WO2022253202A1 - 一种glp-1r和gipr双重靶向激动作用的多肽衍生物及其制备方法和用途 - Google Patents

一种glp-1r和gipr双重靶向激动作用的多肽衍生物及其制备方法和用途 Download PDF

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WO2022253202A1
WO2022253202A1 PCT/CN2022/096109 CN2022096109W WO2022253202A1 WO 2022253202 A1 WO2022253202 A1 WO 2022253202A1 CN 2022096109 W CN2022096109 W CN 2022096109W WO 2022253202 A1 WO2022253202 A1 WO 2022253202A1
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pharmaceutically acceptable
polypeptide derivative
acceptable salt
stereoisomer
geometric isomer
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PCT/CN2022/096109
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English (en)
French (fr)
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丁伟
张哲峰
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南京知和医药科技有限公司
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Priority to EP22815247.6A priority Critical patent/EP4345105A1/en
Priority to CN202280035107.2A priority patent/CN117440964A/zh
Priority to CA3222051A priority patent/CA3222051A1/en
Publication of WO2022253202A1 publication Critical patent/WO2022253202A1/zh

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    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/26—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
    • A61P3/08—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
    • 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

Definitions

  • the invention belongs to the technical field of medicine, and in particular relates to a polypeptide derivative with double targeting agonism of GLP-1R and GIPR, its preparation method and application.
  • Glucose-dependent insulinotropic polypeptide is a polypeptide composed of 42 amino acids, secreted by K cells mainly located in the proximal small intestine, and is the first incretin discovered.
  • GIP Gastric Inhibitor Polypeptide
  • GIP can act on bone and adipose tissue, inhibit bone reabsorption, and promote lipid synthesis in adipocytes, thereby affecting lipid metabolism and fat distribution, and its effect is independent of insulin action; GIP may also activate hypothalamus GIPR+ neurons, reduced food intake, and weight loss; GIP-treated mice have improved learning and memory abilities, increased levels of brain growth factors that protect nerve cell function, reduced amyloid protein levels associated with Alzheimer's disease, and chronic inflammation And reduce oxidative stress, slow down the apoptosis of nerve cells.
  • Glucagon-like peptide-1 (GLP-1, Glucagon-like peptide 1) is a peptide hormone secreted by human intestinal L cells, which can promote the secretion of insulin and inhibit the secretion of glucagon.
  • the effect of blood glucose concentration is used in the treatment of type 2 diabetes.
  • GLP-1 exerts physiological effects by binding to its specific receptor GLP-1R, and GLP-1R is widely distributed in multiple organs or tissues throughout the body.
  • GLP-1R agonism GLP-1RA
  • GLP-1R GLP-1R agonism
  • GLP-1R is widely distributed and can cooperate with other receptors to regulate the metabolism of key target tissues.
  • DPP-IV dipeptidyl peptidase-IV
  • Eli Lilly's Tirzepatide (LY3298176), as a new drug with dual targeting agonism of GLP-1R and GIPR, is undergoing phase III clinical studies for various indications.
  • Other new peptide drugs with dual-target effects are also under study, such as CN105849122A, CN104470948A, CN105209485A and so on.
  • the purpose of the present invention is to find a polypeptide derivative or its salt and corresponding pharmaceutical composition with dual targeting agonism of GLP-1R and GIPR through in-depth research and creative work, and its long-term effect in vivo far exceeds that of similar products.
  • Peptide compounds at the same time, have significantly stronger drug activity and better clinical value in some aspects at effective doses, making them useful in the treatment of diabetes, weight loss, cardiovascular diabetes, metabolic syndrome, NASH, neurodegenerative diseases such as AD and PD have huge potential.
  • the present invention provides a GLP-1R and GIPR dual-targeted agonist polypeptide derivative or a salt thereof.
  • the GLP-1R and GIPR dual-targeted agonist polypeptide derivative is represented by formula (I), the main chain Xaa 20 in Xaa is Lys and its side chain ⁇ -amino group is connected to X 1 in the branched chain (II) in the form of an amide bond;
  • Aib is aminoisobutyric acid, which is connected with front and back amino acids with amide bond (peptide bond);
  • Xaa 13 is Aib, Tyr or Ala
  • n in formula (II) is 16 or 18; X 1 and X 2 are simultaneously or x2 for At the same time X1 is
  • n in formula (II) is 16.
  • n in formula (II) is 18
  • X 2 is At the same time X1 is
  • polypeptide derivative is the following formula (1):
  • polypeptide derivative is the following formula (2)
  • polypeptide derivative is the following formula (3)
  • polypeptide derivative is the following formula (4)
  • polypeptide derivative is the following formula (5)
  • polypeptide derivative is the following formula (6):
  • polypeptide derivative is the following formula (7)
  • polypeptide derivative is the following formula (8)
  • polypeptide derivative is the following formula (9)
  • polypeptide derivative is the following formula (10)
  • the glutamine in the branched chain (II) is in the (L)-configuration.
  • the compounds of the invention may exist in particular geometric or stereoisomeric forms.
  • the present invention contemplates all such compounds, including cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers isomers, (D)-isomers, (L)-isomers, and their racemic and other mixtures, such as enantiomerically or diastereomerically enriched mixtures, all of which are subject to the present within the scope of the invention. All such isomers, as well as mixtures thereof, are included within the scope of the present invention.
  • the amino acid in the polypeptide derivative is preferably an L-type amino acid.
  • the present invention provides a method for preparing the above-mentioned polypeptide derivative, the preparation method comprising the following step A:
  • the present invention provides another preparation method of the above-mentioned polypeptide derivative, and the preparation method comprises the following step B:
  • the specific preparation process step A is described as follows:
  • the scheme for synthesizing branched chain fragments is as follows:
  • the scheme for synthesizing branched chain fragments is as follows:
  • X 1 is X2 is When Xaa 20 is K mounting branch chain,
  • TFA removes Boc protection
  • Fmoc-Glu-OtBu is condensed and coupled with the previous step product
  • Pip solution or other alkaline solution removes Fmoc protection
  • tBu group monoprotected octadecane The diacid is condensed and coupled to the amino position of Glu in the previous step; finally, the benzyl ester is removed by hydrogen hydrolysis under the catalysis of palladium carbon, exposing the carboxyl group that needs to be combined with the peptide chain.
  • the scheme of the synthetic main chain is as follows:
  • the Fmoc protected amino acids are sequentially coupled to the solid-phase synthetic resin in reverse order, the Pip solution or other alkaline solution is used to protect the Fmoc, and the cycle is completed until all the amino acids of the main chain of the peptide chain are completed; wherein the side chains to be connected
  • the Lys of the fragment is protected by Alloc (optionally, using the raw material Fmoc-Lys(Alloc)-OH) or Dde protected (optionally, using the raw material Fmoc-Lys(Dde)-OH); the terminal filament of the molecular structure of the present invention
  • Amino acid amide use Rink Amide-AM resin, Rink Amide-MBHA resin, Sieber resin, PAM resin, Rink Amide resin and other resins that can build amide amino acids.
  • the scheme for coupling the branched chain to the peptide chain is as follows:
  • Depeptidation resin Lys side chain protection such as Xaa 20 when the side chain protecting group is Alloc, adopt tetrakis (triphenylphosphine) palladium and PhSiH 3 to remove the Alloc protection; such as Xaa 20 when the side chain protecting group is Dde, Use hydrazine hydrate to remove the Dde protection; couple the branched chain fragments to the peptide resin; Pip solution or other basic solution to remove the Fmoc protection of the first amino acid.
  • side chain protection such as Xaa 20 when the side chain protecting group is Alloc
  • tetrakis (triphenylphosphine) palladium and PhSiH 3 to remove the Alloc protection
  • Xaa 20 when the side chain protecting group is Dde Use hydrazine hydrate to remove the Dde protection
  • couple the branched chain fragments to the peptide resin Pip solution or other basic solution to remove the Fmoc protection of the first amino acid.
  • Cleavage and cleavage remove the protective group and resin mount, and obtain the crude product of the dual-target polypeptide derivative.
  • the lysis protocol is as follows:
  • TFA lysate in a certain ratio, add the fully protected peptide resin to the lysate, cut it from the resin and remove the side chain. Vacuum rotary evaporation removes TFA, ethers or alkane solvents are added to the concentrated solution to precipitate a white solid, which is the target product.
  • Chromatographic purification perform multi-step reverse-phase chromatography purification or ion chromatography purification, and finally turn salt into a certain acid salt solution or free form solution; and 6) freeze-drying.
  • the scheme for synthesizing the main chain in step B is the same as the scheme for synthesizing the main chain in step A, and the amino terminal of the first amino acid Tyr is protected with Boc.
  • the step B solid-phase method is to sequentially couple each module of the branched chain to the peptide resin as follows:
  • This step is carried out by means of polypeptide solid-phase synthesis, and the structural sequence of each branched chain module of the present invention is sequentially and reversely connected to the main chain of polypeptide on the resin.
  • Tetrakis(triphenylphosphine)palladium and PhSiH 3 remove the Alloc protection of the main peptide chain X 2 (Lys) or the Dde protection with hydrazine hydrate; the condensation reagent preactivates 3,6-dioxa-suberic acid (X 1 ), then coupled with the main peptide chain reaction coupling upload; then add Fmoc mono-protected 3,6-dioxa-1,8-octanediamine (X 2 ) condensation coupling; Pip/DMF solution or other Alkaline solution removes Fmoc protection of X2 ; condensation reagent preactivates Fmoc-Glu-OtBu, and then condensation coupling; Pip/DMF solution or other alkaline solution removes Fmoc protection; tBu group monoprotected octadecane (or Eicosane) dioic acid is condensed and coupled to the ⁇ -amino position of Glu; so far,
  • the condensation coupling mentioned in the article refers to the condensation coupling reaction between carboxylic acid and amino group, and DCC/HOBt, DIC/Cl-HOBt, TBTU/DIEA, HBTU/DIEA, PyBOP can be used according to specific conditions (such as solid-phase method of polypeptide synthesis) /DIEA, EDC/HOBt, HATU/DIEA, T3P/DIEA, DEPBT/TEA and other condensing agents in organic synthesis.
  • HBTU O-benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate
  • HATU 2-(7-Azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate
  • DEPBT 3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4-one
  • T3P 1-Propylphosphoric Anhydride
  • Pd[P(C 6 H 5 ) 3 ] 4 Tetrakis(triphenylphosphine)palladium
  • the present invention discloses a pharmaceutical composition, which comprises the compound of the present invention or a pharmaceutically acceptable salt thereof as an active ingredient or main active ingredient, and a pharmaceutically acceptable carrier.
  • a part of the present invention is a pharmaceutically acceptable salt
  • suitable "pharmaceutically acceptable salt” includes the free compound of the present invention, the conventional non-toxic salt of the compound of the present invention formed by the reaction of an inorganic acid or an organic acid, and an inorganic base Or the conventional non-toxic salts of the compounds of the present invention formed by the reaction with organic bases.
  • salts derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, etc.
  • 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, ethane Salts of disulfonic acid, oxalic acid, isethionic acid, trifluoroacetic acid, etc.
  • 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
  • salts derived from inorganic bases such as sodium, potassium, calcium, magnesium, zinc, iron, etc.
  • salts derived from organic bases such as ammonia, arginine, lysine, citrulline, histidine, etc. Salt.
  • the salts stated in the present invention include salts containing acids or bases formed by the reaction of small amounts of acid or base compounds used for pharmaceutical purposes.
  • Pharmaceutically acceptable salts are preferably sodium-containing salts.
  • the formulation may further contain buffers, preservatives, isotonic agents, solubilizers, tonicity agents, chelating agents, stabilizers, antioxidants, surfactants, acid-base regulators and the like.
  • concentration is usually 0.01 mg/ml to 50 mg/ml, wherein the formulation has a pH of 3.0 to 9.0.
  • the pharmaceutical formulation is an aqueous formulation, ie an aqueous solution, usually a solution, emulsion or suspension.
  • the pharmaceutical preparation is a freeze-dried preparation, which is prepared by adding a solvent and/or diluent to fully dissolve it before use.
  • the pharmaceutical preparation is a ready-to-use dry preparation that does not need to be pre-dissolved, such as spray inhalation freeze-dried powder and the like.
  • the selection of the pH range of the pharmaceutical preparation is very important, which will affect the solubility and stability of the polypeptide derivatives with dual targeting agonism of GLP-1R and GIPR. Physical aggregation or adsorption of peptides will occur.
  • the pH value of the pharmaceutical preparation is 3.0-5.0.
  • the pH value of the pharmaceutical preparation is 7.0-8.0.
  • the pH of the pharmaceutical formulation is 7.5-8.5.
  • the pH of the pharmaceutical formulation is 5.0-7.5.
  • the buffering agent is selected from disodium hydrogen phosphate, sodium acetate, etc.
  • the preservative is selected from phenol, o-cresol, m-cresol, p-cresol, etc.
  • the isotonic agent is selected from sodium chloride Salt, sugar or sugar alcohol, amino acid, propylene glycol, mannitol, etc., co-solvent selected from mannitol, propylene glycol, PEG, glycerin, Tween, ethanol, etc., tonicity agent selected from sodium chloride salt, propylene glycol, glycerin, manna Alcohol, etc.
  • the chelating agent is selected from EDTA, citrate, etc.
  • the stabilizer is selected from creatinine, glycine, nicotinamide, PEG, etc.
  • the antioxidant is selected from sodium bisulfite, sodium sulfite, cysteine, methionine, etc.
  • the surfactant is selected from polysorb
  • compositions can be present in the pharmaceutical preparation of the polypeptide derivative of the present invention according to the needs of the pharmaceutical preparation (such as long-term stability), including emulsifiers, metal ions, oily carriers, proteins (such as human serum albumin, gelatin or protein, etc. ) and zwitterions (such as arginine, glycine, lysine, histidine, betaine and taurine, etc.) and other pharmaceutical preparation additives.
  • pharmaceutically acceptable carrier refers to any preparation carrier or medium that can deliver an effective amount of the active substance of the present invention, does not interfere with the biological activity of the active substance, and has no toxic side effects on the host or patient.
  • Representative carriers include water, oil , liposomes, etc.
  • the term "effective amount” or “therapeutically effective amount” refers to a non-toxic but sufficient amount of the drug or agent to achieve the desired effect.
  • active ingredient refers to a chemical entity that is effective in treating the disorder, disease or condition of interest.
  • the diseases include metabolic syndrome, obesity, diabetes, obesity-related diseases, and diabetes-related diseases.
  • Diabetes mellitus includes a group of metabolic diseases characterized by hyperglycemia due to defects in insulin secretion, insulin action, or both. Diabetes is divided into type I diabetes, type II diabetes and gestational diabetes according to the disease mechanism.
  • the GLP-1R and GIPR dual-targeted agonistic polypeptide derivatives of the present invention and pharmaceutically acceptable salts thereof can combine the effects of GIP (for example, the effects on fat metabolism and weight loss and blood sugar) with the effects of GLP-1 (e.g., effects on blood glucose levels and food intake) combination. Therefore, it can be used to accelerate the elimination of excess adipose tissue, cause sustained weight loss, and improve blood sugar control.
  • Dual targeted agonism of GLP-1R and GIPR can also be used to reduce cardiovascular risk factors such as high cholesterol, such as high LDL cholesterol.
  • the GLP-1R and GIPR dual-targeted agonistic polypeptide derivatives of the present invention and their pharmaceutically acceptable salts can also be used for the treatment of insulin resistance, impaired glucose tolerance, prediabetes, elevated fasting blood sugar, type II diabetes, hypertensive Blood pressure, 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 effects 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.
  • the GLP-1R and GIPR dual-targeted agonistic polypeptide derivatives of the present invention and pharmaceutically acceptable salts thereof can be used in diseases such as obesity-related inflammation, obesity-related gallbladder disease, and obesity-induced sleep apnea, etc.
  • Obesity-related diseases play a therapeutic role.
  • it also includes the application in the preparation of drugs for delaying the effect of treating type II diabetes and/or preventing the exacerbation of type II diabetes, and a method for improving blood sugar control in adults with type II diabetes, including administering an effective amount of Use of the above-mentioned polypeptide derivatives as dietary and sports supplements.
  • the GLP-1R and GIPR dual-targeted agonist polypeptide derivatives and the pharmaceutically acceptable salts thereof in the present invention have active prevention and treatment significance for NASH, which is related to multiple metabolic factors and has complicated pathogenesis. Insulin resistance and disturbances in fat metabolism constitute early damage to the liver, resulting in the formation of fat deposits in liver cells (NAFLD). With the development of the disease and the formation of the body's immune regulation, liver cells produce an inflammatory response, which in turn promotes the formation of fibrosis, eventually leading to symptoms of end-stage liver disease such as cirrhosis.
  • the GLP-1R and GIPR dual-targeted agonistic polypeptide derivatives of the present invention can effectively regulate blood sugar levels and participate in metabolism, and can fully or partially mediate NASH, or can be independent of the above effects.
  • the metabolic syndrome of the present invention may be diabetes or diabetes-related diseases, or obesity or obesity-related diseases, or NAFLD, NASH or related diseases.
  • the obesity-diabetes link is well known, so these conditions are not independent or mutually exclusive, but may combine.
  • NAFLD, NASH or related diseases may not be independent or mutually exclusive because of complex mechanisms but also involve and correlate with obesity and diabetes.
  • the GLP-1R and GIPR dual-targeted agonistic polypeptide derivatives of the present invention and pharmaceutically acceptable salts thereof are used for the prevention of neurodegenerative diseases such as AD (Alzheimer's syndrome) or PD (Parkinson's syndrome) Positive with treatment.
  • AD Alzheimer's syndrome
  • PD Parkinson's syndrome
  • the experimental research of the present invention found that the GLP-1R and GIPR dual-targeting agonistic polypeptide derivatives of the present invention unexpectedly have very good in vivo long-term growth after conservative or non-conservative amino acid substitutions and branched chain modifications in the prior art. Effectiveness, its half-life was significantly better than the control drug Tirzepatide with statistical significance.
  • the published data of the control drug Tirzepatide shows that the average clinical half-life of the human body is 5.0 days. According to the results of the SD rat pharmacokinetic experiment in this experiment, the human half-life of the polypeptide derivative (1) can be deduced to be 7.88 days, which means that patients can be administered for half a month.
  • One-time medicine greatly facilitates clinical application.
  • the experimental research of the present invention found that the GLP-1R and GIPR dual targeting agonist polypeptide derivatives of the present invention can effectively activate GIPR and GLP- 1R, which has dual-target agonistic activity and appropriate activity matching and pharmacodynamic effect, is similar to the control drug Tirzepatide, and unexpectedly found that some active pharmacodynamic effects are significantly stronger than Tirzepatide and have statistical significance, such as TC (total cholesterol) reduction.
  • TC total cholesterol
  • Embodiment 1 prepares polypeptide derivative (1)
  • Select Rink Amide-MBHA resin to sequentially couple the Fmoc protected amino acids to the solid-phase synthetic resin according to the amino acid sequence of the peptide chain of the polypeptide derivative (1) from the C-terminus to the N-terminus, deprotect the Fmoc with 20% Pip/NMP solution, and cycle until GLP-1 main chain amino acids are all completed; Fmoc-Lys(Dde)-OH is selected when condensing the Lys of the branched chain fragments to be connected to the peptide resin.
  • step 1) side chain fragment and step 2) peptide resin coupling using condensing agent HATU/DIEA; 20% Pip/DMF solution to remove first amino acid Fmoc protection.
  • the trifluoroacetic acid polypeptide derivative (1) was obtained as a white or off-white loose solid.
  • Embodiment 2 prepares polypeptide derivative (1)
  • Fmoc protected amino acids were coupled to Rink Amide-AM resin in reverse order, and the condensation coupling reagent was PyBOP/DIEA or DIC/Cl-HOBt, 20% piperidine/DMF solution Deprotect Fmoc, and cycle until all amino acids complete the peptide chain; the Lys of the side chain fragment to be connected is protected by Alloc, that is, Fmoc-Lys(Alloc)-OH. Boc-Tyr(tBu)-OH was selected as the first amino acid Tyr position.
  • the acetic acid polypeptide derivative (1) was obtained as a white or off-white loose solid.
  • Embodiment 3 SD rat pharmacokinetic experiment
  • the peak integration method of the spectrum sample is automatic integration; the ratio of the peak area of the sample to the peak area of the internal standard is used as the index, and the concentration of the sample is used for regression. Regression method: linear regression, the weight coefficient is 1/X 2 .
  • Pharmacokinetic parameters were analyzed with WinNonlin Professional v6.3 (Pharsight, USA) using non-compartmental models.
  • C max is the measured maximum blood drug concentration, the area under the blood drug concentration-time curve AUC (0 ⁇ t) is calculated by the trapezoidal method, and T max is the peak time of blood drug concentration after administration.
  • the pharmacokinetic parameters after administration were calculated using the WinNonlin V6.3 non-compartmental model, as shown in the table below.
  • mice 32 ob/ob mice were randomly divided into 4 groups according to fasting body weight, fasting serum TC and LDL, which were model control group, test sample group (1.35mg/kg) and positive control group (Tirzepatide, 1.35mg/kg). kg), 8 in each group. The other 8 conventionally fed mice were set as normal control group (vehicle).
  • Body weight before grouping, fasting and water should be measured once for 12 hours, and random grouping was carried out according to body weight, TC and LDL balance. After grouping, start from D0 and measure once a day.
  • Body length was measured once before the first administration and on D23. Body length is the maximum straight line length from the tip of the nose to the anus. Adjust the body position of the mouse so that its body is in a stretched state, and read the body length of the mouse with a ruler scale.
  • Lee's index [body weight (g) ⁇ 10 3 /body length (cm)] 1/3 .
  • Fat-body weight coefficient After the mice were euthanized, abdominal subcutaneous fat, perigonadal fat, and scapular subcutaneous fat were removed and weighed to calculate the fat-body weight coefficient.
  • the initial body weight of the polypeptide compound (1) group was lower than that of the model control group (D0, p ⁇ 0.05), and there was no statistical difference with the Tirzepatide group. From D1 onwards, the weight of the animals in the polypeptide compound (1) and Tirzepatide groups decreased, and the body weight increased slowly during the experiment (D1-D23), which were significantly lower than the model control group (p ⁇ 0.01); the weight of the polypeptide compound (1) group The average value was slightly higher than that of Tirzepatide group, but there was no statistical difference.
  • Lee's index is an effective index to reflect the degree of obesity in rats.
  • the Lee's index of all animals increased, and the increase of the animals in the normal group was the least.
  • the Lee's index of animals in each administration group decreased slightly. The results show that both the test drug and the positive control drug can inhibit the obesity of ob/ob mice, and the control ability of the test group drug and the positive control drug is close, and there is no statistical difference.
  • TC The initial value of the polypeptide compound (1) before the experiment (D0) was higher than that of the model group compared with other mice of the same strain. Compared with self-administration at different times, the TC of obese mice in the polypeptide compound (1) and Tirzepatide groups decreased significantly after administration, indicating that the test drug and the positive control drug can significantly reduce the TC content. From the results of D11 and D23, it can be seen that the ability of the polypeptide compound (1) to reduce TC is similar to that of Tirzepatide. And when the initial value of the polypeptide compound (1) group was higher, the ability to reduce TC was stronger, approaching the level of the Tirzepatide group on D23, and the statistical significance was significantly improved from ##p ⁇ 0.01 to #p ⁇ 0.05.
  • the polypeptide compound (1) can significantly reduce the abdominal subcutaneous and perigonadal fat of ob/ob mice, and the effect is not significantly different from that of the positive control drug Tirzepatide. In addition, the drug had no effect on the scapular subcutaneous fat-to-body mass coefficient.
  • the drinking water of the experimental animals in each group is quite different, and the drinking water of the treatment group is lower than that of the normal group and the model control group. It can be clearly observed that the water consumption of animals decreases after administration, and the effect is most obvious after the first administration.
  • the water consumption of animals in each group increased significantly on D11, which may be related to the subsequent fasting operation, but the water consumption of mice in each group increased.
  • the experimental results show that both the test drug and the positive control drug have obvious effects on drinking water control.

Abstract

一种GLP-1R和GIPR双重靶向激动作用的多肽衍生物及其制备方法和用途，如式(Ⅰ)所示，(Ⅱ)为肽链的支链修饰结构。所述多肽衍生化合物及其药学上可接受的盐可用于治疗糖尿病、减肥、NASH等代谢性相关疾病、神经退行性疾病等领域。 Tyr-Aib-Asp-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-Xaa 13-Leu-Asp-Lys-Ile-Ala-Gln-Xaa 20-Glu-Phe-Val-Gln-Trp-Leu-Leu-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2 (I)

Description

一种GLP-1R和GIPR双重靶向激动作用的多肽衍生物及其制备方法和用途 技术领域
本发明属于医药技术领域，尤其涉及一种作为GLP-1R和GIPR双重靶向激动作用的多肽衍生物及其制备方法和用途。
背景技术
葡萄糖依赖性促胰岛素多肽(抑胃多肽，GIP，Gastric Inhibitor Polypeptide)是一种42个氨基酸组成的多肽，由主要位于近端小肠的K细胞分泌，是第一个被发现的肠促胰素。在早期的研究中，T2DM患者急性输注GIP引发的促胰岛素分泌效应减弱，限制了其作为药物开发靶点的可能性。但近年来的研究发现，T2DM患者在接受降糖药物治疗，改善血糖控制后，GIP的促胰岛素作用可以得到恢复。此外，GIP能作用于骨和脂肪组织，抑制骨的重吸收，并促进脂肪细胞的脂质合成，进而影响脂代谢与脂肪分布，其效应独立于胰岛素作用；GIP还可能通过激活下丘脑中的GIPR+神经元，减少食物摄入，减轻体重；GIP治疗小鼠的学习记忆能力得到改善，保护神经细胞功能的大脑生长因子水平上升、和阿尔茨海默症相关的淀粉样蛋白数量减少、慢性炎症和氧化应激减少、神经细胞凋亡速度减慢。
胰高血糖素样肽-1(GLP-1，Glucagon-like peptide 1)是由人肠道L细胞分泌的一种肽类激素，能够促进胰岛素的分泌、抑制胰高血糖素的分泌，具有降低血糖浓度的功效，被用于II型糖尿病的治疗。GLP-1通过与其特异性受体GLP-1R结合发挥生理效应，而GLP-1R广泛分布于全身多个器官或组织，循证医学证据也证实GLP-1R激动作用(GLP-1RA)除了治疗糖尿病和肥胖外，对心血管系统、肾脏、中枢神经系统均有保护作用。GLP-1R分布广泛，可以与其他受体共同调节关键靶组织的代谢。
然而天然GLP-1和GIP在体内不稳定，易被二肽基肽酶-IV(DPP-IV)快速降解。
礼来制药公司的Tirzepatide(LY3298176)作为GLP-1R和GIPR双重靶向激动作用的新药正在进行各种适应症的临床三期研究。2022年5月14日，FDA批准礼来(Eli Lilly)公司开发的Mounjaro(tirzepatide)上市，用于与控制饮食和锻炼联用，改善成人2型糖尿 病患者的血糖控制。其它双靶点作用的多肽新药也在研究中，如CN105849122A、CN104470948A、CN105209485A等等。
已有的研究结果发现，GLP-1R和GIPR双重靶向激动活性的合理搭配是此类药物的核心，不同的活性搭配产生了不同的药理作用与不良反应。同时不同的支链修饰对这类GLP-1R和GIPR双重靶向激动活性和药效半衰期等产生不同的效果。本领域仍然渴望对GLP-1R和GIPR双重靶向激动作用的多肽在治疗糖尿病、减肥、心血管合并糖尿病、NASH、神经退行性疾病如AD和PD等具有巨大的治疗潜能。
发明内容
本发明的目的在于通过深入的研究和创造性的劳动，发现一种GLP-1R和GIPR双重靶向激动作用的多肽衍生物或其盐以及对应的药物组合物，其体内长效性远远超过同类多肽化合物，同时有效剂量下在某些方面具有明显更强的药物活性与更好的临床价值，使其在治疗糖尿病、减肥、心血管合并糖尿病、代谢综合征、NASH、神经退行性疾病如AD和PD等领域具有巨大的潜能。
本发明提供了一种GLP-1R和GIPR双重靶向激动作用的多肽衍生物或其盐，所述GLP-1R和GIPR双重靶向激动作用的多肽衍生物为式(I)所示，主链中Xaa 20为Lys且其侧链ε-氨基以酰胺键形式连接支链(II)式中的X 1；
Tyr-Aib-Asp-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-Xaa 13-Leu-Asp-Lys-Ile-Ala-Gln-Xaa 20-Glu-Phe-Val-Gln-Trp-Leu-Leu-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2
(Ⅰ)
Figure PCTCN2022096109-appb-000001
其中，Aib为氨基异丁酸，与前后氨基酸以酰胺键(肽键)连接；
Xaa 13为Aib，Tyr或Ala；
另有，(II)式中n为16或18；X 1与X 2同时为
Figure PCTCN2022096109-appb-000002
或X 2为
Figure PCTCN2022096109-appb-000003
同时X 1为
Figure PCTCN2022096109-appb-000004
在本发明的一种实施方案中，所述多肽衍生物中，(II)式中n为16。
在本发明的一种实施方案中，所述多肽衍生物中，(II)式中n为18
在本发明的一种实施方案中，所述多肽衍生物中，(II)式中X 1与X 2同时为
Figure PCTCN2022096109-appb-000005
在本发明的一种实施方案中，所述多肽衍生物中，(II)式中X 2为
Figure PCTCN2022096109-appb-000006
同时X 1为
Figure PCTCN2022096109-appb-000007
在本发明的一种实施方案中，所述多肽衍生物为下列式(1)：
Figure PCTCN2022096109-appb-000008
在本发明的一种实施方案中，所述多肽衍生物为下列式(2)
Figure PCTCN2022096109-appb-000009
在本发明的一种实施方案中，所述多肽衍生物为下列式(3)
Y(Aib)DGTFTSDYSI(Aib)LDKIAQKAFVQWLLAGGPSSGAPPPS-NH2
Figure PCTCN2022096109-appb-000010
在本发明的一种实施方案中，所述多肽衍生物为下列式(4)
Figure PCTCN2022096109-appb-000011
在本发明的一种实施方案中，所述多肽衍生物为下列式(5)
Figure PCTCN2022096109-appb-000012
在本发明的一种实施方案中，所述多肽衍生物为下列式(6)
Figure PCTCN2022096109-appb-000013
在本发明的一种实施方案中，所述多肽衍生物为下列式(7)
Figure PCTCN2022096109-appb-000014
在本发明的一种实施方案中，所述多肽衍生物为下列式(8)
Figure PCTCN2022096109-appb-000015
在本发明的一种实施方案中，所述多肽衍生物为下列式(9)
Figure PCTCN2022096109-appb-000016
在本发明的一种实施方案中，所述多肽衍生物为下列式(10)
Figure PCTCN2022096109-appb-000017
在本发明的一种实施方案中，支链(II)式中谷氨酰胺为(L)-构型。
本发明的化合物可以存在特定的几何或立体异构体形式。本发明设想所有的这类化合物，包括顺式和反式异构体、(-)-和(+)-对映体、(R)-和(S)-对映体、非对映异构体、(D)-异构体、(L)-异构体，及其外消旋混合物和其他混合物，例如对映异构体或非对映体富集的混合物，所有这些混合物都属于本发明的范围之内。所有这些异构体以及它们的混合物，均包括在本发明的范围之内。在本发明的实施方案中，作为本发明的优选实施方式，所述的多肽衍生物其中的氨基酸优选为L型氨基酸。
除非天然氨基酸外，文中表述的天然氨基酸均为国际通用单字母或三字母缩写，如S或Ser代表丝氨酸，P或Pro代表脯氨酸等。
另一方面，本发明提供了一种上述多肽衍生物的制备方法，所述的制备方法包括如下步骤A：
1)固相或液相法合成支链片段；
2)固相法合成肽链；
3)将支链片段与肽树脂偶联；
4)裂解除去保护基团与树脂挂载，得到多肽衍生物。
本发明提供了另一种上述多肽衍生物的制备方法，所述的制备方法包括如下步骤B：
1)固相法合成肽链；
2)固相法将支链各模块逐次与肽树脂偶联；
3)裂解除去保护基团与树脂挂载，得到多肽衍生物。
在一些实施例中，所述具体制备过程步骤A描述如下：
1)固相或液相法合成支链片段；
在一些实施例中，所述合成支链片段的方案如下所示：
将Fmoc-AEEA-OH(2-(2-(芴甲氧羰基-2-氨基乙氧基)乙氧基)乙酸缩合偶连到2-CTC树脂上；Pip(哌啶)/DMF(N,N-二甲基甲酰胺)溶液或其它碱性溶液脱Fmoc保护；将第二个Fmoc-AEEA-OH缩合偶联到树脂上；Pip(哌啶)/DMF(N,N-二甲基甲酰胺)溶液或其它碱性溶液脱Fmoc保护；将Fmoc-Glu-OtBu缩合偶联到树脂上；tBu基团单保护的十八烷二酸偶联到树脂上；采用1％TFA(三氟乙酸)的DCM溶液将片段切落，暴露出需要与肽链结合的羧基。
在一些实施例中，所述合成支链片段的方案如下所示：
X 1为
Figure PCTCN2022096109-appb-000018
X 2为
Figure PCTCN2022096109-appb-000019
Xaa 20为K挂载支链时，
将
Figure PCTCN2022096109-appb-000020
与
Figure PCTCN2022096109-appb-000021
在弱有机碱条件下缩合偶联，TFA脱除Boc保护；将Fmoc-Glu-OtBu与前一步产物缩合偶联，Pip溶液或其它碱性溶液脱Fmoc保护；tBu基团单保护的十八烷二酸缩合偶联到前一步Glu的氨基位置；最后采用钯碳催化作用下氢气水解脱除苄酯，暴露出需要与肽链结合的羧基。
2)固相法合成肽链；
在一些实施例中，所述合成主链的方案如下所示：
按本发明分子结构的氨基酸顺序将Fmoc保护氨基酸依次逆序偶联到固相合成树脂上，Pip溶液或其它碱性溶液去保护Fmoc，循环直至肽链主链氨基酸全部完成；其中将待连接侧链片段的的Lys采用Alloc保护(可选地，采用原料Fmoc-Lys(Alloc)-OH)或Dde保护(可选地，采用原料Fmoc-Lys(Dde)-OH)；本发明分子结构的末端丝氨酰胺，采用Rink Amide-AM树脂、Rink Amide-MBHA树脂、Sieber树脂、PAM树脂、Rink Amide树脂等可构建酰胺型氨基酸的树脂。
3)将支链片段与肽树脂偶联；
在一些实施例中，所述支链与肽链偶联的方案如下：
脱肽树脂Lys侧链保护，如Xaa 20为侧链保护基为Alloc时，采用四(三苯基膦)钯和PhSiH 3脱去的Alloc保护；如Xaa 20为侧链保护基为Dde时，采用水合肼脱去的Dde保护；将支链片段与肽树脂偶联；Pip溶液或其它碱性溶液脱首位氨基酸Fmoc保护。
4)裂解除去保护基团与树脂挂载
裂解切割除去保护基团与树脂挂载，得到双靶点多肽衍生物粗品。
在一些实施例中，所述裂解的方案如下：
按一定比例配制TFA裂解液，将全保护肽树脂加入裂解液，从树脂上切割下来并脱除侧链。真空旋蒸除去TFA，将醚类或烷烃类溶剂加入浓缩液析出白色固体即目标产物。
可选地，进一步包括：
5)色谱纯化：进行多步反相色谱纯化或离子色谱纯化，最后转盐成某种酸的盐溶液或游离型溶液；和6)冻干。
在一些实施例中，所述步骤B合成主链的方案与步骤A合成主链的方案相同，首位氨基酸Tyr采用Boc保护氨基端。
在一些实施例中，所述步骤B固相法将支链各模块逐次与肽树脂偶联的方案如下：
该步骤采用多肽固相合成的方式进行，按本发明支链各模块结构顺序依次逆序连接到树脂上的多肽主链。先脱去Xaa 20的侧链保护，将X 1连接到Xaa 20，将X 2连接到X 1，将谷氨酸侧链连接到X 2，最后连接十八烷(或二十烷)二酸单叔丁酯完成整个支链的上载与合成。
如X 1和X 2为
Figure PCTCN2022096109-appb-000022
结构，Xaa 20为K挂载支链时，
采用四(三苯基膦)钯和PhSiH 3脱去主肽链Xaa 20(Lys)的Alloc保护或采用水合肼脱去的Dde保护；用缩合试剂激活Fmoc-AEEA-OH(X 1)的羧酸，缩合偶联到肽链，Pip溶液或其它碱性溶液脱Fmoc保护；用缩合试剂激活Fmoc-AEEA-OH(X 2)的羧酸，缩合偶联到X 1，Pip溶液或其它碱性溶液脱首位氨基酸Fmoc保护；用缩合试剂将Fmoc-Glu-OtBu的羧酸激活，缩合偶联到X 2，Pip溶液或其它碱性溶液脱Fmoc保护；tBu基团单保护的十八烷(或二十烷)二酸缩合偶联到Glu的α氨基位置；至此完成整个支链的上载与合成。
如X 1为
Figure PCTCN2022096109-appb-000023
X 2为
Figure PCTCN2022096109-appb-000024
Xaa 20为K挂载支链时，
采用四(三苯基膦)钯和PhSiH 3脱去主肽链X 2(Lys)的Alloc保护或采用水合肼脱去的Dde保护；缩合试剂预活化3,6-二氧杂-辛二酸(X 1)，然后与主肽链反应偶联上载；然后加入Fmoc单保护的3,6-二氧杂-1,8-辛二胺(X 2)缩合偶联；Pip/DMF溶液或其它碱性溶液脱去X 2的Fmoc保护；缩合试剂预活化Fmoc-Glu-OtBu，再缩合偶联；Pip/DMF溶液或其它碱性溶液脱Fmoc保护；tBu基团单保护的十八烷(或二十烷)二酸缩合偶联到Glu的α氨基位置；至此完成整个支链的上载与合成。
文中所述缩合偶联是指羧酸与氨基的缩合偶联反应，可根据具体情况(如固相法多肽合成)使用DCC/HOBt，DIC/Cl-HOBt，TBTU/DIEA，HBTU/DIEA，PyBOP/DIEA，EDC/HOBt，HATU/DIEA，T3P/DIEA，DEPBT/TEA等有机合成中的缩合剂。
文中部分缩写符号释义如下所示。
2-CTC树脂：
Figure PCTCN2022096109-appb-000025
Sieber树脂：
Figure PCTCN2022096109-appb-000026
Rink Amide-AM树脂：
Figure PCTCN2022096109-appb-000027
Fmoc：芴甲氧羰基
EDC：1-(3-二甲胺基丙基)-3-乙基碳二亚胺
DIC：N,N'-二异丙基碳二亚胺
HOBt：1-羟基苯并三氮唑
DCC：N,N'-二环丙基碳二亚胺
Cl-HOBt：6-氯-1-羟基苯并三氮唑
HBTU：O-苯并三氮唑-N,N,N',N'-四甲基脲六氟磷酸盐
TBTU：O-苯并三氮唑-N,N,N',N'-四甲基脲四氟硼酸酯
PyBop：六氟磷酸苯并三唑-1-基-氧基三吡咯烷基磷
HATU：2-(7-氮杂苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯
TIS：三异丙基硅烷
DIEA：二异丙基乙胺
Pip：哌啶(六氢吡啶)
OtBu：叔丁酯
DCM：二氯甲烷
MTBE：甲基叔丁基醚
Et 2O：乙醚
NMP：N-甲基吡咯烷酮
Bzl：苄基
TEA：三乙胺
DEPBT：3-(二乙氧基磷酰氧基)-1,2,3-苯并三嗪-4-酮
Dde：1-(4,4-二甲基-2,6-二氧代环己亚基)乙基
T3P：1-丙基磷酸酐
Alloc：烯丙氧羰基
tBu：叔丁基
Pd[P(C 6H 5) 3] 4：四(三苯基膦)钯
PhSiH 3：苯基硅烷
第三方面，本发明公开了一种药物组合物，其包含本发明所述的化合物或其药学上可 接受的盐为活性成分或主要活性成分，以及药学上可接受的载体。
构成本发明的一部分是药学上可接受的盐，适当的“药学上可接受的盐”包括本发明的游离型化合物、无机酸或有机酸反应形成的本发明化合物的常规无毒盐、无机碱或有机碱反应形成的本发明化合物的常规无毒盐。例如，包括得自无机酸例如盐酸、氢溴酸、硫酸、磷酸、硝酸等的盐，也包括得自有机酸例如乙酸、丙酸、琥珀酸、乙醇酸、硬脂酸、乳酸、苹果酸、酒石酸、柠檬酸、抗坏血酸、马来酸、羟基马来酸、苯乙酸、谷氨酸、苯甲酸、水杨酸、对氨基苯磺酸、富马酸、甲苯磺酸、甲磺酸、乙烷二磺酸、草酸、羟乙基磺酸、三氟乙酸等的盐。又例如，包括得自无机碱例如钠、钾、钙、镁、锌、铁等的盐，也包括得自有机碱例如氨水、精氨酸、赖氨酸、瓜氨酸、组氨酸等的盐。本发明陈述的盐包括因药学目的而采用的少量酸或碱化合物反应形成的含酸或碱的盐。药学上可接受的盐优选为含钠的盐。
本发明的其中一个目标是根据本发明的GLP-1R和GIPR双重靶向激动作用的多肽衍生物研究开发成为临床可用的药物制剂。该制剂可进一步包含缓冲剂、防腐剂、等渗剂、助溶剂、张度剂、螯合剂、稳定剂、抗氧化剂、表面活性剂、酸碱调节剂等。浓度通常为0.01mg/ml至50mg/ml，其中所述的制剂具有3.0至9.0的pH。
在本发明的一个实施方案中，药物制剂是含水制剂，即水溶液，通常是溶液、乳液或混悬液。
在另一个实施方案中，药物制剂是一种冻干制剂，在使用前加入溶剂和/或稀释剂至其中充分溶解后备用。
在另一个实施方案中，药物制剂是不需预先溶解的即用型干燥制剂，例如喷雾吸入冻干粉等。
在本发明的另一个实施方案中，药物制剂pH值的范围选择至关重要，会影响到GLP-1R和GIPR双重靶向激动作用的多肽衍生物的溶解性和稳定性，在某些特定条件下会产生多肽物理性聚集或吸附。在本发明的一个实施方案中，药物制剂的pH值为3.0～5.0。在本发明的一个实施方案中，药物制剂的pH值为7.0～8.0。在本发明的一个实施方案中，药物制剂的pH值为7.5～8.5。在本发明的另一个实施方案中，药物制剂的pH值为5.0～7.5。
在本发明的进一步实施方案中，缓冲剂选自磷酸氢二钠、乙酸钠等，防腐剂选自苯酚、邻甲酚、间甲酚、对甲酚等，等渗剂选自氯化钠类盐、糖或糖醇、氨基酸、丙二醇、甘露 醇等，助溶剂选自甘露醇、丙二醇、PEG、甘油、吐温、乙醇等，张度剂选自氯化钠类盐、丙二醇、甘油、甘露醇等，螯合剂选自EDTA、柠檬酸盐等，稳定剂选自肌酐、甘氨酸、烟酰胺、PEG等，抗氧化剂选自亚硫酸氢钠、亚硫酸钠、半胱氨酸、甲硫氨酸等，表面活性剂选自聚山梨酯、甘油、甘露醇等，酸碱调节剂选自盐酸、磷酸、硫酸、氢氧化钠、氢氧化钾等。
其它成分可根据药物制剂的需要(例如长期稳定性)存在于本发明的多肽衍生物的药物制剂中，包括乳化剂、金属离子、油质载体、蛋白质(如人血清白蛋白、明胶或蛋白质等)以及两性离子(例如精氨酸、甘氨酸、赖氨酸、组氨酸、甜菜碱和牛磺酸等)等和其它药用制剂添加剂。
术语“药学上可接受的载体”是指能够递送本发明有效量活性物质、不干扰活性物质的生物活性并且对宿主或者患者无毒副作用的任何制剂载体或介质，代表性的载体包括水、油、脂质体等。
针对药物或药理学活性剂而言，术语“有效量”或“治疗有效量”是指无毒的但能达到预期效果的药物或药剂的足够用量。
术语“活性成分”、“治疗剂”，“活性物质”或“活性剂”是指一种化学实体，它可以有效地治疗目标紊乱、疾病或病症。
“任选”或“任选地”指的是随后描述的事件或状况可能但不是必需出现的，并且该描述包括其中所述事件或状况发生的情况以及所述事件或状况不发生的情况。
第四方面，本发明所述的GLP-1R和GIPR双重靶向激动作用多肽衍生物与其药学上可接受的盐在预防和或治疗疾病中的用途，其优选的技术方案，所述疾病包括代谢综合征、肥胖、糖尿病、肥胖相关疾病和糖尿病相关疾病。糖尿病包括以因胰岛素分泌、胰岛素作用或其二者的缺陷引起的高血糖为特征的一组代谢疾病。根据病症机理将糖尿病分为Ⅰ型糖尿病、Ⅱ型糖尿病和妊娠糖尿病。
本发明所述GLP-1R和GIPR双重靶向激动作用多肽衍生物与其药学上可接受的盐，可将GIP的作用(例如，对脂肪代谢和体重减轻以及血糖的作用)与GLP-1的作用(例如，对血糖水平和食物摄取的作用)组合。因此，其可用于加速消除过量的脂肪组织，引起持续的体重减轻，并改善血糖控制。GLP-1R和GIPR双重靶向激动作用还可用于降低心血管风险因素，例如高胆固醇，如高LDL胆固醇。
本发明所述GLP-1R和GIPR双重靶向激动作用多肽衍生物与其药学上可接受的盐，还可用于治疗胰岛素抵抗、糖耐量受损、前驱糖尿病、空腹血糖升高、Ⅱ型糖尿病、高血压、血脂异常(或这些代谢风险因素的组合)、动脉粥样硬化、动脉硬化、冠心病、外周动脉疾病和卒中。这些都是可能与肥胖有关的病症。但是，本发明所述使用的化合物对这些病症的作用可通过对体重的作用来全面或部分地介导，或者可独立于所述作用。
在某些实施方案中，本发明所述GLP-1R和GIPR双重靶向激动作用多肽衍生物与其药学上可接受的盐可在如肥胖相关炎症、肥胖相关胆囊疾病和肥胖诱发的睡眠呼吸暂停等肥胖相关疾病起到治疗作用。
作为优选，还包括在制备治疗II型糖尿病药效延迟和/或预防II型糖尿病恶化的药物中的应用，以及改善II型糖尿病成人中血糖控制的方法、包括向有需要的患者施用有效量的上述多肽衍生物作为饮食和运动补充的应用。
本发明所述GLP-1R和GIPR双重靶向激动作用多肽衍生物与其药学上可接受的盐，对于与多个代谢相关的因素有关、发病机理复杂的NASH有积极预防与治疗意义。胰岛素抵抗及脂肪代谢紊乱构成了早期对肝脏的损伤，从而在肝脏细胞内形成脂肪堆积(NAFLD)。随着疾病的发展，机体免疫调节的形成，肝脏细胞产生炎症反应，继而推动形成纤维化，最终导致肝硬化等终末期肝病症状。而本发明所述GLP-1R和GIPR双重靶向激动作用多肽衍生物能有效调节血糖水平参与代谢，对于NASH可全面或部分地介导，或者可独立于所述作用。
本发明所述代谢综合征可以是糖尿病或糖尿病相关疾病，或者肥胖或肥胖相关疾病，或者NAFLD、NASH或相关疾病。肥胖、糖尿病之间的联系是众所周知的，因此这些病症并非独立或互相排斥的，而是可能结合在一起。NAFLD、NASH或相关疾病因为机理复杂但也涉及并相关肥胖与糖尿病，可能并非独立或互相排斥的。
本发明所述GLP-1R和GIPR双重靶向激动作用多肽衍生物与其药学上可接受的盐，对于神经退行性疾病如AD(阿尔茨海默综合症)或PD(帕金森综合症)的预防与治疗有积极意义。
第五方面，本发明实验研究发现，本发明的GLP-1R和GIPR双重靶向激动多肽衍生物在现有技术上通过保守或非保守氨基酸替换与支链修饰后出人意料地具有非常好的体内长效性，其半衰期显著优于对照药物Tirzepatide且有统计学意义。对照药物Tirzepatide 已公开数据显示其人体临床平均半衰期为5.0天，按本实验SD大鼠药代实验结果可推导多肽衍生物(1)的人体半衰期为7.88天，意即可实现患者半个月给药一次，极大地方便临床应用。
第六方面，本发明实验研究发现，本发明所述GLP-1R和GIPR双重靶向激动多肽衍生物在现有技术上通过保守或非保守氨基酸替换与支链修饰后能有效激活GIPR和GLP-1R，起到双靶点激动活性与适宜的活性搭配以及药效作用，与对照药物Tirzepatide药效作用相似，并意外地发现某些活性药效作用明显强于Tirzepatide且有统计学意义，如TC(总胆固醇)的降低。
具体实施方式
实施例1制备多肽衍生物(1)
Figure PCTCN2022096109-appb-000028
MALDI-TOF-MS：[M+H]4800.51
1)液相法合成支链片段；
将
Figure PCTCN2022096109-appb-000029
与
Figure PCTCN2022096109-appb-000030
在三乙胺条件下使用氯甲酸异丁酯缩合偶联，TFA脱除Boc保护；将Fmoc-Glu-OtBu与前一步产物用TBTU/DIEA缩合偶联，20％Pip/NMP溶液脱Fmoc保护；tBu基团单保护的十八烷二酸缩合偶联到前一步Glu的氨基位置；最后采用钯碳催化氢气水解脱除苄酯，得到支链片段。
Figure PCTCN2022096109-appb-000031
2)固相法合成肽链；
选用Rink Amide-MBHA树脂按多肽衍生物(1)肽链氨基酸序列从C端到N端逆序将Fmoc保护氨基酸依次偶联到固相合成树脂上，20％Pip/NMP溶液脱保护Fmoc，循环直 至GLP-1主链氨基酸全部完成；其中将待连接支链片段的的Lys缩合到肽树脂上时选用Fmoc-Lys(Dde)-OH。
3)将支链片段与肽树脂偶联；
采用2％水合肼/乙醇胺溶液脱去的Dde保护；将步骤1)侧链片段与步骤2)肽树脂使用缩合剂HATU/DIEA偶联；20％Pip/DMF溶液脱首位氨基酸Fmoc保护。
4)裂解除去保护基团与树脂挂载，
按一定比例(TFA:TIS:H 2O:EDT＝96:2:1:1)配制裂解液，将全保护肽树脂加入裂解液，将目标化合物从树脂上切割下来并脱除侧链。真空旋蒸除去TFA，将Et 2O加入浓缩液析出白色固体即目标产物粗品。
5)色谱纯化
进行多步反相色谱纯化或离子色谱纯化，最后转盐成含钠的盐溶液；
6)冻干
冻干后得到三氟乙酸多肽衍生物(1)白色或类白色疏松状固体。
实施例2制备多肽衍生物(1)
Figure PCTCN2022096109-appb-000032
MALDI-TOF-MS：[M+H]4800.53
1)固相法合成肽链；
按多肽衍生物(1)肽链的氨基酸序列将Fmoc保护氨基酸依次逆序偶联到Rink Amide-AM树脂上，缩合偶联试剂采用PyBOP/DIEA或DIC/Cl-HOBt，20％哌啶/DMF溶液脱保护Fmoc，循环直至氨基酸全部完成肽链；其中待连接侧链片段的Lys为Alloc保护，即Fmoc-Lys(Alloc)-OH。首位氨基酸Tyr位置选用Boc-Tyr(tBu)-OH。
2)固相法将支链各模块逐次与肽树脂偶联；
四(三苯基膦)钯Pd[P(C 6H 5) 3] 4和苯基硅烷PhSiH 3/DMF溶液脱肽树脂Lys侧链Alloc 保护；采用HATU/DIEA预先激活3,6-二氧杂辛二酸，然后与肽树脂偶联上载；加入Fmoc单保护的3,6-二氧杂-1,8-辛二胺缩合偶联；20％Pip/DCM脱去Fmoc保护；缩合剂HATU/DIEA预活化Fmoc-Glu-OtBu，再缩合偶联；20％Pip/DCM脱去Fmoc保护；tBu基团单保护的二十烷二酸缩合偶联到Glu的α氨基位置完成整个支链的上载与合成。
3)裂解除去保护基团与树脂挂载，得到多肽衍生物。
按一定比例(TFA:TIS:H 2O:苯酚＝96:1.5:1.5:1)配制裂解液，将全保护肽树脂加入裂解液，将目标化合物从树脂上切割下来并脱除侧链。真空旋蒸除去TFA，将MTBE加入浓缩液析出白色固体即目标产物粗品。
4)色谱纯化
进行多步反相色谱纯化纯化，最后转盐成醋酸的盐溶液；
5)冻干
冻干后得到醋酸多肽衍生物(1)白色或类白色疏松状固体。
参考实施例1与实施例2与专利中所述的合成步骤分别合成了发明中所述部分其它化合物：
多肽衍生物(2)MALDI-TOF-MS：[M+H]4878.59；
多肽衍生物(3)MALDI-TOF-MS：[M+H]4786.52；
多肽衍生物(4)MALDI-TOF-MS：[M+H]4772.41；
多肽衍生物(5)MALDI-TOF-MS：[M+H]4786.44；
多肽衍生物(6)MALDI-TOF-MS：[M+H]4864.55；
多肽衍生物(7)MALDI-TOF-MS：[M+H]4786.56；
多肽衍生物(8)MALDI-TOF-MS：[M+H]4800.57；
多肽衍生物(9)MALDI-TOF-MS：[M+H]4864.50；
多肽衍生物(10)MALDI-TOF-MS：[M+H]4786.43。
实施例3 SD大鼠药代实验
多肽化合物(1)与Tirzepatide在雄性大鼠体内单剂量皮下给药后，于不同时间点采 集血样，用LC-MS/MS测定血浆中化合物的浓度并计算相关药代参数，考察化合物在大鼠体内药代动力学特征。
16只雄性SD大鼠按体重随机分为2组，每组8只。给药前1天禁食不禁水12～14h，给药后4h给食。
Figure PCTCN2022096109-appb-000033
样品采集。于给药前及给药后异氟烷麻醉经眼眶取血0.1ml，置于EDTAK2离心管中并放置冰浴上。5000rpm，4℃离心10min，收集血浆。
SC组采血时间点：0,5,15,30min,1,2,4,6,8,24,28,32,48h，分析检测前，所有血浆样品存于-80℃。
数据采集及控制系统软件为Analyst1.5.1软件(Applied Biosystem)。图谱样品峰积分方式为自动积分；采用样品峰面积和内标峰面积的比值作为指标，和样品的浓度进行回归。回归方式：线性回归，权重系数为1/X 2。药代动力学参数用WinNonlin Professional v6.3(Pharsight,USA)用非房室模型分析处理。C max为实测的最大血药浓度，血药浓度-时间曲线下面积AUC (0→t)由梯形法计算得到，T max为给药后血药浓度达峰时间。实验数据用“均数±标准差”(Mean±SD,n≥8)或“均数”(Mean,n＝8)表示。
根据血药浓度数据，使用WinNonlin V6.3非房室模型计算给药后的药代参数，见下表。
多肽化合物(1)组
Figure PCTCN2022096109-appb-000034
Figure PCTCN2022096109-appb-000035
Tirzepatide组
Figure PCTCN2022096109-appb-000036
实施例4ob/ob小鼠降糖、减重降脂实验
32只ob/ob小鼠根据空腹体重，空腹血清TC、LDL，均衡随机分成4组，分别为模型对照组，受试样品组(1.35mg/kg)和阳性对照组(Tirzepatide，1.35mg/kg)，每组8只。另8只常规饲喂小鼠设为正常对照组(溶媒)。
每3天背部皮下注射给药1次，共给药8次(D0、D3、D6、D9、D12、D15、D18、D21)，模型对照组和正常对照组按生理盐水0.15mL/20g的剂量给药，受试样品组和阳性对照组按0.312mg/kg的剂量给药。
1)体重：分组前禁食不禁水12h测定1次，根据体重和TC、LDL均衡随机分组。分组后自D0始，每天测定1次。
2)Lee’s指数：首次给药前和D23各测定1次体长。体长为鼻尖至肛门的最大直线长度。调整小鼠体位使其身体呈舒展状态，用直尺刻度读出小鼠体长。
Lee’s指数＝[体重(g)×10 3/体长(cm)] 1/3。
3)血液中葡萄糖(GLU)的测定：每3天测定一次。
4)血液生化(TG、TC、LDL、HDL)：分别在分组前、D11和D23各测定一次。
5)脂肪-体重系数：小鼠安乐死后，摘取腹部皮下脂肪、性腺周围脂肪，肩胛骨皮下脂肪并分别称重，计算脂肪-体重系数。
6)摄食量、饮水量：自D0始，每天测定1次(有禁食操作时除外)。
实验结果如下：
1)体重：
多肽化合物(1)组初始体重较模型对照组低(D0，p<0.05)，与Tirzepatide组无统计学差异。从D1开始，多肽化合物(1)和Tirzepatide组动物体重均出现下降，且实验期间(D1-D23)体重增长缓慢，均显著低于模型对照组(p<0.01)；多肽化合物(1)组体重平均值略高于Tirzepatide组，但无统计学差异。
实验动物体重记录表
Figure PCTCN2022096109-appb-000037
Figure PCTCN2022096109-appb-000038
与模型对照组相比，*p<0.05，**p<0.01；与对应的阳性药相比，#p<0.05，##p<0.01。
2)Lee’s指数
Lee’s指数是反应大鼠肥胖程度的有效指标，所有动物的Lee’s指数均有增加，正常组动物增加最少。与模型对照组相比，各给药组动物Lee’s指数略有降低。结果说明受试药与阳性对照药对于ob/ob小鼠的肥胖均能起到抑制作用，受试组药物与阳性对照药的控制能力接近，无统计学差异。
动物Lee’s指数表
Figure PCTCN2022096109-appb-000039
3)随机血糖：
动物在给药后第三天进行初次血糖测定，结果表明各给药组动物的血糖浓度与正常组相比明显降低，在D0-D10期间血糖浓度控制良好。实验期间，动物血糖浓度逐渐上升，可能与其年龄和体重增加有关。与模型对照组相比，正常对照组动物D14-D23血糖值明显低于模型对照组小鼠，表明ob/ob小鼠的基础血糖值较高。与模型对照组小鼠相比，D3-D23受试药与阳性对照药均有不同程度明显的降血糖作用。给药组与阳性对照药(多肽化合物(1)/Tirzepatide)相比无显著差异。
实验动物血糖变化表
Figure PCTCN2022096109-appb-000040
Figure PCTCN2022096109-appb-000041
与模型对照组相比，*p<0.05，**p<0.01；与对应的阳性药相比，#p<0.05，##p<0.01。
4)血液生化
(1)TC：实验开始前(D0)多肽化合物(1)与模型组相较其他同品系小鼠组初始数值较高。与自身给药后不同时间相比，给药后多肽化合物(1)与Tirzepatide组肥胖小鼠的TC降低明显，说明此受试药与阳性对照药能够显著的降低TC含量。从D11和D23的结果可以看出，多肽化合物(1)降低TC的能力与Tirzepatide相似。且多肽化合物(1)组在初始值较高的情况下，降低TC的能力较强在D23时接近Tirzepatide组水平，且统计学意义从##p<0.01到#p<0.05显著改善。
(2)TG：实验开始前(D0)多肽化合物(1)和Tirzepatide相较其他同品系小鼠组初始数值较低。与自身给药后不同时间相比，给药后多肽化合物(1)与Tirzepatide组肥胖小鼠的TG降低明显，说明此受试药与阳性对照药能够显著的降低TG含量。D23，多肽化合物(1)降低TG的能力与Tirzepatide相似(多肽化合物(1)vs Tirzepatide，p<0.01)。
(3)LDL：实验开始前(D0)多肽化合物(1)和Tirzepatide相较其他同品系小鼠组初始数值较高。与自身给药后不同时间相比，给药后D11和D23，多肽化合物(1)与Tirzepatide组肥胖小鼠的LDL水平均明显降低，且显著低于模型对照组(p<0.01)，多肽化合物(1)和Tirzepatide组间比较未见统计学差异。
(4)HDL：实验进行期间，模型对照组小鼠HDL水平均高于正常对照组小鼠。给药前(D0)同品系小鼠HDL水平较为接近。相比于模型对照组，给药后D11和D23，受试组与阳性对照药组均能显著降低HDL水平。
上述结果说明，受试药与阳性对照药物均能降低动物的TG、TC、LDL和HDL浓度。
动物血液TC含量变化表
Figure PCTCN2022096109-appb-000042
与模型对照组相比，*p<0.05，**p<0.01；与对应的阳性药相比，#p<0.05，##p<0.01。
动物血液TG含量表
Figure PCTCN2022096109-appb-000043
与模型对照组相比，*p<0.05，**p<0.01；与对应的阳性药相比，#p<0.05，##p<0.01。
动物血液LDL含量表
Figure PCTCN2022096109-appb-000044
与模型对照组相比，*p<0.05，**p<0.01；与对应的阳性药相比，#p<0.05，##p<0.01。
动物血液HDL含量表
Figure PCTCN2022096109-appb-000045
与模型对照组相比，*p<0.05，**p<0.01；与对应的阳性药相比，#p<0.05，##p<0.01。
5)脂肪-体重系数
在给药23天后，解剖小鼠，分离不同部位脂肪，并根据公式(脂肪/体重×100)计算脂肪-体重系数。由结果可知，模型对照组小鼠各部位脂肪-体重系数均显著高于正常对照组小鼠。多肽化合物(1)能显著降低ob/ob小鼠腹部皮下和性腺周围的脂肪，且效果与阳性对照药物Tirzepatide无明显差异。此外，药物对于肩胛骨皮下脂肪-体重系数无影响。
脂肪-体重系数表
Figure PCTCN2022096109-appb-000046
Figure PCTCN2022096109-appb-000047
与模型对照组相比，*p<0.05，**p<0.01；与对应的阳性药相比，#p<0.05，##p<0.01。
6)摄食量、饮水量
给药后动物的进食量明显下降，动物整体进食趋势与给药频率呈现了一定相关性，表现为给药当天进食量最低，随着时间推移，进食量缓慢增加，但整体增加量低于模型对照组与正常对照组。在实验后期动物的进食量整体增加，可能与动物年龄和体重增加有关。实验结果说明，受试药与阳性对照药均有明显的摄食控制作用。
各组实验动物的饮水差异较大，给药组低于正常组与模型对照组。能明显观察动物给药后饮水量降低，首次给药后作用最为明显。D11各组动物饮水量均明显增加可能与后续禁食操作有关，但各组小鼠的饮水量都有增加。实验结果说明，受试药与阳性对照药均有明显的饮水控制作用。

Claims (12)

  1. 一种如(I)所示的多肽衍生物，或几何异构体，或立体异构体、或其药学上可接受的盐，主链中Xaa 20为Lys且其侧链ε-氨基以酰胺键形式连接支链(II)式中的X 1；
    Tyr-Aib-Asp-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-Xaa 13-Leu-Asp-Lys-Ile-Ala-Gln-Xaa 20-Glu-Phe-Val-Gln-Trp-Leu-Leu-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2
    (Ⅰ)
    Figure PCTCN2022096109-appb-100001
    其中，Aib为氨基异丁酸，与前后氨基酸以酰胺键(肽键)连接；
    Xaa 13为Aib，Tyr或Ala；
    另有，(II)式中n为16或18；X 1与X 2同时为
    Figure PCTCN2022096109-appb-100002
    或X 2为
    Figure PCTCN2022096109-appb-100003
    同时X 1为
    Figure PCTCN2022096109-appb-100004
  2. 根据权利要求1所述的多肽衍生物，或其几何异构体、立体异构体、或其药学上可接受的盐，其特征在于所述的多肽衍生物为多肽主链有且仅有Xaa20位Lys其侧链ε-氨基以酰胺键形式连接含长脂肪酸修饰的支链，X 2为
    Figure PCTCN2022096109-appb-100005
    同时X 1为
    Figure PCTCN2022096109-appb-100006
  3. 如权利要求1所述的多肽衍生物，或其几何异构体、立体异构体、或其药学上可接受的盐，所述的多肽衍生物为：
    Figure PCTCN2022096109-appb-100007
    Figure PCTCN2022096109-appb-100008
  4. 如权利要求1至3中任一项所述的多肽衍生物，或其几何异构体、立体异构体、或其药学上可接受的盐，其中氨基酸为L型，药学上可接受的盐优选为含钠的盐。
  5. 权利要求1至4中任一项所述的多肽衍生物，或其几何异构体、立体异构体、或其药学上可接受的盐的制备方法，其特征在于，包含支链片段合成、全保护多肽主链合成、支链偶联上肽树脂、裂解脱除肽链与侧链全保护、色谱纯化和冻干步骤或又包含固相法合成肽链、固相法将支链各模块逐次与肽树脂偶联、裂解除去保护基团与树脂挂载、色谱纯化和冻干步骤。
  6. 包含权利要求1至4中任一项所述的多肽衍生物、或几何异构体、或立体异构体、或其 药学上可接受的盐的药物组合物，所述的药物组合物还包括药学上可接受的载体。
  7. 权利要求1至4中任一项所述多肽衍生物、或几何异构体、或立体异构体、或其药学上可接受的盐、或者权利要求6所述的药物组合物在治疗和/或预防下列至少一种疾病的药物中的应用：代谢综合征、肥胖、糖尿病、肥胖相关疾病和糖尿病相关疾病。
  8. 权利要求1至4中任一项所述多肽衍生物、或几何异构体、或立体异构体、或其药学上可接受的盐、或权利要求6所述药物组合物在治疗和/或预防下列至少一种疾病的药物中的应用：胰岛素抵抗、糖耐量受损、前驱糖尿病、空腹血糖升高、Ⅱ型糖尿病、高血压、血脂异常(或这些代谢风险因素的组合)、动脉粥样硬化、动脉硬化、冠心病、外周动脉疾病和卒中等可能与肥胖有关的病症。
  9. 权利要求1至4中任一项所述多肽衍生物、或几何异构体、或立体异构体、或其药学上可接受的盐、或权利要求6所述药物组合物在治疗和/或预防下列至少一种疾病的药物中的应用：肥胖相关炎症、肥胖相关胆囊疾病和肥胖诱发的睡眠呼吸暂停等肥胖相关疾病。
  10. 权利要求1至4中任一项所述多肽衍生物、或几何异构体、或立体异构体、或其药学上可接受的盐、或者权利要求6所述的药物组合物在制备减少食物摄入量、减少β细胞凋亡、增加胰岛β细胞功能、增加β-细胞团和/或恢复葡萄糖对β-细胞的敏感性的药物中的应用。
  11. 权利要求1至4中任一项所述多肽衍生物、或几何异构体、或立体异构体、或其药学上可接受的盐、或权利要求6所述药物组合物在治疗和/或预防下列至少一种疾病的药物中的应用：非酒精性单纯性脂肪肝(NAFL)、非酒精性脂肪性肝炎(NASH)及其相关肝硬化和肝细胞癌等疾病。
  12. 权利要求1至4中任一项所述多肽衍生物、或几何异构体、或立体异构体、或其药学上可接受的盐、或权利要求6所述药物组合物在治疗和/或预防下列至少一种疾病的药物中的应用：神经退行性疾病，包括阿尔茨海默症(AD)、帕金森症(PD)等。
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