanolamine/DCM solution; the single protected eicosanedioic acid condensation of the tBu group is coupled to the amino position of Glu; finally, hydrogen is hydrolyzed to remove benzyl ester under the catalysis of palladium carbon;
2) Solid phase method for synthesizing peptide chain
Sequentially coupling Fmoc-protected amino acids to solid phase synthetic resin according to the amino acid sequence of a molecular structure, wherein a solid phase synthetic carrier is CTC resin, 50% ethanolamine/DCM solution is used for deprotection of Fmoc, and a coupling reagent is circulated by DIC+Cl-HOBt until peptide chain amino acids are all completed; wherein the Lys protecting amino acid of the branched chain fragment to be connected adopts Fmoc-Lys (Alloc) -OH as a raw material, and the alpha amino of the final amino acid His adopts Boc group for protecting;
3) Coupling branched fragments to peptide resins
Using tetrakis (triphenylphosphine) palladium and PhSiH 3 Removing the Alloc protection of Lys on the peptide chain; coupling the branched fragment condensation to a peptide resin using dic+c1-HOBt; fmoc protection was removed from 50% ethanolamine/DCM solution;
4) Cleavage to remove protecting group and resin mounting
In proportion (TFA: TIS: EDT: H) 2 O=96:2:1:1), the fully protected peptide resin was added to the lysate, cleaved from the resin and the side chains were removed. Removing TFA by vacuum rotary evaporation, adding petroleum ether into the concentrated solution to separate out white solid, namely a target product;
5) Chromatographic purification and lyophilization
The purification adopts a mode of combining ion chromatography and reverse phase chromatography, and finally salt is transformed into aqueous solution containing sodium for freeze-drying.
Example 4: single dose pharmacokinetic experiments in SD rats
SD rats are taken, both male and female are used, the animals are grouped by random numbers, and the number n of each group is more than or equal to 6.
The preparation method of the test sample solution comprises the following steps: the polypeptide compound (3), the polypeptide compound (2) and the cable Ma Lutai of the test sample are respectively weighed, 0.2+/-0.02 mg of each polypeptide compound is fully dissolved by 2.0mL of water for injection, the test sample solution with the concentration of 0.1mg/mL is prepared before administration, and the test sample solution is stored at the temperature of 2-8 ℃.
The administration dose was 0.2mg/kg, and the administration volume was 2.0mL/kg. Administration is via subcutaneous injection via the back. About 0.3mL of blood is taken through jugular vein in 0.5, 2.5, 5.5, 7.0, 8.0, 9.0, 10.0, 12.0, 16.0, 24.0, 32.0 and 48.0 hours before and after administration, respectively, and the obtained solution is placed in an EDTA-K2 anticoagulant tube placed on an ice bath, centrifuged for 10 minutes at 4 ℃ and 2600g for 2 hours, and blood plasma is taken and temporarily stored in dry ice, and is placed in a refrigerator below-60 ℃ for preservation in 14 hours. Plasma samples were analyzed by established LC-MS/MS methods.
And (3) fitting the obtained original data through instrument software to obtain a standard curve equation and a correlation coefficient, and calculating the drug concentration in the plasma sample at each sampling time point and the accompanying quality control sample concentration.
Counting the original data by using SPSS 21.0; drug concentration and time data pharmacokinetic parameter calculations were performed using winnonlin6.4 software according to the non-compartmental model method. The results of the pharmacokinetic experiments are shown in Table 1 and FIG. 1.
Table 1: results of single drug administration pharmacokinetic experiments on SD rats
Experimental results show that the half-life of the polypeptide compound is remarkably prolonged compared with that of the cable Ma Lutai, and other main pharmacokinetic parameter values also show the trend. For patients who cannot be cured of diabetes or metabolic diseases and need long-term medication, the half-life extension has important significance for reducing clinical medication frequency and improving medication compliance of patients. Meanwhile, because GLP-1 drugs possibly have the risk of hypoglycemia, compared with marketed cable Ma Lutai, the polypeptide compound provided by the invention has similar Cmax, the risk of hypoglycemia is smaller, and the polypeptide compound is a GLP-1 potential drug with ideal drug substitution data.
Example 5: weight-losing and blood-sugar-reducing efficacy experiment of ob/ob mice
The strain of ob/ob mice has ob gene (obese), and obese mice are spontaneous mutation of homozygotes, which can lead to simple obesity with late diabetes, and are classical model animals with good correlation between the research of excessive obesity, endocrine and immune functions.
The 40 ob/ob mice were randomly divided into 4 groups according to fasting body weight, fasting serum TC, LDL, and balance, which are model control group, polypeptide compound (2) group, polypeptide compound (3) group, and cord Ma Lutai group, each group being 10. Another 10 normal fed mice were set as normal control group (vehicle).
The model control group, normal control group and cord Ma Lutai group were subcutaneously administered 1 time every 3 days for the back and 8 times (D0, D3, D6, D9, D12, D15, D18, D21) in total. The polypeptide compound (3) group and the polypeptide compound (2) group were administered 1 time by back subcutaneous injection every 4 days, and 6 times (D0, D4, D8, D12, D16, D20) in total. The model control group and the normal control group were given physiological saline (0.15 mL/20 g), and the polypeptide compound (3) group, the polypeptide compound (2) group and the cable Ma Lutai group were each administered at a dose of 0.312 mg/kg.
(1) Weight of: the pre-grouping fasting, non-water-out, was measured 1 time and randomized groups were equilibrated according to body weight and TC, LDL. After grouping, 1 time per day was measured starting from D0. And simultaneously observing the feed intake and the water intake.
(2) Lee's index: the body length was measured 1 time before the first dose and for D23. The body length is the maximum straight line length from the tip of the nose to the anus. The body position of the mouse is adjusted to enable the body of the mouse to be in a stretching state, and the body length of the mouse is read out by using ruler scales.
Lee's index= [ body weight (g) ×10 3 Body length (cm)] 1/3 。
(3) Measurement of Glucose (GLU) in blood: the measurement was performed every 3 days.
(4) Biochemistry of blood (TG, TC, LDL, HDL): approximately D11 and D23 were each measured once before grouping, respectively.
(5) Fat-body mass coefficient: after euthanasia of the mice, abdominal subcutaneous fat, perigonadal fat, subscapular subcutaneous fat were removed and weighed separately to calculate the fat-body weight coefficient.
The experimental results are as follows:
(1) Weight of body
During the experimental period, the weight of the animals in the model control group grows fastest, the average weight of the animals in the model control group is obviously higher than that of the animals in the normal control group (p is less than 0.01) during the experimental period, the animals in the normal control group grow about 6.2g at the experimental terminal point, the animals in the model control group grow about 12.9g, the animals in the polypeptide compound (3) group grow about 4.5g, the animals in the polypeptide compound (2) group grow about 4.8g, and the animals in the cable Ma Lutai group grow about 5.8g.
Starting from D1, the animals in group (3), compound (2) and group Ma Lutai showed a decrease in body weight and the food intake and water intake decreased (comparison model group and normal group) exhibited a certain regularity, i.e. less food was taken on the day of administration, followed by gradual recovery, which was related to the nerve conduction mechanism of drug activation of GLP-1 target to decrease hunger sensation and enhance satiety. The body weight of the administration group (D1-D23) in the experimental period is slowly increased, which is obviously lower than that of the model control group (p < 0.01), the body weight average value of the polypeptide compound (3) group and the polypeptide compound (2) group is slightly lower than that of the cable Ma Lutai group, and the statistical difference (p < 0.05) exists.
(2) Lee's index
The Lee's index is an effective index in reflecting the degree of obesity in rats, and the Lee's index is increased in all animals with minimal increase in normal animals. Animals in each dosing group had slightly lower Lee's index compared to the model control group, with statistical differences. The results show that the polypeptide compound (2), the polypeptide compound (3) and the positive medicine cable Ma Lutai can inhibit the obesity of ob/ob mice, and the Lee's index control capacity of each medicine is close. The results are shown in Table 2:
table 2: lee's index results in rats
Note that: p < 0.05, < p < 0.01 compared to model control; compared with the corresponding positive medicine, #p < 0.05, #p < 0.01
(3) Random blood sugar
During the experiment, compared with the mice in the model control group, the polypeptide compound (2), the polypeptide compound (3) and the positive medicine cable Ma Lutai all have obvious blood sugar reducing effect in the D3-D23 process. The group of polypeptide compound (2) and polypeptide compound (3) were not significantly different from the positive drug line Ma Lutai. The results are shown in Table 3 (unit mmol/L):
table 3: random blood sugar change results of experimental animals
Note that: p < 0.05, < p < 0.01 compared to model control; compared with the corresponding positive drug, #p < 0.05, #p < 0.01.
(4) Biochemical treatment of blood
(1) TC: the initial TC levels for each group of animals prior to dosing (D0) were relatively close. After administration, TC of the polypeptide compound (2), the polypeptide compound (3) and the cable Ma Lutai are lower than that of a model control group, and compared with the model control group, TC levels of the polypeptide compound (2) and the polypeptide compound (3) can be obviously reduced (p is smaller than 0.05), and the TC reducing capacity of the polypeptide compound (2) and the cable Ma Lutai is obviously better than that of the cable Ma Lutai (p is smaller than 0.05).
(2) TG: pre-dose (D0) TG levels were closer to those of mice of the same strain. During the experimental period, the TG level of the mice in the model control group was higher than that of the mice in the normal control group. The TG levels (p < 0.05) were significantly reduced in the group of polypeptide compound (2), group of polypeptide compound (3) and group of cord Ma Lutai after administration compared to the model control group. The polypeptide compound (2) group and the polypeptide compound (3) group are superior to the cord Ma Lutai group in terms of the TG reduction value, but are not statistically different from each other.
(3) LDL: pre-dose (D0) TG levels were closer to those of mice of the same strain. After administration, the LDL levels (p < 0.01) were significantly reduced for polypeptide compound (2), polypeptide compound (3) and cord Ma Lutai compared to the model control group. However, there was no statistical difference between the group of polypeptide compounds (2), the group of polypeptide compounds (3) and the group of cord Ma Lutai.
(4) HDL: pre-dose (D0) HDL levels were closer to the mice of the same strain. During the experimental period, the HDL level of the mice in the model control group is lower than that of the mice in the normal control group. Compared with the model control group, D11 and D23 after the administration, the HDL level of the polypeptide compound (2), the HDL level of the polypeptide compound (3) and the HDL level of the positive medicine are obviously increased, but the HDL average value of the polypeptide compound (2) and the HDL average value of the polypeptide compound (3) are lower than that of the positive medicine, and the HDL level of the polypeptide compound (2) and the HDL average value of the polypeptide compound (3) are not statistically different from each other.
(5) Fat-body mass coefficient:
the mice were dissected 23 days after administration, fat at different sites was separated, and fat-body weight coefficients were calculated according to the formula (fat/body weight×100). From the results, the fat-weight coefficient of each part of the mice in the model control group is obviously higher than that of the mice in the normal control group. The polypeptide compound (2) and the polypeptide compound (3) can obviously reduce fat under the abdomen and around the gonad of the ob/ob mice, and the effect is not obviously different from that of the positive medicine cable Ma Lutai. In addition, the drug had no effect on the scapula subcutaneous fat-body weight coefficient. The results are shown in Table 4:
table 4: fat-body weight coefficient results
Note that: compared with the model control group, p is less than 0.05, and p is less than 0.01; compared with the positive medicine, #p < 0.05, #p < 0.01.
The comprehensive analysis of the experimental data shows that the polypeptide derivative has quite good GLP-1 receptor agonism activity, achieves GLP-1 receptor agonism similar to or better than a positive control drug cable Ma Lutai, achieves lipid-reducing and blood sugar-reducing effects of ob/ob model mice, and has better treatment benefit in certain indexes of blood fat.

Claims (19)

1. An ultra-long acting GLP-1 polypeptide derivative or a pharmaceutically acceptable salt thereof, wherein the ultra-long acting GLP-1 polypeptide derivative or the salt thereof consists of a main chain shown in a formula (I) and a branched chain shown in a formula (II), and the main chain X 2 Is linked to a branched acyl group of formula (II) and forms an amide:
7 H-Aib-X 1 - 10 G-T-F-T-S-D-V-S-S-Y- 20 L-E-G-Q-A- 25 A-X 2 -E-F-I- 30 A-W-L-V-X 3 - 35 G-R-G
(I)
wherein Aib is amino isobutyric acid, X 1 Is D aspartic acid, X 2 Is K lysine, X 3 Selected from R arginine or K lysine, X 2 Is linked to a branched acyl group of formula (II) and forms an amide; b is octadecanedioic acid or eicosanedioic acid, one end of which forms an amide structure with the alpha amino group of glutamic acid, and the other end of which is a carboxylic acid structure.
2. The polypeptide derivative or a pharmaceutically acceptable salt thereof according to claim 1, wherein the polypeptide chain has Asp at position 9 and X in the main chain 2 The side chain epsilon-amino group of the Lys is connected with a branched chain containing long fatty acid, PEG linker and glutamic acid modification shown in the formula (II) in an amide bond form.
3. The very long acting GLP-1 derivative or a pharmaceutically acceptable salt thereof according to claim 1, wherein the very long acting GLP-1 derivative is represented by formula (III):
4. the ultra-long acting GLP-1 derivative or a pharmaceutically acceptable salt thereof according to claim 1, wherein the ultra-long acting GLP-1 derivative is represented by formula (IV):
5. the ultra-long acting GLP-1 derivative or a pharmaceutically acceptable salt thereof according to claim 1, wherein the ultra-long acting GLP-1 derivative is represented by formula (V):
6. the very long acting GLP-1 derivative or a pharmaceutically acceptable salt thereof according to claim 1, wherein the very long acting GLP-1 derivative is represented by formula (VI):
7. the very long acting GLP-1 derivative or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that the amino acids in the polypeptide compound are all L-type amino acids.
8. The method for preparing a very long acting GLP-1 polypeptide derivative according to any one of claims 1 to 6, characterized by comprising the following step a:
1) Synthesizing branched chain fragments by a solid phase or liquid phase method;
2) Synthesizing peptide chains by a solid phase method;
3) Coupling the branched fragment to a peptide resin;
4) Splitting off and removing the protecting group and mounting the protecting group on resin to obtain the ultra-long-acting GLP-1 polypeptide derivative;
or comprises the following step B:
1) Synthesizing peptide chains by a solid phase method;
2) Coupling each module of the branched chain with peptide resin successively by a solid phase method;
3) And (3) splitting off and removing the protecting group and mounting the protecting group on resin to obtain the ultra-long-acting GLP-1 polypeptide derivative.
9. A pharmaceutical composition characterized by comprising the ultra-long acting GLP-1 derivative of any one of claims 1 to 6 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable formulation.
10. The pharmaceutical composition according to claim 9, wherein the pharmaceutically acceptable salt is a free form compound of the ultralong acting GLP-1 derivative, a conventional non-toxic salt formed by reacting it with an inorganic acid, which is hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, or an organic acid, which is acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, sulfanilic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, hydroxyethanesulfonic acid, trifluoroacetic acid, or an inorganic base, which is sodium, potassium, calcium, magnesium, zinc, iron, or an organic base, which is ammonia, arginine, lysine, citrulline, histidine.
11. The pharmaceutical composition of claim 9, wherein the pharmaceutically acceptable formulation is an aqueous formulation, or a lyophilized formulation or a ready-to-use dry formulation, the formulation comprising any one or more of buffers, preservatives, isotonic agents, cosolvents, tonicity agents, chelating agents, stabilizers, antioxidants, surfactants, acid-base modifiers, emulsifiers, metal ions, oleaginous carriers, and zwitterionic and other pharmaceutical formulation additives.
12. The pharmaceutical composition according to claim 11, wherein the pharmaceutically acceptable formulation has a concentration of 0.01mg/ml to 50mg/ml and a pH of 3.0 to 9.0.
13. Use of the ultra-long acting GLP-1 polypeptide derivative of any one of claims 1 to 6 or a pharmaceutically acceptable salt thereof or the pharmaceutical composition of any one of claims 9 to 12 for the manufacture of a medicament for the treatment and/or prevention of at least one of the following diseases: type II diabetes, impaired glucose tolerance, type I diabetes, obesity, metabolic syndrome.
14. Use of the ultra-long acting GLP-1 polypeptide derivative of any one of claims 1 to 6 or a pharmaceutically acceptable salt thereof or the pharmaceutical composition of any one of claims 9 to 12 for the manufacture of a medicament for the treatment of delayed potency of type II diabetes and/or for the prevention of exacerbation of type II diabetes.
15. Use of the ultra-long acting GLP-1 polypeptide derivative of any one of claims 1 to 6 or a pharmaceutically acceptable salt thereof or the pharmaceutical composition of any one of claims 9 to 12 for the manufacture of a medicament for the treatment and/or prevention of at least one of the following diseases associated with obesity: obesity, insulin resistance, impaired glucose tolerance, prediabetes, elevated fasting glucose, type II diabetes, hypertension, dyslipidemia, atherosclerosis, arteriosclerosis, coronary heart disease, peripheral arterial disease and stroke.
16. Use according to claim 15, characterized in that the ultra-long acting GLP-1 polypeptide derivative according to any one of claims 1 to 6 or a pharmaceutically acceptable salt thereof or the pharmaceutical composition according to any one of claims 9 to 12 for the manufacture of a medicament for the treatment of obesity-related inflammation, obesity-induced sleep apnea.
17. Use of the ultra-long acting GLP-1 polypeptide derivative of any one of claims 1 to 6 or a pharmaceutically acceptable salt thereof or the pharmaceutical composition of any one of claims 9 to 12 for the manufacture of a medicament for the treatment and/or prevention of at least one of the following diseases: nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD).
18. Use according to claim 17, characterized in that the ultra-long acting GLP-1 polypeptide derivative or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 9 or the pharmaceutical composition according to claim 11 is for the manufacture of a medicament for the treatment of insulin resistance, fat metabolism disorders.
19. Use of the ultra-long acting GLP-1 polypeptide derivative of any one of claims 1 to 6 or a pharmaceutically acceptable salt thereof or the pharmaceutical composition of any one of claims 9 to 12 for the manufacture of a medicament for the treatment and/or prevention of at least one of the following diseases: alzheimer's Disease (AD), parkinson's Disease (PD).
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