CN116891522A - Long-acting glucagon-like peptide-1 derivative and preparation method and application thereof - Google Patents

Long-acting glucagon-like peptide-1 derivative and preparation method and application thereof Download PDF

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CN116891522A
CN116891522A CN202211481125.1A CN202211481125A CN116891522A CN 116891522 A CN116891522 A CN 116891522A CN 202211481125 A CN202211481125 A CN 202211481125A CN 116891522 A CN116891522 A CN 116891522A
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acid
derivative
peptide
acting glucagon
long acting
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丁伟
张哲峰
侯雯
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Nanjing Zhihe Medical Technology Co ltd
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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
    • 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
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • 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
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Abstract

The invention discloses a long-acting glucagon-like peptide-1 derivative, a preparation method and application thereof, wherein the long-acting glucagon-like peptide derivative is shown as a formula (I), and X is in a main chain 1 Wherein only one amino group of the side chain is connected with a branched acyl group shown as a formula (II) to form an amide, and the long-acting glucagon-like peptide-1 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‑E‑ 10 G‑T‑F‑T‑S‑D‑V‑S‑S‑Y‑ 20 L‑E‑G‑Q‑A‑ 25 A‑X 1 ‑E‑F‑I‑ 30 A‑W‑L‑V‑X 2 ‑ 35 G‑R‑G(I)

Description

Long-acting glucagon-like peptide-1 derivative and preparation method and application thereof
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a long-acting glucagon-like peptide-1 derivative, and 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 Bjerre Knudsen details the course of research on modification of the side chains of polypeptides differently engineered to achieve half-life prolongation while maintaining polypeptide affinity, with cable Ma Lutai and other GLP-1 derivatives content being presented in Chinese patent CN101133082B and world patent WO2006/097537 A2, respectively. New side chain modification schemes have been attempted by Jinhua Zhang et al (Design, synthesis and biological evaluation of double fatty chain-modified glucagon-like peptide-1conjugates, bioorg. Med. Chem.,2021, 44:116291) and by Jing Ha et al (Novel fatty chain-modified glucagon-like peptide-1conjugates with enhanced stability and prolonged in vivo activity,Biochem.Pharmacol, 2013, 86:297-308).
GLP-1 drugs are popular in research and development due to their unique target advantages. There are approximately 291 GLP-1 drugs under investigation in different stages worldwide. How to extend half-life while increasing or maintaining polypeptide affinity is a difficulty in drug development in the art is also a goal of the technological worker cumin. The average increase rate of the number of people suffering from diabetes mellitus is 51%, the number of people suffering from diabetes mellitus in China is up to 1.164 hundred million, and the people are first in the world. Such huge patients determine the clinical rigidity requirements of the domestic market for GLP-1 drugs.
Disclosure of Invention
The invention provides a long-acting glucagon-like peptide-1 derivative or a pharmaceutically acceptable salt thereof, wherein the long-acting glucagon-like peptide-1 derivative is shown as a formula (I), and X is in a main chain 1 Wherein only one amino group of the side chain thereof is linked to a branched acyl group represented by formula (II) and forms an amide:
7 H-Aib-E- 10 G-T-F-T-S-D-V-S-S-Y- 20 L-E-G-Q-A- 25 A-X 1 -E-F-I- 30 A-W-L-V-X 2 - 35 G-R-G(I)
wherein Aib is amino isobutyric acid, X 1 Selected from the group consisting of K amino acids, X 2 Selected from K or R amino acids, X 1 Amino groups having only one side chain thereof are linked to the branched acyl group represented by formula (II) and form an amide; n is 7 or 8.
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 long acting glucagon-like peptide-1 derivative is of the formula (III):
formula (III) is expressed as polypeptide compound (1): x is X 1 K is that epsilon amino group of amino acid side chain is connected with branched structure in form of amide bond, X 2 R and n are 7.
In one embodiment of the invention, the long acting glucagon-like peptide-1 derivative is of the formula (IV):
formula (IV) is expressed as polypeptide compound (2): x is X 1 K is that epsilon amino group of amino acid side chain is connected with branched structure in form of amide bond, X 2 R and n are 8.
In one embodiment of the invention, the long acting glucagon-like peptide-1 derivative is of the formula (V)
Formula (V) is expressed as polypeptide compound (3): x is X 1 K is that epsilon amino group of amino acid side chain is connected with branched structure in form of amide bond, X 2 K, n is 7;
in one embodiment of the invention, the long acting glucagon-like peptide-1 derivative is of the formula (VI)
Formula (VI) is expressed as polypeptide compound (4): x is X 1 K is that epsilon amino group of amino acid side chain is connected with branched structure in form of amide bond, X 2 K and n is 8.
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 and (L) -isomers, as well as 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 acid in the polypeptide compound is preferably an L-type amino acid.
The amino acids expressed herein are all international standard single or three letter abbreviations.
In another aspect, the present invention provides a method for preparing the long-acting glucagon-like peptide-1 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 the protective group and mounting the protective group on resin to obtain the long-acting glucagon-like peptide-1 derivative.
The invention provides a preparation method of the long-acting glucagon-like peptide-1 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 the protective group and mounting the protective group on resin to obtain the long-acting glucagon-like peptide-1 derivative.
In some embodiments, the protocol for synthesizing branched fragments in step a is as follows:
according to the structure sequence of the branched chain fragment, the single-protected long fatty diacid is connected in sequence or in reverse sequence, and finally the active group protection required to be combined with the peptide chain is removed. The step can be carried out by adopting a solid phase synthesis mode of polypeptide or a liquid phase synthesis mode.
The specific implementation steps are as follows:
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 long fatty diacid is condensed and coupled to the amino position of E; finally, hydrogen is hydrolyzed to remove benzyl ester under the catalysis of palladium carbon to expose B which needs to be combined with peptide chain 2 Carboxyl groups of (a) are provided.
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); peptide chain synthesis was performed using resins capable of constructing carboxylic amino acids such as CTC resin, wang resin, HMPA-MBHA resin, HMBA-AM resin, hydroxymethyl resin, rink Acid resin, etc.
In some embodiments, the protocol for coupling the branched chain to the peptide chain of step a is as follows:
side chain protection of specific modification sites of the depeptidation resin; coupling the branched fragment to a peptide resin; finally, the protective group is removed by cleavage and cleavage, and the long-acting glucagon-like peptide-1 derivative is obtained by mounting with resin.
The specific implementation steps are as follows:
x removal using tetrakis (triphenylphosphine) palladium and PhSiH3 1 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.
The specific implementation steps are as follows:
using tetrakis (triphenylphosphine) palladium and PhSiH 3 Removing main peptide chain X 1 Alloc protection of (Lys); pre-activation of 3, 6-dioxa-suberic acid (B) by condensing agent 2 ) Then, the polypeptide is coupled and uploaded with main peptide chain reaction; then Fmoc mono-protected 3, 6-dioxa-1, 8-octanediamine is added for condensation coupling; removing Fmoc protection by Pip/DMF solution or other alkaline solution; 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 long fatty diacid condensation protected by the tBu group is 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 long-acting GLP-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 GLP-1 polypeptide derivative. All such embodiments are intended to be included within the scope of the present invention.
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 GLP-1 polypeptide derivatives according to the invention into clinically useful pharmaceutical formulations. 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 3.0-5.0. In one embodiment of the invention, the pH of the pharmaceutical formulation is 7.0-8.0. In one embodiment of the invention, the pH of the pharmaceutical formulation is 7.5-8.5. In another embodiment of the invention, the pH of the pharmaceutical formulation is between 5.0 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 G LP-1 polypeptide derivatives of the present 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 agent" 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 long acting glucagon-like peptide-1 derivative as a GLP-1 receptor agonist, said clinical use including, but not limited to, the use in the manufacture of a medicament for the treatment or prevention of at least one of 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 long-acting glucagon-like peptide-1 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 long-acting glucagon-like peptide-1 derivative of 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 these 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 free compound or salt which is acceptable in pharmacy of the GLP-1 receptor agonist polypeptide derivative has positive preventive and therapeutic significance for NAFLD and NASH with complex pathogenesis and related to a plurality of factors related to 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, the present invention has found that the polypeptide derivative has better in vivo long-acting property, and unexpectedly found that the polypeptide derivative structure of the present invention has significantly better pharmaceutical potential in terms of Cmax and AUC, etc., due to the difference from the prior art, especially in one embodiment, the replacement of eicosanoids greatly prolongs half-life (almost twice), and is obviously better than the relevant literature values, and is shown in the content of pharmaceutical effects to some extent.
In the fifth aspect, in the existing GLP-1 research literature, slight changes in the branched structure tend to result in poor activity or poor drug substitution results. The present inventors have surprisingly found that the polypeptide derivatives, despite the change in branched structure (and conservative amino acid substitutions in the peptide chain), have quite good GLP-1 receptor agonistic activity, achieving GLP-1 receptor agonism similar to or better than that of cable 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.
FIG. 2 shows the TC content change in the blood of ob/ob mice in the blood glucose-lowering, weight-lowering and lipid-lowering test.
FIG. 3 shows the variation of TG content in blood of ob/ob mice in the blood glucose-lowering, weight-lowering and lipid-lowering test.
FIG. 4 shows the change in blood LDL levels in the ob/ob mice in the hypoglycemic, weight-reducing and lipid-lowering assays.
FIG. 5 shows the blood HDL level change in ob/ob mice in the blood glucose and weight reducing and lipid lowering test.
Detailed Description
The following examples will provide those skilled in the art with a more complete understanding of the present invention and are not intended to limit the invention in any way.
Example 1: preparation of polypeptide Compound (3)
ESI-MS：[M+5H]818.16，[M+4H]1021.43，[M+3H]1362.88。
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% of morpholine/DCM solution; the octadecanedioic acid condensation of the single protection 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 the solid phase synthetic carrier is Rink Acid resin, and 50% piperazine/DCM solution is used for deprotection of Fmoc, and the Fmoc-protected amino acids are circulated 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 branched fragment condensation to peptide resin using TBTU/DIEA; removing Fmoc protection from 50% piperazine/DCM solution;
4) Cleavage to remove protecting group and resin mounting
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 petroleum ether was added to the concentrate to precipitate the desired product as a white solid.
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 2 preparation of polypeptide Compound (1)
ESI-MS：[M+5H]823.75，[M+4H]1029.43，[M+3H]1372.25
1) Solid phase synthesis of branched fragments;
3, 6-dioxa-suberic acid was dissolved in DCM and mounted to 2-CTC resin under DIEA catalysis; fmoc-3, 6-dioxa-1, 8-octanediamine was coupled to the resin using HATU/DIEA; removing Fmoc protection by 15% pip/DMF solution; condensing Fmoc-Glu-OtBu onto resin with PyBoP/DIEA; removing Fmoc protection by 15% pip/DMF solution; finally, condensation coupling of octadecanedioic acid with single protection of tBu group to amino position of Glu; cleavage with 30% TFE/DCM gives a branched fragment intermediate.
2) Synthesizing peptide chains by a solid phase method;
sequentially coupling Fmoc-protected amino acids to solid phase synthetic resin according to the amino acid sequence of a molecular structure, wherein the solid phase synthetic carrier is Wang resin, and a 15% PIP/DMF solution is used for deprotection of Fmoc, and the Fmoc-protected amino acids are circulated until all peptide chain amino acids are 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 the branched fragment to a peptide resin;
using tetrakis (triphenylphosphine) palladium and PhSiH 3 Removing the Alloc protection of Lys on the peptide chain; the branched fragment intermediates were condensed onto peptide resins using HATU/DIEA coupling.
4) And (3) splitting off the protective group and mounting the protective group on resin to obtain the long-acting glucagon-like peptide-1 derivative.
In proportion (TFA: phenol: 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 diethyl ether was added to the concentrate to precipitate the desired product as an off-white solid. The purification adopts a mode of combining ion chromatography and reverse phase chromatography, and finally the solution is freeze-dried after salt conversion into a free polypeptide compound solution.
Example 3 preparation of polypeptide Compound (4)
ESI-MS：[M+4H]1029.48，[M+3H]1372.26。
1) Synthesizing peptide chains by a solid phase method;
sequentially coupling Fmoc-protected amino acids to solid-phase synthetic resin according to the amino Acid sequence of a molecular structure, wherein the solid-phase synthetic carrier is Rink Acid resin, and 50% ethanolamine/DCM solution is used for removing Fmoc protection; the condensing reagent adopts TBTU/DIEA, and the cycle is carried out until the amino acid of the peptide chain is completely completed; wherein the Lys protecting amino acid of the branched chain fragment to be connected adopts Fmoc-Lys (Dde) -OH as a raw material, and the alpha amino of the final amino acid His adopts Boc group protection;
2) Coupling each module of the branched chain with peptide resin successively by a solid phase method;
removal of the main peptide chain X with 2% hydrazine hydrate/DMF 1 Dde protection of (Lys); pre-activation of 3, 6-dioxa-suberic acid (B) by condensing agent 2 ) Then, the polypeptide is coupled and uploaded with main peptide chain reaction; then Fmoc mono-protected 3, 6-dioxa-1, 8-octanediamine is added for condensation coupling; removing Fmoc protection from 50% ethanolamine/DCM solution; the condensing reagent TBTU/DIEA pre-activates Fmoc-Glu-OtBu, and then is condensed and coupled to the peptide resin; removing Fmoc protection from 50% ethanolamine/DCM solution; the tBu group is singly protected, and octadecanedioic acid is condensed and coupled to the alpha amino position of Glu; thus, the uploading and the synthesis of each module of the whole branched chain are completed.
3) And (3) splitting off the protective group and mounting the protective group on resin to obtain the long-acting glucagon-like peptide-1 derivative.
The cleavage process was similar to that of example 1, and isopropyl ether was used for crystallization during cleavage. The purification adopts a mode of combining ion chromatography and reverse phase chromatography, and finally salt conversion is carried out to form a trifluoroacetate solution 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: taking a proper amount of the test sample medicine, fully dissolving the test sample medicine with water for injection to obtain a test sample solution with the concentration of 0.09mg/m L, preparing the test sample solution before administration, and preserving the test sample solution at the temperature of 2-8 ℃.
The administration dose was 0.18mg/kg, and the administration volume was 2.0mL/kg. Administration is via subcutaneous injection via the back. 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, 0.1ml of blood is taken through the eye socket, placed in an EDTA-K2 anticoagulant tube placed on an ice bath, centrifuged at 4 ℃ and 5000rpm for 10 minutes within 2 hours, blood plasma is taken, temporarily stored in dry ice, and placed in a refrigerator below-60 ℃ for preservation within 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 under the same dosage conditions, cmax and AUC of the compounds (1), (3) and (4) are obviously higher than those of the positive control drug cable Ma Lutai, which means that the compounds can realize or achieve the drug effect with lower or smaller dosage in the research of drug exposure-effect relationship, thereby better ensuring the safety and effectiveness of the drug. In addition, the half life of the compound (4) is obviously prolonged by almost one time, and the long-acting property is obvious, so that the guarantee is provided for improving the medication compliance of patients by reducing the number of times of medication in future clinic.
Example 9ob/ob mice blood glucose and weight reducing and lipid lowering experiments
The 40 ob/ob mice were randomly divided into 5 groups according to fasting body weight, fasting serum TC, LDL, and balance, which are model control group, polypeptide compound (3) group, polypeptide compound (4) group, and cord Ma Lutai group, 8 each. Another 8 normal fed mice were set as normal control group (vehicle).
1 subcutaneous injection every 3 days (except for compound (4)) for 8 times (D0, D3, D6, D9, D12, D15, D18, D21); compound (4) was administered subcutaneously 1 time every 4 days back, and physiological saline (0.15 mL/20 g) was administered to 6 total (D0, D4, D8, D12, D16, D20) model control and normal control groups, and each of the polypeptide compound (3) group, the polypeptide compound (4) group and the cord Ma Lutai group was 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.
(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:
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 smaller than 0.01) during the experimental period, the animals in the normal control group grow 7.1g, the animals in the model control group grow 12.4g, the animals in the polypeptide compound (4) group grow 4.8g, the animals in the polypeptide compound (3) group grow 5.6g, and the animals in the cable Ma Lutai group grow 6.5g.
Starting from D1, the weight of animals in groups (4), (3) and Ma Lutai was reduced, and the weight gain during the experiment (D1-D23) was slow, which was significantly lower than that of the model control group (p < 0.01), and the average of the weights of the groups (4) and (3) was lower than that of the group Ma Lutai, but the effect of certain drug generation advantages on the drug effect was reflected, although there was no statistical difference. The specific results are shown in Table 2.
Table 2: weight results of experimental animals
Note that: compared with the model control group, p is less than 0.05, p is less than 0.01; compared with the positive medicine, #p < 0.05, #p < 0.01.
(2) Lee's index
The Lee's index is an effective index in reflecting the degree of obesity in rats, and there is an increase in Lee's index 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 tested drugs and the positive drugs can inhibit the obesity of the o b/o b mice, the control capacity of the tested group drugs and the positive drugs is close, and no statistical difference exists. The specific results are shown in Table 3.
Table 3: lee's index results in rats
Note that: compared with the model control group, p is less than 0.05, p is less than 0.01; compared with the positive medicine, #p < 0.05, #p < 0.01.
(3) Random blood glucose:
during the experiment, the random blood glucose concentration of the animals increased gradually, possibly in relation to their age and weight gain. Compared with the model control group, the blood glucose value of the normal control group animals D14-D23 is obviously lower than that of the model control group mice, which indicates that the basal blood glucose value of the ob/ob mice is higher. Compared with the mice in the model control group, the D3-D23 polypeptide compound (3) and the polypeptide compound (4) have obvious hypoglycemic effects with different degrees and most have obvious differences. The polypeptide compound (3) and the polypeptide compound (4) were not significantly different from each other in comparison with the cord Ma Lutai. The specific results are shown in Table 4 (mmol/L).
Table 4: random blood sugar change results of experimental animals
Note that: compared with the model control group, p is less than 0.05, p is less than 0.01; compared with the positive medicine, #p < 0.05, #p < 0.01.
(4) Biochemical treatment of blood
(a) TC: the initial TC levels for each group of animals prior to dosing (D0) were relatively close. Following dosing, D11 and D23, the polypeptide compound (3) and polypeptide compound (4) significantly reduced TC levels (p < 0.05) compared to the model control, and their ability to reduce TC was significantly better than cord Ma Lutai, whereas the cord Ma Lutai group was not statistically different from the model group despite the reduced values. The specific results are shown in FIG. 2.
(b) 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. After administration, D11 and polypeptide compound (4) can obviously reduce TG level (p is less than 0.05), and the effect is obviously better than that of the group of the cable Ma Lutai. Similarly, polypeptide compound (3) also significantly reduced TG levels, but no statistical differences compared to the cord Ma Lutai group. Post-dose D23, both the polypeptide compound (3) group and the polypeptide compound (4) group and cord Ma Lutai reduced TG levels, but were not statistically different compared to the model control group. The specific results are shown in FIG. 3.
(c) LDL: pre-dose (D0) TG levels were closer to those of mice of the same strain. After administration, D11 significantly reduced LDL levels (p < 0.01) in both the group of polypeptide compound (3) and polypeptide compound (4) and in cable Ma Lutai compared to the model control group. Following administration of D23, the LDL-lowering effect of the polypeptide compound (3) and the polypeptide compound (4) was better than that of the cable Ma Lutai group (p < 0.05), whereas the cable Ma Lutai group was not statistically different from the model control group. The specific results are shown in FIG. 4.
(d) 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 higher than that of the mice in the normal control group. Both the test and positive drug groups significantly reduced HDL levels (p < 0.05) after dosing with D11 and D23 compared to the model control group. However, the values of the polypeptide compound (3) and the polypeptide compound (4) are lower than those of the positive drug group. Because HDL has an "anti-atherosclerosis" effect, lower values mean that the group of polypeptide compounds (3) and (4) is inferior to the positive group in terms of H D L. The specific results are shown in FIG. 5.
(5) Fat-body weight coefficient
23 days after administration, the mice were dissected, 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 tested drug polypeptide compound (1) and the polypeptide compound (3) can obviously reduce fat under abdomen and around gonads of ob/ob mice, and the effect is not statistically different from the positive drug cable Ma Lutai. In addition, the drug had no effect on the scapula subcutaneous fat-body weight coefficient. The specific results are shown in Table 5.
Table 5: fat-body weight coefficient results
Note that: compared with the model control group, p is less than 0.01
The comprehensive analysis of the experimental data shows that the polypeptide derivative has quite good GLP-1 receptor agonistic activity, achieves G LP-1 receptor agonistic effect similar to or better than that of a positive control medicine cable Ma Lutai, realizes lipid-lowering and blood glucose-lowering effects on ob/ob model mice, and has better therapeutic benefit in certain indexes of blood fat.

Claims (21)

1. A long-acting glucagon-like peptide-1 derivative or pharmaceutically acceptable salt thereof, wherein the long-acting glucagon-like peptide derivative is shown as a formula (I), and X is arranged in a main chain 1 Wherein only one amino group of the side chain thereof is linked to a branched acyl group represented by formula (II) and forms an amide:
7 H-Aib-E- 10 G-T-F-T-S-D-V-S-S-Y- 20 L-E-G-Q-A- 25 A-X 1 -E-F-I- 30 A-W-L-V-X 2 - 35 G-R-G
(I)
wherein Aib is amino isobutyric acid, X 1 Selected from the group consisting of K amino acids, X 2 Selected from K or R amino acids, X 1 Amino groups having only one side chain thereof are linked to the branched acyl group represented by formula (II) and form an amide; n is 7 or 8.
2. The polypeptide derivative or a pharmaceutically acceptable salt thereof according to claim 1, wherein the polypeptide backbone has and only X 1 The side chain epsilon-amino group of the Lys is connected with a branched chain modified by long fatty acid, PEG linker and glutamic acid in an amide bond mode.
3. The long acting glucagon-like peptide-1 derivative of claim 1, wherein said long acting glucagon-like peptide-1 derivative is represented by formula (III):
4. the long acting glucagon-like peptide-1 derivative of claim 1, wherein said long acting glucagon-like peptide-1 derivative is represented by formula (IV):
5. the long acting glucagon-like peptide-1 derivative of claim 1, wherein said long acting glucagon-like peptide-1 derivative is represented by formula (V):
6. the long acting glucagon-like peptide-1 derivative of claim 1, wherein said long acting glucagon-like peptide-1 derivative is represented by formula (VI):
7. a process for the preparation of a long acting glucagon-1 derivative according to any one of claims 1 to 6, characterized in that it 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 the protective group and mounting the protective group on resin to obtain the long-acting glucagon-like peptide-1 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 the protective group and mounting the protective group on resin to obtain the long-acting glucagon-like peptide-1 derivative.
8. A pharmaceutical composition comprising a long acting glucagon-like peptide-1 derivative of any one of claims 1 to 6, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
9. The pharmaceutical composition of claim 8, wherein the pharmaceutically acceptable salt is a free compound of the long acting glucagon-like peptide-1 derivative, or a salt formed by reaction with an inorganic or organic acid, or a salt formed by reaction with an inorganic or organic base; salts with inorganic acids are preferred, salts with hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid are more preferred, or salts with organic acids are preferred, salts with 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 are more preferred; salts with inorganic bases are preferred, salts with sodium, potassium, calcium, magnesium, zinc, iron, etc. are more preferred, or salts with organic bases are preferred, salts with ammonia, arginine, lysine, citrulline, histidine, etc. are more preferred.
10. The pharmaceutical composition of claim 8, wherein the pharmaceutical composition is an aqueous formulation, or a lyophilized formulation, or a ready-to-use dry formulation, and the formulation may comprise one or more of buffers, preservatives, isotonic agents, cosolvents, tonicity agents, chelating agents, stabilizers, antioxidants, surfactants, acid-base modifiers, emulsifiers, metal ions, oleaginous carriers, proteins, and zwitterionic, other pharmaceutical formulation additives, and the like.
11. The pharmaceutical composition of claim 8, wherein the pharmaceutical composition is generally at a concentration of 0.01mg/ml to 50mg/ml and has a pH of 3.0 to 9.0.
12. Use of a long acting glucagon derivative of any one of claims 1 to 6 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition of any one of claims 8 to 11 for the manufacture of a medicament for the treatment and/or prophylaxis of at least one of the following diseases: type II diabetes, impaired glucose tolerance, type I diabetes, obesity, metabolic syndrome.
13. Use of a long acting glucagon derivative of any one of claims 1 to 6 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition of any one of claims 8 to 11 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.
14. Use according to claim 12, characterized in that the use of a long acting glucagon derivative according to any one of claims 1 to 6, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to any one of claims 8 to 11, for the preparation of a medicament for reducing food intake, reducing beta cell apoptosis, increasing islet beta cell function, increasing beta-cell mass and/or restoring glucose sensitivity to beta cells.
15. Use of a long acting glucagon derivative of any one of claims 1 to 6 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition of any one of claims 8 to 11 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 (or a combination of these metabolic risk factors), atherosclerosis, arteriosclerosis, coronary heart disease, peripheral arterial disease and stroke.
16. Use according to claim 15, characterized in that the long acting glucagon 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 8 to 11, is used in obesity-related diseases such as obesity-related inflammation, obesity-related gallbladder diseases and obesity-induced sleep apnea.
17. Use of a long acting glucagon derivative of any one of claims 1 to 6 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition of any one of claims 8 to 11 for the manufacture of a medicament for the treatment and/or prophylaxis 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 long acting glucagon 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 8 to 11 is used in the treatment of diseases related to insulin resistance, fat metabolism disorders and the like.
19. Use of a long acting glucagon derivative of any one of claims 1 to 6 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition of any one of claims 8 to 11 for the manufacture of a medicament for the treatment and/or prophylaxis of at least one of the following diseases: neurodegenerative diseases including Alzheimer's Disease (AD), parkinson's Disease (PD), and the like.
20. Use according to claim 19, characterized in that the use of a long acting glucagon derivative according to any one of claims 1 to 6 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition according to any one of claims 8 to 11 for the preparation of a medicament for reducing beta amyloid plaque precipitation in the brain, reducing neuronal damage caused by oxidative stress, modulating the delivery of a nerve synapse, increasing synaptic plasticity, stimulating axonal ropes, affecting long term potentiation.
21. Use according to claim 19, characterized in that the use of a long acting glucagon derivative according to any one of claims 1 to 6, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to any one of claims 8 to 11, for the preparation of a medicament for activating GLP-1 receptors of the brain, enhancing the function of dopamine attachment, exerting an anti-inflammatory effect, improving energy production and opening cell survival signals.
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