WO2024098718A1 - Nouveau composé polypeptidique à action prolongée, composition et utilisation associée - Google Patents

Nouveau composé polypeptidique à action prolongée, composition et utilisation associée Download PDF

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WO2024098718A1
WO2024098718A1 PCT/CN2023/096029 CN2023096029W WO2024098718A1 WO 2024098718 A1 WO2024098718 A1 WO 2024098718A1 CN 2023096029 W CN2023096029 W CN 2023096029W WO 2024098718 A1 WO2024098718 A1 WO 2024098718A1
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compound
iva
aib
γglu
egtftsdysi
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PCT/CN2023/096029
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Chinese (zh)
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胡菲菲
李春艳
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内蒙古博睿精创科技有限公司
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/18—Peptides; Protein hydrolysates
    • 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
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/06—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/06—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents
    • C07K1/08—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents using activating agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/20—Partition-, reverse-phase or hydrophobic interaction chromatography
    • 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 present invention belongs to the field of biochemical technology. Specifically, the present invention relates to a novel long-acting polypeptide compound, composition and application thereof that can be used to treat or prevent diabetes or obesity.
  • Diabetes is a group of clinical syndromes caused by the interaction of genetic and environmental factors, in which the body is in a state of high blood sugar levels for a long time. Diabetes is manifested in absolute or relative insufficiency of insulin secretion and decreased sensitivity of target tissue cells to insulin, accompanied by a series of metabolic disorders such as sugar, protein, fat, water and electrolytes.
  • ADA American Diabetes Association
  • type I diabetes accounts for 5-10% of all diabetic patients
  • type II diabetes accounts for about 90-95% of all diabetic patients.
  • Type I diabetes is caused by autoimmune ⁇ -cell destruction, which usually leads to absolute insulin deficiency, including latent autoimmune diabetes in adults (LADA).
  • Type II diabetes patients gradually lose insulin secretion from ⁇ cells on the basis of insulin resistance. It was once called “non-insulin-dependent diabetes” or "adult-onset diabetes”. Its significant pathophysiological characteristics are a decrease in insulin's ability to regulate glucose metabolism (insulin resistance) accompanied by a decrease in insulin secretion due to defective pancreatic ⁇ cell function.
  • insulin resistance is mainly caused by obesity, dyslipidemia, and an unhealthy lifestyle, resulting in a relative lack of insulin secretion (Report of a WHO Consultation. 1999).
  • GLP-1 is a pancreatic hormone secreted by intestinal L cells, which has pharmacological effects such as promoting insulin secretion, inhibiting the release of glucagon, stimulating pancreatic B cell regeneration, improving insulin sensitivity and increasing glucose utilization (Modern Medicine and Clinic, 2020.).
  • GLP-1 and its related receptors have been clinically studied as important targets for the treatment of type 2 diabetes, and have shown strong and broad application prospects in the field of diabetes treatment (GLP-1 receptor Expert guidance on the clinical application of agonists. Chinese Journal of Diabetes, 2018, 26(05): 353-361.).
  • GLP-1RA GLP-1 receptor agonists
  • multi-target agonists have been launched on the market. From the initial twice-daily administration to the recent once-a-week administration, the clinical practice has lasted for more than 10 years, and a large amount of clinical evidence has been accumulated for hypoglycemic, cardiovascular benefits and weight loss. With the launch of weekly preparations, it has greatly facilitated patients. At the same time, it has a good effect on the treatment of cardiovascular diseases, which has also led to the recognition of this type of drug in recent years.
  • Ultra-long-acting peptide drug molecule modification technology is the key to research and development in this field, and it is also a bottleneck to be broken through internationally. Therefore, how to further design and improve the hypoglycemic effect of compounds, reduce toxic side effects, and increase the duration of drug effect and/or half-life is still an important technical problem to be solved by technicians in this field, which has great social and clinical needs!
  • the object of the present invention is to provide a new type of long-acting polypeptide compound.
  • Another object of the present invention is to provide a composition containing the above-mentioned long-acting polypeptide compound.
  • Another object of the present invention is to provide the application of the long-acting polypeptide compound.
  • amino acid sequence of the long-acting polypeptide compound is as follows:
  • X3 is selected from E, Q or N; X27 is selected from L or I; X28 is selected from A or D; X35 is selected from A or Aib; X20 is K, K( Gx (SG) Z - ⁇ Glu-CO ( CH2 ) aCO2H ), K(( PEG2 ) b - ⁇ Glu-CO( CH2 ) cCO2H ) or K((AEEA) d - ⁇ Glu-CO( CH2 ) eCO2H ), wherein x is an integer of 0-5 , z is an integer of 1-5, a is an integer of 12-20, b is an integer of 1-8, c is an integer of 12-20, d is an integer of 1-8, and e is an integer of 12-20; R1 is selected from OH or NH2 ;
  • X2 is selected from Aib, Iva or Cba:
  • X1 is selected from H or Y;
  • X13 is selected from Aib, Iva or Cba;
  • X2 is D-Ser: X1 is H; X13 is selected from Aib, Iva or Cba.
  • X20 is selected from K( Gx (SG) Z - ⁇ Glu-CO( CH2 ) aCO2H ) , K(( PEG2 ) b - ⁇ Glu-CO( CH2 ) cCO2H ) or K((AEEA) d - ⁇ Glu-CO( CH2 ) eCO2H )
  • Gx (SG) Z - ⁇ Glu-CO( CH2 ) aCO2H, ( PEG2 ) b - ⁇ Glu-CO( CH2 ) cCO2H or (AEEA) d - ⁇ Glu-CO( CH2 ) eCO2H is the "sidearm" structure of the long-acting polypeptide compound described in the present application, and the "sidearm" structure is connected to the main peptide chain of the long-acting polypeptide compound by forming an amide bond with the side chain amino group of amino acid K on the main peptide chain of the long-acting polypeptide compound.
  • the 9th amino acid D and the 16th amino acid K on the long-acting polypeptide compound are connected via an amide bond.
  • X20 is K(G x (SG) Z - ⁇ Glu-CO(CH 2 ) a CO 2 H), wherein x is 2, z is 2 or 3, and a is 16 or 18.
  • X20 is K((PEG 2 ) b - ⁇ Glu-CO(CH 2 ) c CO 2 H), wherein b is 2, and c is 16 or 18.
  • X20 is K((AEEA) d - ⁇ Glu-CO(CH 2 ) e CO 2 H), wherein d is 2, and e is 16 or 18.
  • X35 is A.
  • X1 is Y
  • X2 is selected from Aib, Iva or Cba
  • X3 is E
  • X13 is selected from Aib, Iva or Cba.
  • X2 is Iva
  • X13 is Aib or Iva
  • X2 is Aib
  • X13 is Iva
  • X1 is H
  • X2 is selected from Aib, Iva or Cba
  • X3 is selected from E or Q
  • X13 is selected from Aib, Iva or Cba.
  • X2 is Aib or Iva
  • X13 is Iva
  • X2 is Aib
  • X1 is Y
  • X3 is E
  • X13 is selected from Iva or Cba
  • X27 is selected from L or I
  • X28 is selected from A or D
  • X35 is A.
  • X2 is selected from Iva or Cba, and X1 is Y; X3 is E; and X13 is selected from Aib, Iva or Cba; X27 is selected from L or I; X28 is selected from A or D; X35 is A.
  • X2 is selected from Aib, Iva or Cba, and X1 is H; X3 is selected from E or Q; X13 is selected from Aib, Iva or Cba; X27 is selected from L or I; X28 is selected from A or D; and X35 is A.
  • X2 is Aib, and X1 is Y; X3 is E; X13 is Iva; X20 is K( Gx (SG) Z - ⁇ Glu-CO( CH2 ) aCO2H ) , wherein x is 2, z is 2 or 3, and a is 16 or 18; X27 is selected from L or I; X28 is selected from A or D; and X35 is A.
  • X2 is Iva
  • X1 is Y
  • X3 is E
  • X13 is selected from Aib or Iva
  • X20 is K( Gx (SG) Z - ⁇ Glu-CO( CH2 ) aCO2H ), wherein x is 2, z is 2 or 3, and a is 16 or 18
  • X27 is selected from L or I
  • X28 is selected from A or D
  • X35 is A.
  • X2 is selected from Aib or Iva, and X1 is H; X3 is selected from E or Q; X13 is Iva; X20 is K( Gx (SG) Z - ⁇ Glu-CO ( CH2 ) aCO2H ), wherein x is 2, z is 2 or 3, and a is 16 or 18; X27 is selected from L or I; X28 is selected from A or D; and X35 is A.
  • the long-acting polypeptide compound is selected from any one of the following compounds:
  • Y-Iva-EGTFTS D
  • YSI-Iva-LD K
  • IAQKAFVQWLIAGGPSSGAPPPS-OH IAQKAFVQWLIAGGPSSGAPPPS-OH, wherein the 9th amino acid D is linked to the 16th amino acid K through an amide bond;
  • Y-Aib-EGTFTS D
  • YSI-Iva-LD K
  • IAQKAFVQWLLDGGPSSGAPPPS-OH IAQKAFVQWLLDGGPSSGAPPPS-OH, wherein the 9th amino acid D is linked to the 16th amino acid K through an amide bond;
  • Y-Iva-EGTFTS D
  • YSI-Iva-LD K
  • IAQKAFVQWLLDGGPSSGAPPPS-OH IAQKAFVQWLLDGGPSSGAPPPS-OH, wherein the 9th amino acid D is linked to the 16th amino acid K through an amide bond;
  • H-Aib-QGTFTS D
  • YSI-Aib-LD K
  • IAQKAFVQWLLDGGPSSGAPPPS-OH IAQKAFVQWLLDGGPSSGAPPPS-OH, wherein the 9th amino acid D is linked to the 16th amino acid K through an amide bond;
  • H-Iva-EGTFTS D
  • YSI-Aib-LD K
  • IAQKAFVQWLLDGGPSSGAPPPS-OH IAQKAFVQWLLDGGPSSGAPPPS-OH, in which the 9th amino acid D is linked to the 16th amino acid K through an amide bond;
  • Y-Iva-EGTFTS D
  • YSI-Iva-LD K
  • IAQK(GGSGSGSG- ⁇ Glu-CO(CH 2 ) 18 CO 2 H)AFVQWLIAGGPSSGAPPPS-NH 2 wherein the 9th amino acid D is linked to the 16th amino acid K via an amide bond;
  • Y-Aib-EGTFTS D
  • YSI-Iva-LD K
  • IAQK(GGSGSGSG- ⁇ Glu-CO(CH 2 ) 18 CO 2 H)AFVQWLLDGGPSSGAPPPS-NH 2 wherein the 9th amino acid D is linked to the 16th amino acid K via an amide bond;
  • Y-Iva-EGTFTS D
  • YSI-Iva-LD K
  • IAQK(GGSGSGSG- ⁇ Glu-CO(CH 2 ) 18 CO 2 H)AFVQWLLDGGPSSGAPPPS-NH 2 wherein the 9th amino acid D is linked to the 16th amino acid K via an amide bond;
  • H-Aib-QGTFTS D
  • YSI-Aib-LD K
  • IAQK(GGSGSGSG- ⁇ Glu-CO(CH 2 ) 18 CO 2 H)AFVQWLLDGGPSSGAPPPS-OH IAQK(GGSGSGSG- ⁇ Glu-CO(CH 2 ) 18 CO 2 H)AFVQWLLDGGPSSGAPPPS-OH, wherein the 9th amino acid D is linked to the 16th amino acid K via an amide bond;
  • H-Iva-EGTFTS D
  • YSI-Aib-LD K
  • IAQK(GGSGSGSG- ⁇ Glu-CO(CH 2 ) 18 CO 2 H)AFVQWLLDGGPSSGAPPPS-OH IAQK(GGSGSGSG- ⁇ Glu-CO(CH 2 ) 18 CO 2 H)AFVQWLLDGGPSSGAPPPS-OH, wherein the 9th amino acid D is linked to the 16th amino acid K via an amide bond;
  • Compound 60 (SEQ ID NO. 60):
  • the -NH2 at the C-terminus of the compounds of the present invention means that the terminal amino acid is amidated to form a C-terminal primary amide.
  • the -OH at the end is the structure of the terminal amino acid itself.
  • the long-acting polypeptide compound is a pharmaceutically acceptable salt.
  • the preparation method of the above series of long-acting polypeptide compounds comprises the following steps:
  • Step 1 According to the Fmoc/t-Bu strategy, the main peptide resin corresponding to the main peptide chain of the polypeptide analog is synthesized.
  • Step 2 Based on the main peptide resin, according to the Fmoc/t-Bu strategy, coupling the corresponding "sidearm" structure to obtain the corresponding polypeptide resin; wherein the "sidearm" structure is PEG 2 -PEG 2 - ⁇ Glu-CO(CH 2 ) 18 CO 2 H, AEEA-AEEA- ⁇ Glu-CO(CH 2 ) 18 CO 2 H, GGSGSG- ⁇ Glu-CO(CH 2 ) 18 CO 2 H or GGSGSGSG- ⁇ Glu-CO(CH 2 ) 18 CO 2 H.
  • the "sidearm” structure is PEG 2 -PEG 2 - ⁇ Glu-CO(CH 2 ) 18 CO 2 H, AEEA-AEEA- ⁇ Glu-CO(CH 2 ) 18 CO 2 H, GGSGSG- ⁇ Glu-CO(CH 2 ) 18 CO 2 H or GGSGSGSG- ⁇ Glu-CO(CH 2 ) 18 CO 2 H.
  • Step 3 adding a cleavage solution to the polypeptide resin to perform a cleavage reaction, removing the polypeptide from the entire protection, and extracting a crude compound; and purifying the crude compound.
  • the coupling agents used are 1-hydroxybenzotriazole (HOBt) and N, N-diisopropylcarbodiimide (DIC), the solvent is N, N-dimethylformamide (DMF), and the Fmoc group is removed with a 20% piperidine/N, N-dimethylformamide solution;
  • the cleavage solution is composed of trifluoroacetic acid (TFA), 2,2'-(1,2-ethylenedioxy)bis(ethylenethiol) (DODT), m-cresol and H2O in a volume ratio of 92.5:2.5:2.5:2.5;
  • the crude compound extraction method includes filtration, precipitation and/or methyl tert-butyl ether extraction.
  • the purity of the obtained compounds 31, 35 and 38 is greater than 96%.
  • Another object of the present invention is to provide a composition, which contains a long-acting polypeptide compound and a pharmaceutically acceptable carrier or excipient.
  • a carrier that can reduce drug degradation and loss and reduce side effects such as micelles, microemulsions, gels and other carriers
  • excipients refer to materials added to make the drug into a suitable dosage form, such as buffers, lyophilization excipients, etc., which can make the pharmaceutical composition containing the compound of the present invention into a solution or lyophilized powder for parenteral administration.
  • the lyophilized powder can be added with an appropriate solvent or other pharmaceutically acceptable carrier to reconfigure the powder.
  • the liquid formula is generally a buffer solution, an isotonic solution and an aqueous solution.
  • the buffer solution can be a phosphate buffer solution
  • the isotonic solution can be a 0.9% sodium chloride solution
  • the aqueous solution is a solution obtained by directly dissolving with purified water.
  • the pharmaceutical composition prepared by adding the long-acting polypeptide compound as an active ingredient to a pharmaceutically acceptable carrier and/or excipient is suitable for various modes of administration, such as oral administration, transdermal administration, intravenous administration, intramuscular administration, topical administration, nasal administration, etc.
  • the pharmaceutical composition of the present invention can be prepared into various suitable dosage forms, which contain at least one effective dose of the compound of the present invention and at least one pharmaceutically acceptable Examples of suitable dosage forms are tablets, capsules, sugar-coated tablets, granules, oral solutions and syrups, ointments and patches for skin surfaces, aerosols, nasal sprays, and sterile solutions that can be used for injection.
  • the dosage of the pharmaceutical composition of the present invention can vary within a wide range, and those skilled in the art can determine it based on objective factors, such as the type of disease, severity of the disease, patient weight, dosage form, route of administration, and the like.
  • Another object of the present invention is to provide applications of long-acting polypeptide compounds and compositions.
  • the present invention obtains a series of long-acting polypeptide compounds, and studies the pharmacodynamic effects of this series of drugs. Studies have shown that the long-acting polypeptide compounds of the present invention have a longer half-life, have insulinotropic activity, have no adverse reactions, can be used to treat diabetes and obesity, and can potentially be used as a new generation of drugs for treating diabetes and obesity.
  • the applications of the long-acting polypeptide compound and composition of the present invention specifically include:
  • the polypeptide compounds involved in the present invention not only have a primary structure, but also have a secondary/tertiary structure that is crucial to the activity of the polypeptide compounds.
  • Various proteins/polypeptides have specific secondary/tertiary spatial conformations, and these specific spatial conformations are related to their specific biological functions, and the structure and function are highly unified; the secondary and tertiary spatial structures have a significant impact on whether the compound can bind to the target, how it binds to the target, and the strength of the binding, that is, the secondary/tertiary structure is crucial for the polypeptide compounds to exert their biological functions.
  • side chains in the present invention greatly affects the secondary and tertiary spatial structures of the polypeptide compounds, so in the present invention, different
  • the addition of side chains causes a great change in the spatial conformation of the main peptide chain of the polypeptide compound involved in the present application, and the change in the polypeptide conformation will affect the performance of its biological function.
  • the biological properties of the polypeptide are unpredictable. Whether it is effective for the long-term effect of a specific polypeptide or protein requires a large number of biopharmaceutical tests and experiments to know.
  • the design and synthesis of the novel long-acting polypeptide compound described in the present invention and the applied long-acting modification technology are only effective for the polypeptide compound described in the present invention or the undefined polypeptide compound, and are unpredictable.
  • the results of the test (Example 6) to further verify the creativity and novelty of the invention show that the long-acting polypeptide compound of the present invention greatly prolongs the half-life, and the drug half-life in rats can reach more than 22 hours, realizing the ultra-long-acting polypeptide drug, and achieving the frequency of drug administration once every 2 weeks or more for human use.
  • the drug half-life of the polypeptide drug in rats currently reported in this field is basically less than 10 hours, and the frequency of drug administration can only be achieved once a week.
  • the -NH 2 at the C-terminus of compound 61 indicates that the terminal amino acid is amidated, and the -OH at the C-terminus of compounds 62-64 is the terminal amino acid structure itself.
  • lixisenatide can bind to the extracellular domain and transmembrane pocket of GLP-1R, and form a suitable steric hindrance with the GLP-1R receptor.
  • the two modified peptides (compounds 63 and 64) have a change in conformation due to the entanglement of the long-acting side chain, thereby forming a large steric hindrance with GLP-1R, hindering ligand-receptor binding, and only showing entanglement with the extracellular domain of GLP-1R, but there is no binding site in the transmembrane pocket, which not only fails to prolong its hypoglycemic effect, but also causes the original parent peptide to lose its hypoglycemic effect.
  • novel long-acting polypeptide compound of the present invention utilizes lipophilic substituents to bind to albumin in the blood, protecting it from enzymatic degradation, thereby increasing the half-life.
  • the helical structure of the molecule is stabilized by an intramolecular bridge, thereby increasing the efficacy and/or selectivity against the target.
  • novel long-acting polypeptide compound of the present invention has high synthesis yield, good stability, is easy to scale up production and has low cost.
  • the novel long-acting polypeptide compound of the present invention has a better pharmacological effect of reducing body weight.
  • the long-acting polypeptide compound can be used to prevent weight gain or promote weight loss by causing a decrease in food intake and/or an increase in energy consumption. Therefore, the novel long-acting polypeptide compound of the present invention can also be used to directly or indirectly treat other diseases caused by or characterized by overweight, such as treating and/or preventing obesity, morbid obesity, obesity-related inflammation, obesity-related gallbladder disease, and sleep apnea caused by obesity.
  • the effect of the present invention in these diseases may be due to the direct or indirect effect of the novel long-acting polypeptide compound on body weight, or the effect on other aspects of the body other than body weight.
  • DCM dichloromethane
  • DMF is N, N-dimethylformamide
  • MeOH is methanol
  • Piperidine is piperidine
  • HOBt is 1-hydroxybenzotriazole
  • DIC is N, N'-diisopropylcarbodiimide
  • Fmoc is fluorenylmethoxycarbonyl
  • resin is resin
  • FBS is fetal bovine serum
  • H and His are histidine
  • Y and Tyr are tyrosine
  • E and Glu are glutamic acid
  • Q and Gln are glutamine
  • N and Asn are asparagine
  • G and Gly are glycine
  • T and Th are r is threonine
  • F and Phe are phenylalanine
  • S and Ser are serine
  • D and Asp are aspartic acid
  • I and IIe are isoleucine
  • L and Leu are leucine
  • K and Lys are lysine
  • AEEA 8-amino-3,6-dioxaoctanoic acid
  • TFA trifluoroacetic acid
  • DODT 2,2'-(1,2-ethylenedioxy)bis(ethanediol)
  • ACN is acetonitrile.
  • FIG1 is a graph showing the time-blood glucose results of an OGTT experiment in mice 0.5 h after administration in Example 2;
  • FIG2 is a graph showing the area under the blood glucose curve (AUC) in FIG1 ;
  • FIG3 is a graph showing the time-blood glucose results of an OGTT experiment in mice 1 hour after administration in Example 2;
  • FIG4 is a graph showing the area under the blood glucose curve (AUC) in FIG3 ;
  • FIG5 is a graph showing the time-blood glucose results of an OGTT experiment in mice 24 hours after administration in Example 3;
  • FIG6 is a graph showing the area under the blood glucose curve (AUC) in FIG5 ;
  • FIG. 7 is a graph showing the time-blood glucose results of the OGTT experiment in Example 3 for mice 48 hours after administration.
  • FIG8 is a graph showing the area under the blood glucose curve (AUC) in FIG7 ;
  • FIG. 9 is a graph showing the time-blood glucose results of the OGTT experiment in Example 3 in mice 72 hours after administration.
  • FIG10 is a graph showing the area under the blood glucose curve (AUC) in FIG9 ;
  • FIG. 11 is a graph showing the time-blood glucose results of the OGTT experiment in Example 3 in mice 96 hours after administration.
  • FIG12 is a graph showing the area under the blood glucose curve (AUC) in FIG11 ;
  • FIG. 13 is a graph showing the time-blood glucose results of the OGTT experiment in Example 3 in mice 120 hours after administration.
  • FIG14 is a graph showing the area under the blood glucose curve (AUC) in FIG13 ;
  • FIG. 15 is a graph showing the time-blood glucose results of the OGTT experiment in Example 3 in mice 144 hours after administration.
  • FIG16 is a graph showing the area under the blood glucose curve (AUC) in FIG15 ;
  • FIG. 17 is a graph showing the time-blood glucose results of the OGTT experiment in Example 3 in mice 168 hours after administration.
  • FIG18 is a graph showing the area under the blood glucose curve (AUC) in FIG17 ;
  • Figure 19 is a statistical diagram of the weight monitoring data of mice in Example 4.
  • Figure 20 is the fasting blood glucose monitoring data statistics of mice in Example 4.
  • FIG21 is a graph showing the time-blood glucose results of an OGTT experiment in mice 24 hours after administration in Example 5;
  • FIG. 22 is a graph showing the area under the blood glucose curve (AUC) in FIG. 21 .
  • Boc-His(Trt)-OH and Fmoc-Aib-OH were purchased from Shanghai Jier, and mono-tert-butyl eicosanedioate was self-made.
  • the remaining amino acids were purchased from Chengdu Zhengyuan Company, and the condensation agent was purchased from Suzhou Haofan Company. Unless otherwise specified, all other reagents were analytically pure, and the solvents were purchased from Shanghai Titan Company.
  • the centrifuge was purchased from Lu Xiangyi.
  • a 5.0 cm reverse phase C 18 preparative column (46 mm x 250 mm) was used to purify the peptide.
  • the high performance liquid chromatograph was a product of Thermo Fisher Scientific. Mass spectrometry was performed using a Waters mass spectrometer.
  • Step 1 Synthesize the main peptide resin corresponding to the main peptide chain
  • the synthesis scale was 0.5 mmol, and the following main peptide resin was synthesized:
  • the main peptide chain peptide resin was obtained: Boc-Tyr(tBu)-Iva-Glu(tBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-Aib-Leu-Asp(OtBu)-Lys-Ile-Ala-Gln(Trt)-Lys-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Rink Amide AM Resin.
  • Coupling Fmoc-Gly-OH Add Fmoc-Gly-OH, HOBt, and an appropriate amount of DMF to the main peptide resin product, stir evenly with nitrogen, add DIC, stir with nitrogen for 2 hours, and use hydrated ninhydrin to detect the coupling effect. If it is colorless and transparent, the reaction is over. Draw off the reaction solution, wash it with N, N-dimethylformamide (DMF) 3 times, remove the Fmoc group with 20% Piperidine/DMF solution (twice 5min + 7min), wash it with DMF 6 times after removing Fmoc, take a sample for ninhydrin detection, and if it is positive, proceed to the subsequent coupling step.
  • DMF N-dimethylformamide
  • Lysis solution TFA, DODT, m-cresol, and H 2 O were prepared in advance at a volume ratio of 92.5:2.5:2.5:2.5 and frozen in a refrigerator for 2 h.
  • the filtrate was collected by filtration, and the resin was washed 3 times with a small amount of lysis solution. After the filtrate was combined, it was concentrated under reduced pressure to about 1/4 of the original volume, and slowly poured into ice methyl tert-butyl ether under stirring, and the residue in the bottle was washed into methyl tert-butyl ether with a small amount of lysis solution. Let it stand for more than 2 hours until the precipitation is complete. The supernatant was removed, the precipitate was centrifuged, washed 3 times with methyl tert-butyl ether, centrifuged, and the solid was blown dry with nitrogen.
  • the HPLC purification was repeated once, starting with 29% ACN/H 2 O (containing 0.1% acetic acid), the column was eluted with a gradient (increasing the proportion of ACN at a rate of 0.33%/min) at a flow rate of 80 mL/min for 60 minutes, and the fractions containing the polypeptide were collected and freeze-dried to obtain 440 mg of refined peptide with a purity of greater than 98.96% and a total yield of 17%.
  • the isolated product peptide was identified by LC-MS, starting with 5% ACN/H 2 O (containing 0.1% formic acid), with a gradient (increasing the proportion of ACN at a rate of 6%/min), a flow rate of 0.4 mL/min, and elution analysis for 15 minutes.
  • the target compound 31 was determined to have a calculated [M+H] + value of 5083.73 and a measured [M+3H] 3+ value of 1695.20.
  • step 1 synthesizes the following main peptide resin different from compound 31:
  • the purification and product confirmation methods of compound 35 were the same as those of compound 31, and 332 mg of refined peptide was obtained with a purity greater than 98.46% and a total yield of 13%.
  • the separated product was identified by LC-MS and confirmed to be the target compound 35, with a calculated value of [M+H] + of 5097.76 and a measured value of [M+3H] 3+ of 1699.90.
  • the synthesis scale was 0.5 mmol and the following main peptide resin was synthesized manually: Boc-Tyr(tBu)-Aib-Glu(tBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-Iva-Leu-Asp(OtBu)-Lys-Ile-Ala-Gln(Trt)-Lys(Alloc)-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Leu-Asp(OtBu)-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Ser(tBu)-Rink Amide AM Resin.
  • the purification and product confirmation methods of compound 38 were the same as those of compound 31, and 385 mg of refined peptide was obtained with a purity greater than 98.35% and a total yield of 15%.
  • the separated product was identified by LC-MS and confirmed to be the target compound 38, with a calculated value of [M+H] + of 5128.69 and a measured value of [M+3H] 3+ of 1709.50.
  • mice 8-week-old C57BL/6J male mice (purchased from Guangdong Sijia Jingda Biotechnology Co., Ltd.) were used in this experiment, 7 mice/group, and the dosage of all compounds and Semaglutide (injection, purchased from Guangzhou Tonghui Pharmaceutical Co., Ltd.) was 50 ⁇ g/kg.
  • the weight and random blood glucose of mice were measured before the experiment, and the mice were regrouped according to the weight and random blood glucose to ensure that the average weight and random blood glucose of each group were similar.
  • the drugs were prepared on the day of administration, and the corresponding drugs were subcutaneously injected according to the groups.
  • OGTT tests were performed 0.5h and 1h after administration (parallel experiments, one group of mice underwent 0.5h OGTT test, and another group of mice underwent 1h OGTT test).
  • Glucose was administered by gavage at a dose of 2g/kg, and blood was collected from the tail vein to detect blood glucose values at 0, 15, 30, 60, 90 and 120min after gavage.
  • the data were processed using the software GraphPadPrism, and the time-blood glucose curve was plotted to calculate the area under the blood glucose curve AUC.
  • One-way ANOVA analysis was performed with the PBS control group without medication to calculate the significant difference. The test results are shown in Figures 1-4, where * indicates p ⁇ 0.05; ** indicates p ⁇ 0.01; *** indicates p ⁇ 0.001; **** indicates p ⁇ 0.0001.
  • the weight and random blood glucose of mice were measured before the experiment, and the mice were regrouped according to the weight and random blood glucose to ensure that the average weight and random blood glucose of each group were similar.
  • the drugs were prepared on the day of administration and the corresponding drugs were subcutaneously injected according to the group.
  • Glucose OGTT test was performed 24h, 48h, 72h, 96h, 120h, 144h, and 168h after administration. Glucose was gavaged at a dose of 2g/kg, and blood was collected from the tail vein to detect the blood glucose values at 0, 15, 30, 60, 90 and 120min after gavage. The data were processed using the software GraphPadPrism, the time-blood glucose curve was drawn, and the area under the blood glucose curve AUC was calculated. One-way ANOVA analysis was performed with the PBS control group without medication to calculate the significant difference. The test results are shown in Figure 5-18, where * indicates p ⁇ 0.05; ** indicates p ⁇ 0.01; *** indicates p ⁇ 0.001; **** indicates p ⁇ 0.0001.
  • Example 4 Therapeutic effects of compounds 31, 33, 35, 38, 39, 43, 44, 49, 58, 60 and Semaglutide on BKS-db diabetic mice
  • mice 8-week-old BKS-db diabetic mice with the genotype of Lepr KO/KO (purchased from Guangdong Yaokang Biotechnology Co., Ltd.). Blood glucose and body weight were measured, and the mice were randomly divided into each compound group, positive control group (Semaglutide) and model control group (PBS) according to body weight and blood glucose.
  • Semaglutide positive control group
  • PBS model control group
  • Each group of mice was subcutaneously injected with compounds 31, 33, 35, 38, 39, 43, 44, 49, 58, 60, and Semaglutide (injection purchased from Guangzhou Tonghui Pharmaceutical Co., Ltd.) at a dose of 120 ⁇ g/kg, once every other day, and the control group was injected with an equal volume of saline.
  • the experimental period was 4 weeks.
  • Compound 62 synthesized by the present invention is the marketed GLP-1 receptor agonist polypeptide drug lixisenatide
  • compound 63 is a compound obtained by modifying the 12th Lys of lixisenatide with the side chain Gly-Gly-Ser-Gly-Ser-Gly-Ser-Gly- ⁇ -Glu-CO(CH 2 ) 18 CO 2 H of the present invention
  • compound 64 is a compound obtained by modifying the 26th Lys of lixisenatide with the side chain Gly-Gly-Ser-Gly-Ser-Gly-Ser-Gly- ⁇ -Glu-CO(CH 2 ) 18 CO 2 H of the present invention.
  • OGTT oral glucose tolerance
  • mice of about 8 weeks old Male C57BL/6J mice of about 8 weeks old (purchased from Guangdong Sijia Jingda Biotechnology Co., Ltd.) were fed for one week to adapt to the environment and randomly divided into groups according to blood sugar, with 8 mice in each group.
  • Each peptide compound was administered at a dose of 80ug/kg.
  • the drug was administered by subcutaneous injection, and the control group was given an equal volume of PBS.
  • the subjects were fasted for 16 hours on the evening of the administration.
  • the data were processed using the software GraphPadPrism, and the time-blood glucose curve was plotted to calculate the area under the blood glucose curve AUC.
  • One-way ANOVA analysis was performed with the PBS control group that was not administered, and significant differences were calculated
  • Example 6 Pharmacokinetic study of compound 33, compound 35, compound 38, compound 39, compound 43, compound 44, compound 58 and compound 61 in SD rats
  • SD rats (SPF grade, source: Sbefor (Beijing) Biotechnology Co., Ltd., experimental animal production license number: SCXK (Beijing) 2019-0010, weight: 180-200g, age: 6-8 weeks) were raised for one week to adapt to the environment. The general condition of the animals was checked during the adaptation period. Unqualified animals were not included in this experiment.
  • the feeding environment control system used WINCC7.3 EMS series computer room environment monitoring system, and the feed used SPF mice and rats to maintain the feed. 20 qualified SD rats were randomly divided into groups according to their weight, with 4 rats in each group (half male and half female), for a total of 5 groups (animals were grouped and statistically analyzed using software Stata 15).
  • the compound was administered subcutaneously to the skin of the back of the neck at a dose of 0.15 mg/kg or 0.2 mg/kg, a dosing volume of 2 mL/kg, a dosing concentration of 0.075 mg/mL or 0.1 mg/mL, and a solvent of PBS.
  • Blood samples were collected from the jugular vein of rats in the single subcutaneous administration group before administration (0h) and 0.5h, 1h, 2h, 4h, 6h, 8h, 24h, 48h, 72h, 96h, 120h, 144h, 168h, and 192h after administration.
  • About 0.2mL of whole blood was collected at each time point and placed in an EDTA- K2 anticoagulant tube.
  • the blood was centrifuged at 4°C and 1800g for 10min, and the supernatant was taken.
  • the separated plasma was transferred to a -80°C refrigerator for storage.
  • UPLC-MS/MS was used to establish the chemical and molecular weight distribution of plasma in SD rats.
  • the concentration analysis method of the compound was used to determine the drug concentration of the compound in plasma.
  • the data were processed using WinNonlin 8.1 software to calculate the pharmacokinetic parameters. The experimental results are shown in Table 2.

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Abstract

L'invention concerne un nouveau composé polypeptidique à action prolongée, une composition et une utilisation associée. Dans le composé polypeptidique à action prolongée, la substitution d'un acide aminé à une position particulière clé améliore la stabilité, l'activité et la résistance à l'hydrolyse d'un polypeptide. La substitution de la position d'acide aminé 2 et/ou 3 du squelette du composé polypeptidique par un acide aminé non naturel AiB, Iva ou Cba, après plusieurs essais, peut améliorer de manière significative la stabilité, l'activité et la résistance à l'hydrolyse de molécules polypeptidiques, et la substitution respective de la position d'acide aminé 27 et/ou 28 par un acide aminé L et/ou D peut également améliorer de manière significative l'activité des molécules polypeptidiques. Le composé polypeptidique à action prolongée a une longue durée de demi-vie, et a de bons effets pharmacodynamiques de traitement du diabète et de réduction du poids.
PCT/CN2023/096029 2022-11-07 2023-05-24 Nouveau composé polypeptidique à action prolongée, composition et utilisation associée WO2024098718A1 (fr)

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Citations (6)

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WO2016065090A1 (fr) * 2014-10-24 2016-04-28 Merck Sharp & Dohme Corp. Co-agonistes des récepteurs du glucagon et du glp-1
CN107207576A (zh) * 2015-01-09 2017-09-26 伊莱利利公司 Gip和glp‑1共激动剂化合物
WO2021169512A1 (fr) * 2020-02-24 2021-09-02 中山大学 Composé polypeptidique et son application dans la prévention ou le traitement du diabète ou d'une complication du diabète
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WO2021260530A1 (fr) * 2020-06-22 2021-12-30 Sun Pharmaceutical Industries Limited Agonistes doubles de glp-1/gip à action prolongée
WO2022079639A1 (fr) * 2020-10-17 2022-04-21 Sun Pharmaceutical Industries Limited Doubles agonistes de glp-1/gip

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WO2016065090A1 (fr) * 2014-10-24 2016-04-28 Merck Sharp & Dohme Corp. Co-agonistes des récepteurs du glucagon et du glp-1
CN107108715A (zh) * 2014-10-24 2017-08-29 默沙东公司 胰高血糖素和glp‑1受体的共激动剂
CN107207576A (zh) * 2015-01-09 2017-09-26 伊莱利利公司 Gip和glp‑1共激动剂化合物
WO2021169512A1 (fr) * 2020-02-24 2021-09-02 中山大学 Composé polypeptidique et son application dans la prévention ou le traitement du diabète ou d'une complication du diabète
WO2021260530A1 (fr) * 2020-06-22 2021-12-30 Sun Pharmaceutical Industries Limited Agonistes doubles de glp-1/gip à action prolongée
WO2022079639A1 (fr) * 2020-10-17 2022-04-21 Sun Pharmaceutical Industries Limited Doubles agonistes de glp-1/gip
CN113429471A (zh) * 2021-07-19 2021-09-24 青岛博睿精创科技有限公司 长效glp-1多肽类似物及其制备方法和应用

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