CN115073582A - Polypeptide compound and application thereof in preventing or treating diabetes or diabetic complications - Google Patents

Polypeptide compound and application thereof in preventing or treating diabetes or diabetic complications Download PDF

Info

Publication number
CN115073582A
CN115073582A CN202110280107.6A CN202110280107A CN115073582A CN 115073582 A CN115073582 A CN 115073582A CN 202110280107 A CN202110280107 A CN 202110280107A CN 115073582 A CN115073582 A CN 115073582A
Authority
CN
China
Prior art keywords
ser
gly
peg
aib
lys
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202110280107.6A
Other languages
Chinese (zh)
Inventor
蒋先兴
赵倩
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shenzhen Turier Biotech Co ltd
Original Assignee
Shenzhen Turier Biotech Co ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shenzhen Turier Biotech Co ltd filed Critical Shenzhen Turier Biotech Co ltd
Priority to CN202110280107.6A priority Critical patent/CN115073582A/en
Publication of CN115073582A publication Critical patent/CN115073582A/en
Pending legal-status Critical Current

Links

Images

Classifications

    • 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
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • 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
    • 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
    • C07K14/57563—Vasoactive intestinal peptide [VIP]; Related peptides
    • 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
    • C07K14/57572—Gastrin releasing peptide
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Diabetes (AREA)
  • Endocrinology (AREA)
  • Veterinary Medicine (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • General Chemical & Material Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Public Health (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Obesity (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Emergency Medicine (AREA)
  • Child & Adolescent Psychology (AREA)
  • Vascular Medicine (AREA)
  • Mycology (AREA)
  • Nutrition Science (AREA)
  • Food Science & Technology (AREA)
  • Polymers & Plastics (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

The invention discloses a polypeptide compound and application thereof in preventing or treating diabetes or diabetic complications, wherein the polypeptide compound shows good long-acting hypoglycemic effect and diabetic nephropathy treatment effect; meanwhile, the compound enzyme has the effects of high enzymolysis stability, high biological activity, no adverse reaction and the like, and can be used for preparing medicines for treating hyperphagia, obesity, overweight, cholesterol increase, diabetes and diabetic nephropathy.

Description

Polypeptide compound and application thereof in preventing or treating diabetes or diabetic complications
Technical Field
The invention belongs to the technical field of biochemistry, and particularly relates to a polypeptide compound and application thereof in preventing or treating diabetes or diabetic complications.
Background
In recent years, the prevalence of diabetes has also rapidly increased worldwide with rapid economic development, changes in dietary structure and lifestyle, and an increase in the aging rate of the population. Statistically, nearly 9% of adults worldwide suffer from type II diabetes. And the Global prevalence of Diabetes has been estimated by the International Diabetes Federation (IDF) to increase from 4.15 million people in 2015 to 6.42 million people in 2040 years (Unnikrishnan R, Pradepa R, Joshi SR, Mohan V.type 2 Diabetes: Demystifying the Global epidemic. Diabetes,2017.66: 1432-inch 1442). Diabetes has become another important chronic non-infectious disease seriously endangering human health after cardiovascular and cerebrovascular diseases and tumors.
In recent years, many studies have shown that the diabetes control status is not optimistic in both developed countries in europe and america and in developing countries such as china. One of the most serious consequences of diabetes is the development of diabetic vasculopathy, which is clinically manifested as microvascular and macrovascular complications. Diabetic nephropathy is a microvascular complication and the most common cause of end-stage renal disease in developed countries.
Approximately 40% of type I diabetic patients will develop diabetic nephropathy, and while there are relatively few cases where type II diabetic nephropathy develops into end stage renal disease, the rapid increase in the number of type II diabetic patients means that the incidence of diabetic nephropathy will increase dramatically if there is no effective measure of diabetes management. Therefore, there is an urgent need for effective and long-acting hypoglycemic agents for the treatment of diabetic nephropathy. Approximately 40% of patients with type I diabetes develop diabetic nephropathy. Due to the considerable ethnic variability in the incidence of diabetic nephropathy, only about one third of type I diabetic patients progress to diabetic nephropathy and have a strong relationship with genetic factors. Although there are relatively few cases of type II diabetic nephropathy progressing to end stage renal disease, with the rapidly increasing number of type II diabetic patients this means that the incidence of diabetic nephropathy will also increase substantially if there are no effective measures for diabetes management, and there is therefore an urgent need for effective and long-acting hypoglycemic drugs for the treatment of diabetic nephropathy (Chen L, Magliano DJ, Zimmet PZ. the world diabetes epidemiology of type 2diabetes mellitus-present and future surgery. Nat Rev Endocrinol,2011,8(4): 228-.
With the intensive research on diabetes and the treatment thereof, people continuously find new targets of the drug action for treating II-type diabetes. GLP-1 analogues (Meier JJ. GLP-1Receptor Agents for induced Treatment of Type-2 Diabetes mellitus. Nat. Rev. Endocrinol,2012.8(12):728) which are marketed since 2005 have different action targets from the prior medicines and provide a new Treatment option for the Treatment of Diabetes mellitus. GLP-1 analogs that have been currently approved for the treatment of type II diabetes include exenatide, liraglutide and lixisenatide, as well as subsequently produced albiglutide and dolaglutide. In addition, the long-acting GLP-1 analog, somaglutide, by noh & nodel corporation, 1 time per week has also been marketed for the treatment of type II diabetes.
Compared with a homologous ligand single-target agonist, the GLP-1R/GCGR dual agonist has the synergistic effects of reducing blood sugar, blood fat and body weight. In addition, a GLP-1R/GCGR dual-target agonist, the synergistic effect of GCGR with GLP-1, enhances pancreatic beta-cell function while possibly minimizing or preventing type II diabetes progression. These innovations increase the intended effect of GLP-1, and the secondary activity may increase more efficacy, for example: weight reduction, cardiovascular benefits, treatment of diabetic nephropathy, and the like.
For example, chinese patent CN201711194175.0 discloses a glucagon analog for treating metabolic diseases, but it was found through research that the glucagon analog has the effect of improving sugar tolerance equivalent to that of liraglutide, but the effect of improving sugar tolerance of bikureuptatide is poor, and the glucagon analog also cannot meet the clinical use requirements.
In view of the above, despite the existence of many drugs for the treatment of diabetes, the treatment of type II diabetes and associated complications of renal disease remains challenging and the suppression of the global spread of diabetic nephropathy is deemed to be a long and daunting task. The development of novel dual-target or multi-target agonist polypeptide drugs is the main direction for treating diabetes and complications thereof.
Disclosure of Invention
The invention aims to provide a novel polypeptide compound. Through a large number of experimental researches, the inventor proves that the novel polypeptide compound has longer half-life period, insulin promoting activity and no adverse reaction, and can be used for preventing or treating diabetes complications such as diabetes, diabetic nephropathy and the like.
Another object of the present invention is to provide the use of the above novel polypeptide compound, which can potentially be used as a new generation of drugs for preventing or treating diabetic complications such as diabetes and diabetic nephropathy, and can also be used for lowering blood sugar or reducing body weight.
In order to achieve the purpose, the technical scheme of the invention is as follows:
the present invention provides a polypeptide compound comprising a parent peptide represented by the amino acid sequence:
His-Xaa2-Gln-Gly-Thr 5 -Phe-Thr-Ser-Asp-Lys 10 -Ser-Lys-Tyr-Leu-Xaa15 15 -Xaa16-Xaa17-Ala-Ala-Gln 20 -Xaa21-Phe-Xaa23-Xaa24-Trp 25 -Leu-Xaa27-Xaa28-Gly-Gly 30 -Pro-Ser-Ser-Gly-Xaa35 35 -Pro-Pro-Pro-Ser，
wherein:
xaa2 ═ Aib, Ser or D-Ser;
xaa15 ═ Asp or Glu;
xaa16 ═ Aib or Glu;
xaa17 ═ Lys or Arg;
xaa21 ═ Asp or Glu;
xaa23 Val or Iva;
xaa24 ═ Glu or Gln;
xaa27 ═ Leu or Lys;
xaa28 ═ Asp, Glu, or Ala;
xaa35 ═ Ala or Aib.
Preferably, in the amino acid sequence of the parent peptide, Xaa2 ═ Aib or D-Ser.
Preferably, in the amino acid sequence of the parent peptide, Xaa21 ═ Asp.
Preferably, the carboxy terminus of the amino acid sequence of the parent peptide is unmodified or amino-modified to form-CONH 2 A group.
And, preferably, the side chain of the Lys at position 10 or 12 in the amino acid sequence of the parent peptide is linked to the lipophilic substituent via a bridging group which is (PEG) m 、(PEG) m -γGlu、(PEG) m -Asp、(Gly) x -(Gly-Ser) y -(Gly) z -、(Gly) x -(Gly-Ser) y -(Gly) z -gamma Glu and (Gly) x -(Gly-Ser) y -(Gly) z -one of Asp. The connection mode is as follows: the side chain amino group of the Lys at position 10 or 12 and the carboxyl group of the glycine residue at one end of the bridging group or (PEG) m The terminal modified carboxyl group forms an amide bond to be connected to the parent peptide; meanwhile, the lipophilic substituent is connected with the amino of the bridging group at the other end through forming an amido bond by the carboxyl of the lipophilic substituent. The lipophilic substituent is CH 3 (CH 2 ) n C (O) -or HOOC (CH) 2 ) n C (o) -and the acyl group thereof forms an amide bond with the amino group in the bridging group.
In the bridging group of the present invention, m is an integer of 2 to 10; x is an integer of 0 to 5; y is an integer of 1 to 5; z is an integer from 1 to 5; in the lipophilic substituents of the present invention, n is an integer from 14 to 20.
Alternatively, Lys at position 10 or 12 in the amino acid sequence of the parent peptide may be replaced by HomoLys, Orn, Dap or Dab.
Further preferably, in the amino acid sequence of the parent peptide:
xaa2 ═ Aib or D-Ser;
Xaa15＝Glu；
Xaa16＝Aib；
Xaa17＝Lys；
Xaa21＝Asp；
Xaa23＝Val；
Xaa24＝Glu；
Xaa27＝Lys；
Xaa28＝Ala；
Xaa35＝Ala；
alternatively, the first and second electrodes may be,
xaa2 ═ Aib or D-Ser;
Xaa15＝Asp；
Xaa16＝Glu；
Xaa17＝Arg；
Xaa21＝Asp；
Xaa23＝Iva；
Xaa24＝Gln；
Xaa27＝Leu；
xaa28 ═ Asp or Glu;
xaa35 ═ Ala or Aib.
According to a particular embodiment of the invention, the amino acid sequence of the parent peptide is selected from the group consisting of the amino acid sequences shown in SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6, SEQ ID No.7, SEQ ID No.8, SEQ ID No.9, SEQ ID No.10, SEQ ID No.11, SEQ ID No.12, SEQ ID No.13, SEQ ID No.14, SEQ ID No.15 and SEQ ID No. 16.
According to a particular embodiment of the invention, the structure formed by the attachment of a Lys at position 10 or 12, preferably a Lys at position 10, to a lipophilic substituent via a bridging group in the amino acid sequence of the parent peptide is:
Figure BDA0002977865590000051
Figure BDA0002977865590000061
according to a particular embodiment of the invention, the invention provides any one of the following polypeptide compounds:
compound 1:
H-Aib-QGTFTSDK(PEG 2 -PEG 2 -γGlu-CO(CH 2 ) 18 CO 2 H)SKYLE-Aib-KAAQDFVEWLKAGGPSSGAPPPS-NH 2
compound 2:
HsQGTFTSDK(PEG 2 -PEG 2 -CO(CH 2 ) 18 CO 2 H)SKYLE-Aib-KAAQDFVEWLKAGGPSSGAPPPS-NH 2
compound 3:
HsQGTFTSDK(PEG 2 -PEG 2 -γGlu-CO(CH 2 ) 18 CO 2 H)SKYLE-Aib-KAAQDFVEWLKAGGPSSGAPPPS-NH 2
compound 4:
HsQGTFTSDK(PEG 2 -PEG 2 -γGlu-CO(CH 2 ) 16 CO 2 H)SKYLE-Aib-KAAQDFVEWLKAGGPSSGAPPPS-NH 2
compound 5:
HsQGTFTSDK(PEG 2 -PEG 2 -γGlu-CO(CH 2 ) 18 CH 3 )SKYLE-Aib-KAAQDFVEWLKAGGPSSGAPPPS-NH 2
compound 6:
HsQGTFTSDK(PEG 2 -PEG 2 -γGlu-CO(CH 2 ) 18 CO 2 H)SKYLDERAAQDF-Iva-QWLLDGGPSSG-Aib-PPPS-NH 2
compound 7:
HsQGTFTSDK(PEG 2 -PEG 2 -γGlu-CO(CH 2 ) 18 CO 2 H)SKYLDERAAQDF-Iva-QWLLEGGPSSGAPPPS-NH 2
compound 8:
H-Aib-QGTFTSDK(PEG 2 -PEG 2 -γGlu-CO(CH 2 ) 18 CO 2 H)SKYLDERAAQDF-Iva-QWLLDGGPSSG-Aib-PPPS-NH 2
compound 9:
H-Aib-QGTFTSDK(PEG 2 -PEG 2 -γGlu-CO(CH 2 ) 16 CO 2 H)SKYLDERAAQDF-Iva-QWLLDGGPSSG-Aib-PPPS-NH 2
compound 10:
H-Aib-QGTFTSDK(PEG 2 -PEG 2 -γGlu-CO(CH 2 ) 18 CH 3 )SKYLDERAAQDF-Iva-QWLLDGGPSSG-Aib-PPPS-NH 2
compound 11:
H-Aib-QGTFTSDK(PEG 2 -PEG 2 -γGlu-CO(CH 2 ) 18 CH 3 )SKYLDERAAQDF-Iva-QWLLDGGPSSG-Aib-PPPS-NH 2
compound 12:
HsQGTFTSDK(PEG 2 -PEG 2 -γGlu-CO(CH 2 ) 18 CO 2 H)SKYLDERAAQDF-Iva-QWLLDGGPSSG-Aib-PPPS-NH 2
compound 13:
HsQGTFTSDK(GGSGSG-γGlu-CO(CH 2 ) 18 -COOH)SKYLE-Aib-KAAQDFVEWLKAGGPSSGAPPPS
compound 14:
HsQGTFTSDK(GGSGSG-γGlu-CO(CH 2 ) 18 -COOH)SKYLE-Aib-KAAQDFVEWLKAGGPSSGAPPPS-NH 2
compound 15:
H-Aib-QGTFTSDK(GGSGSG-γGlu-CO(CH 2 ) 18 -COOH)SKYLDERAAQDF-Iva-QWLLDGGPSSG-Aib-PPPS
compound 16:
H-Aib-QGTFTSDK(GGSGSG-γGlu-CO(CH 2 ) 18 -COOH)SKYLDERAAQDF-Iva-QWLLDGGPSSG-Aib-PPPS-NH 2 。
the present invention also provides a composition comprising a polypeptide compound of the present invention.
Preferably, the composition is a pharmaceutical composition, optionally further comprising a pharmaceutically acceptable carrier or adjuvant.
The invention further provides an application of the polypeptide compound or the composition in preparing a medicament for preventing or treating diabetes and/or diabetic complications; wherein preferably, the diabetic complication is diabetic nephropathy.
The invention also provides application of the polypeptide compound or the composition in preparing health products or medicines for reducing weight.
The technical scheme of the invention is described in detail as follows.
In a first aspect, the present invention provides a novel agonist polypeptide compound comprising a parent peptide represented by the amino acid sequence:
His-Xaa2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys-Ser-Lys-Tyr-Leu-Xaa15-Xaa16-Xaa17-Ala-Ala-Gln-Xaa21-Phe-Xaa23-Xaa24-Trp-Leu-Xaa27-Xaa28-Gly-Gly-Pro-Ser-Ser-Gly-Xaa35-Pro-Pro-Pro-Ser-COR 1
wherein R is 1 ＝-NH 2 or-OH;
xaa2 ═ Aib, Ser or D-Ser;
xaa15 ═ Asp or Glu;
xaa16 ═ Aib or Glu;
xaa17 ═ Lys or Arg;
xaa21 ═ Asp or Glu;
xaa23 Val or Iva;
xaa24 ═ Glu or Gln;
xaa27 ═ Leu or Lys;
xaa28 ═ Asp, Glu, or Ala;
xaa35 ═ Ala or Aib.
In the amino acid sequence of the parent peptide, the side chain of the Lys at position 10 or 12 is linked to a lipophilic substituent via a bridging group.
The bridging group comprises (PEG) m Or (PEG) m -gamma Glu or (PEG) m -Asp; may also contain (Gly) x -(Gly-Ser) y -(Gly) z -, or (Gly) x -(Gly-Ser) y -(Gly) z -gamma Glu or (Gly) x -(Gly-Ser) y -(Gly) z -Asp. The connection mode is as follows: the side chain amino group of the Lys at position 10 or 12 and the carboxyl group of the glycine residue at one end of the bridging group or (PEG) m The terminally modified carboxyl group forms an amide bond to the parent peptide; whilst the lipophilic substituent is bridged by its carboxyl group to another terminal bridging groupThe amino groups are linked by formation of an amide bond. The Lys at position 10 or 12 to which the lipophilic substituent is attached can be replaced by HomoLys, Orn, Dap, or Dab. Wherein m is an integer from 2 to 10; x is an integer of 0 to 5; y is an integer of 1 to 5; z is an integer of 1 to 5. The lipophilic substituent is selected from CH 3 (CH 2 ) n CO-or HOOC (CH) 2 ) n CO-, wherein n is an integer from 14 to 20, are shown in detail in FIGS. 6 and 7, FIG. 6 is one exemplary modification of the Lys side chain in the parent peptide of the compound of the present invention, and FIG. 7 is another exemplary modification of the Lys side chain in the parent peptide of the compound of the present invention.
Throughout the specification of this application, the natural amino acids are represented using the conventional three-letter code, and other amino acids, such as Aib (aminoisobutyric acid), Orn (ornithine), are represented using the accepted three-letter code. The compounds of the invention stabilize the helical structure of the molecule based on a theoretical intramolecular bridge, thereby increasing potency and/or selectivity for the glucagon-like peptide 1(GLP-1R) receptor and the glucagon (GCGR) receptor.
The compounds of the present invention may bind to albumin in the blood based on a theoretical lipophilic substituent, thereby protecting the compounds from enzymatic degradation and increasing the half-life of the compounds.
Another aspect of the present invention is to provide a pharmaceutical composition containing the novel agonist polypeptide compound of the present invention, wherein the novel agonist polypeptide compound is used as an active ingredient, and a pharmaceutically acceptable carrier and/or an adjuvant is added to prepare the pharmaceutical composition.
It is a further aspect of the present invention to provide for the medical use of the novel agonist polypeptide compounds of the present invention. Cell and animal experiments show that the novel agonist polypeptide compound has long-acting hypoglycemic effect and can be used as a medicament for treating diabetes.
It is a further aspect of the present invention to provide additional pharmaceutical uses of the novel agonist polypeptide compounds of the present invention. Cell and animal experiments show that the novel agonist polypeptide compound has the drug effect of treating diabetic complications such as diabetic nephropathy. Animal experiments show that the treatment effect is better than that of the sold GLP-1 analogue Somalutide, and the Somalutide can be used as a medicament for treating diabetic complications such as diabetic nephropathy.
The novel agonist polypeptide compounds of the present invention can also reduce body weight and have potential use as a medicament for treating obesity.
The parent peptide in the novel agonist polypeptide compounds of the present invention is a homologous polypeptide. The homologous polypeptide in the present invention means that the polypeptide originally has an amino acid sequence of Oxyntomodulin (OXM), Glucagon-like peptide (GLP-1), Exenatide (Exenatide), or Glucagon (Glucagon), but one or more amino acid residues are substituted with different amino acid residues, which are conserved with each other, and the resulting polypeptide can be used for practicing the present invention.
The polypeptide compounds of the present invention may also be used to prevent weight gain or to promote weight loss. The polypeptide compounds may cause a decrease in food intake and/or an increase in energy expenditure, resulting in an observable effect on body weight. Accordingly, the polypeptide compounds of the present invention may be used for the direct or indirect treatment of any condition caused by or characterized by being overweight, such as the treatment and/or prevention of obesity, morbid obesity, obesity-related inflammation, obesity-related gallbladder disease, obesity-induced sleep apnea. The effect of the polypeptide compounds of the invention in these conditions may be caused by or associated with the effect of the polypeptide compounds on body weight, or may be independent of their effect on body weight.
It will be appreciated by those skilled in the art that the pharmaceutical compositions of the present invention are suitable for various modes of administration, such as oral, transdermal, intravenous, intramuscular, topical, nasal, and the like. Depending on the mode of administration employed, the pharmaceutical compositions of the polypeptide compounds of the present invention may be formulated into various suitable dosage forms comprising at least one effective dose of the polypeptide compound of the present invention and at least one pharmaceutically acceptable carrier. Examples of suitable dosage forms are tablets, capsules, sugar-coated tablets, granules, oral solutions and syrups, ointments and patches for the skin surface, aerosols, nasal sprays, and sterile solutions for injection.
Pharmaceutical compositions containing the polypeptide compounds of the present invention may be presented as solutions or lyophilized powders for parenteral administration, which may be reconstituted with a suitable solvent or other pharmaceutically acceptable carrier prior to use, and liquid formulations are typically buffers, isotonic solutions and aqueous solutions.
The amount of the pharmaceutical composition of the present invention may vary over a wide range and can be readily determined by one skilled in the art based on objective factors such as the type of disease, the severity of the condition, the weight of the patient, the dosage form, the route of administration, and the like.
Compared with the prior art, the invention has the following advantages:
1) compared with the natural OXM, the polypeptide compound has better biological activity when the mass number is the same;
2) compared with GLP-1 analogues when the mass number is the same, the polypeptide compound has better drug effect for treating diabetic complications such as diabetic nephropathy;
3) the compound shows remarkably prolonged half-life and stability in a pharmaceutical experiment;
4) high synthesis yield, good stability, easy scale-up production and low cost.
In particular embodiments, the present invention relates to novel agonist polypeptide compounds having the following sequence and side chain modifications:
compound 1(SEQ ID NO. 1):
His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(PEG 2 -PEG 2 -γGlu-CO(CH 2 ) 18 CO 2 H)-Ser-Lys-Tyr-Leu-Glu-Aib-Lys-Ala-Ala-Gln-Asp-Phe-Val-Glu-Trp-Leu-Lys-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2
H-Aib-QGTFTSDK(PEG 2 -PEG 2 -γGlu-CO(CH 2 ) 18 CO 2 H)SKYLE-Aib-KAAQDFVEWLKAGGPSSGAPPPS-NH 2
compound 2(SEQ ID NO. 2):
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(PEG 2 -PEG 2 -CO(CH 2 ) 18 CO 2 H)-Ser-Lys-Tyr-Leu-Glu-Aib-Lys-Ala-Ala-Gln-Asp-Phe-Val-Glu-Trp-Leu-Lys-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2
HsQGTFTSDK(PEG 2 -PEG 2 -CO(CH 2 ) 18 CO 2 H)SKYLE-Aib-KAAQDFVEWLKAGGPS SGAPPPS-NH 2
compound 3(SEQ ID NO. 3):
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(PEG 2 -PEG 2 -γGlu-CO(CH 2 ) 18 CO 2 H)-Ser-Lys-Tyr-Leu-Glu-Aib-Lys-Ala-Ala-Gln-Asp-Phe-Val-Glu-Trp-Leu-Lys-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2
HsQGTFTSDK(PEG 2 -PEG 2 -γGlu-CO(CH 2 ) 18 CO 2 H)SKYLE-Aib-KAAQDFVEWLKAGGPSSGAPPPS-NH 2
compound 4(SEQ ID NO. 4):
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(PEG 2 -PEG 2 -γGlu-CO(CH 2 ) 16 CO 2 H)-Ser-Lys-Tyr-Leu-Glu-Aib-Lys-Ala-Ala-Gln-Asp-Phe-Val-Glu-Trp-Leu-Lys-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2
HsQGTFTSDK(PEG 2 -PEG 2 -γGlu-CO(CH 2 ) 16 CO 2 H)SKYLE-Aib-KAAQDFVEWLKAGGPSSGAPPPS-NH 2
compound 5(SEQ ID NO. 5):
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(PEG 2 -PEG 2 -γGlu-CO(CH 2 ) 18 CH 3 )-Ser-Lys-Tyr-Leu-Glu-Aib-Lys-Ala-Ala-Gln-Asp-Phe-Val-Glu-Trp-Leu-Lys-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2
HsQGTFTSDK(PEG 2 -PEG 2 -γGlu-CO(CH 2 ) 18 CH 3 )SKYLE-Aib-KAAQDFVEWLKAGGPSSGAPPPS-NH 2
compound 6(SEQ ID NO. 6):
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(PEG 2 -PEG 2 -γGlu-CO(CH 2 ) 18 CO 2 H)-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Ala-Ala-Gln-Asp-Phe-Iva-Gln-Trp-Leu-Leu-Asp-Gly-Gly-Pro-Ser-Ser-Gly-Aib-Pro-Pro-Pro-Ser-NH 2
HsQGTFTSDK(PEG 2 -PEG 2 -γGlu-CO(CH 2 ) 18 CO 2 H)SKYLDERAAQDF-Iva-QWLLDGGPS SG-Aib-PPPS-NH 2
compound 7(SEQ ID NO. 7):
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(PEG 2 -PEG 2 -γGlu-CO(CH 2 ) 18 CO 2 H)-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Ala-Ala-Gln-Asp-Phe-Iva-Gln-Trp-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2
HsQGTFTSDK(PEG 2 -PEG 2 -γGlu-CO(CH 2 ) 18 CO 2 H)SKYLDERAAQDF-Iva-QWLLEGGPSSGAPPPS-NH 2
compound 8(SEQ ID NO. 8):
His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(PEG 2 -PEG 2 -γGlu-CO(CH 2 ) 18 CO 2 H)-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Ala-Ala-Gln-Asp-Phe-Iva-Gln-Trp-Leu-Leu-Asp-Gly-Gly-Pro-Ser-Ser-Gly-Aib-Pro-Pro-Pro-Ser-NH 2
H-Aib-QGTFTSDK(PEG 2 -PEG 2 -γGlu-CO(CH 2 ) 18 CO 2 H)SKYLDERAAQDF-Iva-QWLLDGGPSSG-Aib-PPPS-NH 2
compound 9(SEQ ID NO. 9):
His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(PEG 2 -PEG 2 -γGlu-CO(CH 2 ) 16 CO 2 H)-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Ala-Ala-Gln-Asp-Phe-Iva-Gln-Trp-Leu-Leu-Asp-Gly-Gly-Pro-Ser-Ser-Gly-Aib-Pro-Pro-Pro-Ser-NH 2
H-Aib-QGTFTSDK(PEG 2 -PEG 2 -γGlu-CO(CH 2 ) 16 CO 2 H)SKYLDERAAQDF-Iva-QWLLDGGPSSG-Aib-PPPS-NH 2
compound 10(SEQ ID NO. 10):
His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(PEG 2 -PEG 2 -γGlu-CO(CH 2 ) 18 CH 3 )-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Ala-Ala-Gln-Asp-Phe-Iva-Gln-Trp-Leu-Leu-Asp-Gly-Gly-Pro-Ser-Ser-Gly-Aib-Pro-Pro-Pro-Ser-NH 2
H-Aib-QGTFTSDK(PEG 2 -PEG 2 -γGlu-CO(CH 2 ) 18 CH 3 )SKYLDERAAQDF-Iva-QWLLDGGPSSG-Aib-PPPS-NH 2
compound 11(SEQ ID NO. 11):
His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(PEG 2 -PEG 2 -CO(CH 2 ) 18 CH 3 )-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Ala-Ala-Gln-Asp-Phe-Iva-Gln-Trp-Leu-Leu-Asp-Gly-Gly-Pro-Ser-Ser-Gly-Aib-Pro-Pro-Pro-Ser-NH 2
H-Aib-QGTFTSDK(PEG 2 -PEG 2 -γGlu-CO(CH 2 ) 18 CH 3 )SKYLDERAAQDF-Iva-QWLLDGGPSSG-Aib-PPPS-NH 2
compound 12(SEQ ID No. 12):
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(PEG 2 -PEG 2 -γGlu-CO(CH 2 ) 18 CO 2 H)-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Ala-Ala-Gln-Asp-Phe-Iva-Gln-Trp-Leu-Leu-Asp-Gly-Gly-Pro-Ser-Ser-Gly-Aib-Pro-Pro-Pro-Ser-NH 2
HsQGTFTSDK(PEG 2 -PEG 2 -γGlu-CO(CH 2 ) 18 CO 2 H)SKYLDERAAQDF-Iva-QWLLDGGPSSG-Aib-PPPS-NH 2
compound 13(SEQ ID NO. 13):
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(Gly-Gly-Ser-Gly-Ser-Gly-γGlu-CO(CH 2 ) 18 -COOH)-Ser-Lys-Tyr-Leu-Glu-Aib-Lys-Ala-Ala-Gln-Asp-Phe-Val-Glu-Trp-Leu-Lys-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser
HsQGTFTSDK(GGSGSG-γGlu-CO(CH 2 ) 18 -COOH)SKYLE-Aib-KAAQDFVEWLKAGGPSSGAPPPS
compound 14(SEQ ID NO. 14):
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(Gly-Gly-Ser-Gly-Ser-Gly-γGlu-CO(CH 2 ) 18 -COOH)-Ser-Lys-Tyr-Leu-Glu-Aib-Lys-Ala-Ala-Gln-Asp-Phe-Val-Glu-Trp-Leu-Lys-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2
HsQGTFTSDK(GGSGSG-γGlu-CO(CH 2 ) 18 -COOH)SKYLE-Aib-KAAQDFVEWLKAGGPSSGAPPPS-NH 2
compound 15(SEQ ID NO. 15):
His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(Gly-Gly-Ser-Gly-Ser-Gly-γGlu-CO(CH 2 ) 18 -COOH)-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Ala-Ala-Gln-Asp-Phe-Iva-Gln-Trp-Leu-Leu-Asp-Gly-Gly-Pro-Ser-Ser-Gly-Aib-Pro-Pro-Pro-Ser
H-Aib-QGTFTSDK(GGSGSG-γGlu-CO(CH 2 ) 18 -COOH)SKYLDERAAQDF-Iva-QWLLDGGPSSG-Aib-PPPS
compound 16(SEQ ID NO. 16):
His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(Gly-Gly-Ser-Gly-Ser-Gly-γGlu-CO(CH 2 ) 18 -COOH)-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Ala-Ala-Gln-Asp-Phe-Iva-Gln-Trp-Leu-Leu-Asp-Gly-Gly-Pro-Ser-Ser-Gly-Aib-Pro-Pro-Pro-Ser-NH 2
H-Aib-QGTFTSDK(GGSGSG-γGlu-CO(CH 2 ) 18 -COOH)SKYLDERAAQDF-Iva-QWLLDGGPSSG-Aib-PPPS-NH 2 。
in the above sequence, the modification of Lys may be one of the following structures:
Figure BDA0002977865590000151
Figure BDA0002977865590000161
the above Lys attached to the lipophilic substituent may be replaced by:
Figure BDA0002977865590000162
the abbreviations used in the present invention have the following specific meanings:
boc is t-butoxycarbonyl, Fmoc is fluorenylmethoxycarbonyl, t-Bu is t-butyl, ivDDe is 1- (4, 4-dimethyl-2, 6-dioxocyclohexylidene) -3-methyl-butyl, resin, TFA is trifluoroacetic acid, EDT is 1, 2-ethanedithiol, Phenol is Phenol, FBS is fetal bovine serum, BSA is bovine serum albumin, HPLC is high performance liquid, GLP-1R is glucagon-like peptide 1receptor, GCGR is glucagon receptor, GLP-1 is glucagon-like peptide, mPEG is monomethoxypolyethylene glycol, His is histidine, Ser is serine, D-Ser is D-type serine, Gln is glutamine, Gly is glycine, Glu is glutamic acid, Ala is alanine, Thr is threonine, Lys is lysine, Arg is arginine, Tyr is tyrosine, asp is aspartic acid, Trp is tryptophan, Phe is phenylalanine, IIe is isoleucine, Leu is leucine, Cys is cysteine, Pro is proline, Val is valine, Met is methionine, and Asn is asparagine. HomoLys is homolysine, Orn is ornithine, Dap is diaminopimelic acid, Dab is 2, 4-diaminobutyric acid, Aib is 2-aminoisobutyric acid, and Iva is isovaline.
Drawings
Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:
fig. 1 is the area under the curve (AUC), p <0.05, calculated in example 2 based on the initial blood glucose level at 4 hours after administration of the experimental animals and the blood glucose change tracked at 24 hours for re-oral glucose at different time points.
FIG. 2A is a FN expression gel electrophoresis chart of different groups detected by Western blot using in vitro renal fibroblast model constructed by inducing MCS lineage membrane cells and NRK renal epithelial cells with high sugar in example 3.
FIG. 2B is a histogram of FN expression levels <0.05 in different groups, as detected by constructing an in vitro renal fibrotic cell model by high-sugar induction of MCS lineage membrane cells and NRK renal epithelial cells in example 3; *: indicates within 95% confidence (p <0.05) compared to control; **: indicating within 99% confidence (p <0.01) compared to the control.
FIG. 3A is blood glucose and body weight of 8-week-old db/db diabetic mice of example 4 administered daily fasting for 6h, p <0.05, subcutaneously daily or every other day, with saline, Compound 3(80 and 120. mu.g/kg, 2 groups total), Compound 8(80 and 120. mu.g/kg, 2 groups total), Compound 13(80 and 120. mu.g/kg, 2 groups total), Compound 15(80 and 120. mu.g/kg, 2 groups total) and Somaltulip (80 and 120. mu.g/kg, 2 groups total), respectively.
FIG. 3B is blood glucose and body weight of 8 week old db/db diabetic mice in example 4 administered daily or every other day subcutaneously with p <0.05, measured by fasting for 6h every other day after injecting normal saline, Compound 3(80 and 120. mu.g/kg, in 2 groups), Compound 8(80 and 120. mu.g/kg, in 2 groups), Compound 13(80 and 120. mu.g/kg, in 2 groups), Compound 15(80 and 120. mu.g/kg, in 2 groups) and Somaltulide (80 and 120. mu.g/kg, in 2 groups), respectively.
FIG. 4A is the blood glucose change values of 12-week-old db/db diabetic mice of example 4, administered by subcutaneous injection of saline, polypeptide compounds 3, 8, 13, 14, 15, 16 and somaglutide, SAR425899, MED C18-acid, for 6 weeks consecutively.
FIG. 4B is the results of OGTT and the corresponding AUC measured at week 4 of 12-week-old db/db diabetic mice of example 4, subcutaneously injected with saline, the polypeptide compounds 3, 8, 13, 14, 15, 16 and somaglutide, SAR425899, MED C18-acid, respectively.
FIG. 4C is the ITT results and corresponding AUC values determined at week 5 in db/db diabetic mice of 12 weeks of age in example 4, injected subcutaneously with saline, the polypeptide compounds 3, 8, 13, 14, 15, 16 and somaglutide, SAR425899, MED C18-acid, respectively.
FIG. 4D is the results of 12 weeks old db/db diabetic mice in example 4 injected subcutaneously with saline, polypeptide compounds 3, 8, 13, 14, 15, 16 and somaglutide, SAR425899, MED C18-acid, ALT and AST in mouse serum, respectively, with p < 0.05.
FIG. 5A shows, from left to right, the urine protein content, the urine sugar content, and the glycated hemoglobin content, the urea nitrogen content, and the blood creatinine content in the serum of the mouse in example 4 after 8 weeks of the administration of the treatment.
FIG. 5B is the result of H & E staining of the kidney 8 weeks after the mice in example 4 were treated with the drug.
FIG. 5C is the PAS staining results of the kidney 8 weeks after the mice were treated with the drug in example 4 and a statistical chart.
Figure 5D is the results and statistical plots of sirius red staining of the kidneys after 8 weeks of treatment with the mice in example 4.
FIG. 5E is FN staining of the kidneys and statistical plots of the mice of example 4 after 8 weeks of treatment.
Figure 5F is a graph of the staining of α -SMA in the kidney 8 weeks after administration of treatment to mice in example 4 and statistics with p < 0.05.
FIG. 6 is an exemplary modification of the Lys side chain in the parent peptide of the compounds of the present invention.
FIG. 7 is another exemplary modification of the Lys side chain in the parent peptide of the compounds of the present invention.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
Example 1Synthesis of polypeptide Compounds
The polypeptide compounds 1-16, the positive control polypeptide compounds 17-20 (derived from the compound in Chinese patent publication No. CN 104926934A), the positive control polypeptide compounds 21-26 (derived from patent CN201711194175.0), SAR425899 and MED C18-acid are all synthesized by self, wherein the SAR425899 and the MED C18-acid are all compounds reported in the prior art.
The amino acid sequences of the positive control polypeptide compounds 17-26, SAR425899 and MED C18-acid compounds are as follows:
compound 17(SEQ ID NO. 17):
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Lys(PEG 2 -PEG 2 -γGlu-CO(CH 2 ) 14 CH 3 )-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2
HsQGTFTSDYSKYLDK(PEG 2 -PEG 2 -γGlu-CO(CH 2 ) 14 CH 3 )RRAQDFVQWLMNTGGPSSGAPPPS-NH 2
compound 18(SEQ ID No. 18):
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Lys(PEG 2 -PEG 2 -γGlu-CO(CH 2 ) 14 CH 3 )-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Leu-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2
HsQGTFTSDYSKYLDK(PEG 2 -PEG 2 -γGlu-CO(CH 2 ) 14 CH 3 )RRAQDFVQWLLNTGGPSSGAPPPS-NH 2
compound 19(SEQ ID NO. 19):
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys(PEG 2 -PEG 2 -CO(CH 2 ) 16 CO 2 H)-Lys-Leu-Asp-Aib-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2
HsQGTFTSDYSK(PEG 2 -PEG 2 -CO(CH 2 ) 16 CO 2 H)KLD-Aib-RRAQDFVQWLMNTGGPSSGAPPPS-NH 2
compound 20(SEQ ID No. 20):
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Lys(PEG 2 -PEG 2 -γGlu-CO(CH 2 ) 14 CH 3 )-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2
H-(d-S)-QGTFTSDYSKYLDK(PEG 2 -PEG 2 -γGlu-CO(CH 2 ) 14 CH 3 )RRAQDFVQWLMNTGGPSSGAPPPS-NH 2
compound 21(SEQ ID No. 21):
His-Xaa-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys * -Ser-Lys-Tyr-Leu-Asp-Glu-Gln-Ala-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Leu-Asp-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2
HXQGTFTSDK*SKYLDEQAAQDFVQWLLDGPSSGAPPPS-NH 2
compound 22(SEQ ID NO. 22):
His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys * -Ser-Lys-Tyr-Leu-Asp-Ser-Gln-Ala-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2
HSQGTFTSDK*SKYLDSQAAQDFVQWLMNGGPSSGAPPPS-NH 2
compound 23(SEQ ID No. 23):
His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys * -Ser-Lys-Tyr-Leu-Asp-Glu-Glu-Ala-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2
HSQGTFTSDK * SKYLDEEAAQDFVQWLMNGGPSSGAPPPS-NH 2
compound 24(SEQ ID No. 24):
His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys * -Ser-Lys-Tyr-Leu-Asp-Glu-Gln-Ala-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2
HSQGTFTSDK * SKYLDEQAAQDFVQWLMNGGPSSGAPPPS-NH 2
compound 25(SEQ ID No. 25):
His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys * -Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Ala-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-OHHSQGTFTSDK * SKYLDERAAQDFVQWLMNTGPSSGAPPPS-OH
compound 26(SEQ ID No. 26):
His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys * -Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Ala-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-OHHSQGTFTSDK * SKYLDSRAAQDFVQWLMNTGPSSGAPPPS-OH
note: in the table, X in compound 21 is aminoisobutyric acid, K in compounds 21 to 25 * Indicating that this position is a lysine residue and covalently linked to a fatty acid having the structure shown below:
Figure BDA0002977865590000211
MED C18-acid(SEQ ID NO.27)：
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Lys(PEG 2 -PEG 2 -γGlu-(CH 2 ) 17 COOH)-Leu-Asp-Ser-Glu-Arg-Ala-Arg-Asp-Phe-Val-Ala-Trp-Leu-Val-Ala-Gly-Gly-NH 2
HsQGTFTSDYSKK(PEG 2 -PEG 2 -γGlu-CO(CH 2 ) 16 CO 2 H)LDSERARDFVAWLVAGG-NH 2
SAR425899(SEQ ID NO.28)：
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Lys(CO(CH 2 ) 14 CH 3 )-Glu-Ser-Lys-Ala-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2
HsQGTFTSDLSKQK(CO(CH 2 ) 14 CH 3 )ESKAAQDFIEWLKAGGPSSGAPPPS-NH 2
in order to conveniently illustrate the preparation method of the polypeptide compound, the invention takes the polypeptide compound 3 as an example, and the specific operation steps are as follows:
material (one):
all amino acids were purchased from NovaBiochem. All other reagents were analytical grade, purchased from Sigma, unless otherwise specified. CS336X polypeptide synthesizer (C S Bio Inc., USA). Phenomenex Luna C18 preparative columns (46 mm. times.250 mm) were used to purify the polypeptides. The high performance liquid chromatograph is a product of Waters company. Mass spectrometry was performed using an Agilent mass spectrometer.
(II) method:
1. synthesis of polypeptide compound 3:
the structural sequence is as follows:
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(PEG 2 -PEG 2 -γGlu-CO(CH 2 ) 18 CO 2 H)-Ser-Lys-Tyr-Leu-Glu-Aib-Lys-Ala-Ala-Gln-Asp-Phe-Val-Glu-Trp-Leu-Lys-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2
1a) assembling a main peptide chain:
the following polypeptides were synthesized on a 0.25mol scale according to the Fmoc/t-Bu strategy on a CS336X polypeptide synthesizer (C SBio, USA):
Boc-His(Boc)-D-Ser(t-Bu)-Gln(Trt)-Gly-Thr(t-Bu)-Phe-Thr(t-Bu)-Ser(tBu)-Asp(OtBu)-Lys(PEG 2 -PEG 2 -γGlu-CO(CH 2 ) 18 CO 2 H) -Ser (t-Bu) -Lys (Boc) -Tyr (t-Bu) -Leu-Glu (OtBu) -Aib-Lys (Boc) -Ala-Ala-Gln (Trt) -Asp (OtBu) -Phe-Val-Glu (OtBu) -Trp (Boc) -Leu-Lys (Boc) -Ala-Gly-Gly-Pro-Ser (t-Bu) -Ser (t-Bu) -Gly-Ala-Pro-Pro-Ser (t-Bu) -rink amide resin
(1) The first step is as follows: 0.75 g Rink amide resin in dichloromethane swelling, using N, N-two methyl formamide washing resin three times;
(2) the second step is that: taking Rink amide resin as a carrier, taking a mixed solution prepared from benzotriazole-N, N, N ', N' -tetramethylurea hexafluorophosphate, 1-hydroxybenzotriazole and N, N-diisopropylethylamine according to the mass ratio (molar ratio) of 1:1:1 as a coupling agent, taking N, N-dimethylformamide as a solvent, carrying out a programmed reaction, and sequentially carrying out a condensation reaction to connect Fmoc-Ser (t-Bu) -OH, Fmoc-Pro-OH (3x), Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser (t-Bu) -OH (2x), Fmoc-Pro-OH, Fmoc-Gly-OH (2x), Fmoc-Ala-OH, Fmoc-Lys (Boc) -OH and Fmoc-Leu-OH, Fmoc-Trp (Boc) -OH, Fmoc-Glu (OtBu) -OH,Fmoc-Val-OH，Fmoc-Phe-OH，Fmoc-Asp(OtBu)-OH，Fmoc-Gln(Trt)-OH，Fmoc-Ala-OH(2x)，Fmoc-Lys(Boc)-OH，Fmoc-Aib-OH，Fmoc-Glu(OtBu)-OH，Fmoc-Leu-OH，Fmoc-Tyr(t-Bu)-OH，Fmoc-Lys(Boc)-OH，Fmoc-Ser(t-Bu)-OH，Fmoc-Lys(PEG 2 -PEG 2 -γGlu-CO(CH 2 ) 18 CO 2 H) -OH, Fmoc-asp (otbu) -OH, Fmoc-Ser (t-Bu) -OH, Fmoc-Thr (t-Bu) -OH, Fmoc-Phe-OH, Fmoc-Thr (t-Bu) -OH, Fmoc-Gly-OH, Fmoc-gln (trt) -OH, Fmoc-D-Ser (t-Bu) -OH, Boc-his (Boc) -OH, to obtain:
Boc-His(Boc)-D-Ser(t-Bu)-Gln(Trt)-Gly-Thr(t-Bu)-Phe-Thr(t-Bu)-Ser(tBu)-Asp(OtBu)-Lys(PEG 2 -PEG 2 -γGlu-CO(CH 2 ) 18 CO 2 H) -Ser (t-Bu) -Lys (Boc) -Tyr (t-Bu) -Leu-Glu (OtBu) -Aib-Lys (Boc) -Ala-Ala-Gln (Trt) -Asp (OtBu) -Phe-Val-Glu (OtBu) -Trp (Boc) -Leu-Lys (Boc) -Ala-Gly-Pro-Ser (t-Bu) -Ser (t-Bu) -Gly-Ala-Pro-Pro-Ser (t-Bu) -rinkamide resin, wherein the ratio of the amount of the Fmoc-protected amino acid to the amount of the resin in each condensation reaction (molar ratio) is 1:1 to 6:1, and the ratio of the amount of the benzotriazole-N, N, N ', N' -tetramethyluronium hexafluorophosphate to the amount of the Fmoc-protected amino acid in each condensation reaction (molar ratio) is 1:1 3:1.
1b)Lys(PEG 2 -PEG 2 -γGlu-CO(CH 2 ) 18 CO 2 H) Removal and introduction of lipophilic substituents:
the protected peptidyl resin synthesized in 1a) was washed twice in a mixed solution of N-methylpyrrolidone and dichloromethane (vol/vol), a freshly prepared 2.0% hydrazine hydrate N-methylpyrrolidone solution was added, and the reaction mixture was shaken at room temperature for 12.0 minutes and then filtered. The hydrazine treatment step was repeated twice to give:
Boc-His (Boc) -D-Ser (t-Bu) -Gln (Trt) -Gly-Thr (t-Bu) -Phe-Thr (t-Bu) -Ser (tBu) -Asp (OtBu) -Lys-Ser (t-Bu) -Lys (Boc) -Tyr (t-Bu) -Leu-Glu (OtBu) -Aib-Lys (Boc) -Ala-Ala-Gln (Trt) -Asp (OtBu) -Phe-Val-Glu (OtBu) -Trp (Boc) -Leu-Lys Boc-Ala-Gly-Pro-Ser (t-Bu) -Ser (t-Bu) -Gly-Ala-Pro-Pro-Ser (t-Bu) -rink amide resin. Thereafter with dichloromethane and N-methylpyrrolidineThe ketone thoroughly washes the resin. Thereto was added FmocNH-PEG 2 And (3) mixing coupling liquid of-OH, benzotriazole-N, N, N ', N' -tetramethylurea hexafluorophosphate, 1-hydroxybenzotriazole and N-methylpyrrolidone of diisopropylethylamine, shaking for 3.0 hours, filtering, washing and repeating the hydrazine treatment step twice to obtain:
Boc-His(Boc)-D-Ser(t-Bu)-Gln(Trt)-Gly-Thr(t-Bu)-Phe-Thr(t-Bu)-Ser(tBu)-Asp(OtBu)-Lys(PEG 2 -OH) -Ser (t-Bu) -Lys (Boc) -Tyr (t-Bu) -Leu-Glu (OtBu) -Aib-Lys (Boc) -Ala-Ala-Gln (Trt) -Asp (OtBu) -Phe-Val-Glu (OtBu) -Trp (Boc) -Leu-Lys (Boc) -Ala-Gly-Gly-Pro-Ser (t-Bu) -Ser (t-Bu) -Gly-Ala-Pro-Pro-Ser (t-Bu) -rink amide resin, piperidine/N, N-dimethylformamide solution to remove Fmoc group, repeating Fmoc-PEG one time 2 -OH coupling reaction to give:
Boc-His(Boc)-D-Ser(t-Bu)-Gln(Trt)-Gly-Thr(t-Bu)-Phe-Thr(t-Bu)-Ser(tBu)-Asp(OtBu)-Lys(PEG 2 -PEG 2 -OH) -Ser (t-Bu) -lys (boc) -Tyr (t-Bu) -Leu-glu (otbu) -Aib-lys (boc) -Ala-gln (trt) -asp (otbu) -Phe-Val-glu (otbu) -trp (boc) -Leu-lys (boc) -Ala-Gly-Pro-Ser (t-Bu) -Gly-Ala-Pro-Ser (t-Bu) -rink amide resin. Removing Fmoc group from piperidine/N, N-dimethylformamide solution, and then sequentially coupling Fmoc-gamma Glu-OtBu and single-protected eicosapentanoic acid of tBu according to conventional conditions to obtain:
Boc-His(Boc)-D-Ser(t-Bu)-Gln(Trt)-Gly-Thr(t-Bu)-Phe-Thr(t-Bu)-Ser(tBu)-Asp(OtBu)-Lys(PEG 2 -PEG 2 -γGlu-CO(CH 2 ) 18 CO 2 H) -Ser (t-Bu) -Lys (Boc) -Tyr (t-Bu) -Leu-Glu (OtBu) -Aib-Lys (Boc) -Ala-Ala-Gln (Trt) -Asp (OtBu) -Phe-Val-Glu (OtBu) -Trp (Boc) -Leu-Lys (Boc) -Ala-Gly-Gly-Pro-Ser (t-Bu) -Ser (t-Bu) -Gly-Ala-Pro-Pro-Ser (t-Bu) -rink amide resin.
1c) And (3) removing full protection of polypeptide:
will be provided with
Boc-His(Boc)-D-Ser(t-Bu)-Gln(Trt)-Gly-Thr(t-Bu)-Phe-Thr(t-Bu)-Ser(tBu)-Asp(OtBu)-Lys(PEG 2 -PEG 2 -γGlu-CO(CH 2 ) 18 CO 2 H)-Ser(t-Bu)-Lys(Boc)-Tyr(t-Bu)-Leu-Glu(OtBu)-Aib-Lys(Boc)-Ala-Ala-Gln (Trt) -Asp (OtBu) -Phe-Val-Glu (OtBu) -Trp (Boc) -Leu-Lys (Boc) -Ala-Gly-Gly-Pro-Ser (t-Bu) -Ser (t-Bu) -Gly-Ala-Pro-Pro-Ser (t-Bu) -rink amide resin was added to a round-bottomed flask, and under ice bath conditions, the cleavage solution TFA/EDT/Phenol/H was added 2 O (88/2/5/5, volume ratio) is heated, the temperature of the lysate is controlled at 25 ℃, and the reaction is carried out for 120 minutes. Filtration was carried out, the filter cake was washed 3 times with a small amount of trifluoroacetic acid, and the filtrates were combined. The filtrate was slowly poured into ice-cold ether with stirring. Standing for more than 1.0 hour until the precipitate is complete. The supernatant was decanted, the pellet centrifuged and washed 6 times with glacial ethyl ether to give the crude compound:
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(PEG 2 -PEG 2 -γGlu-CO(CH 2 ) 18 CO 2 H)-Ser-Lys-Tyr-Leu-Glu-Aib-Lys-Ala-Ala-Gln-Asp-Phe-Val-Glu-Trp-Leu-Lys-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2
1d) and (3) refining and purifying the polypeptide compound:
the crude product obtained in 1c was dissolved in 5.0% acetic acid in acetonitrile H 2 O1: 1 (vol/vol) solution was purified by 2 semi-preparative HPLC on a reverse phase C18 packed 50mm x 250mm column. With 30-60% acetonitrile-0.1% trifluoroacetic acid/H 2 The column was eluted with an O gradient of 40mL/min for 45.0 minutes, and the peptide-containing fractions were collected, concentrated to remove acetonitrile, and lyophilized. The purity of HPLC is more than 95%. The product separated was analyzed by LC-MS and the m/z values of the ion peaks of the protonated molecules were found to be: 4860.9 theoretical value 4863.5.
TABLE 1 polypeptide compounds of the invention
Figure BDA0002977865590000251
Figure BDA0002977865590000261
Figure BDA0002977865590000271
Figure BDA0002977865590000281
Example 2Effect of polypeptide Compounds 1-16, Positive control polypeptide Compounds 17-26, Liraglutide and Somarlutide on oral glucose tolerance (OGTT)
The somaglutide (Seraglutide) of this example was purchased from Zhejiang surge volume peptide biology, Inc. (CAS No.: 910463-68-2), and the Liraglutide (Liraglutide) was purchased from Jianyuan pharmaceutical science and technology, Inc. (CAS No.: 204656-20-2), Shenzhen.
Male C57BL/6J mice (university of tokyo model animal research center) at 8 weeks of age were randomly grouped into groups of 6 per group according to similar blood glucose (assessed by blood samples taken from the tip of the tail).
After fasting (6 hours), the animals were administered the polypeptide compounds 1-16 of the present invention, the positive control polypeptide compounds 17-26, liraglutide, somaglutide, SAR425899 and MED C18-acid at a dose of 100ug/kg subcutaneously and PBS was administered to the control group. After about 4 hours, an initial blood sample (fasting blood glucose level) was obtained. Subsequently, an oral dose of glucose (2g/kg) was administered and the animals were returned to their home cages (t-0). Blood glucose was measured at t-15 min, t-30 min, t-60 min and t-120 min. Then, at 8 hours, 12 hours and 24 hours after the administration, the oral dose of glucose was repeatedly administered, and the change in blood glucose was detected and followed up to 24 hours. The data were processed using software GraphPadPrism to plot the lines of change in blood glucose and the area under the curve was calculated to plot the AUC, the results of which are shown in figure 1.
Liraglutide and compounds 17-26 showed a significant decrease in AUC (P <0.05) over the first OGTT curve period (0-120min) compared to vehicle (control PBS), while AUC began to increase over the following three OGTT curve periods (4-22 h). In contrast, the AUC of somaglutide was significantly decreased (P <0.05) over the four OGTT curve periods, indicating that it had long-acting hypoglycemic effect. Compared with a carrier (control group PBS), the polypeptide compounds of the invention have the effect of improving the glucose tolerance of mice to different degrees, wherein the polypeptide compounds 1-16 show more excellent and remarkable glucose tolerance improving effect in 4 OGTT curve periods (0-22h), and the results also indicate that the polypeptide compounds 1-16 have more excellent and remarkable long-acting glucose reducing effect compared with the polypeptide compounds 17-26, liraglutide, SAR425899 and MED C18-acid. Wherein polypeptide compounds 1,2, 3, 8, 13, 14 and 15 exhibit more excellent and significantly long-lasting hypoglycemic effects than somaglutide over 4 OGTT curve periods (0-22 h).
Example 3Improving effect of polypeptide compounds 1,2, 3, 8, 13, 14, 15 on renal fibrosis in model of renal fibrosis induced by high glucose
Increased production of hyperglycemia and glycosylation end products causes morphological changes such as proliferation and hypertrophy of mesangial cells, increase of extracellular matrix (ECM), and expansion of mesangial, which finally results in loss of physiological functions. In addition, the renal tubular epithelial cell transdifferentiation is also an important pathological basis of renal fibrosis, and studies have found that high concentration of glucose further induces glomerular fibrosis by activating the RAS system. Therefore, in this example, the high-sugar-induced MCS lineage membrane cells and NRK renal epithelial cells are used to construct an in vitro renal fibrosis cell model for screening drugs for treating diabetic nephropathy.
The mesangial cell lines GMC and the mesangial epithelial cells mTEC used in this example were cells stored in the laboratory of the applicant (both of which were primary mouse-extracted kidney cells), and were cultured in a low-sugar medium containing 10% fetal bovine serum. When the cells were grown to 70% confluence, they were starved for 24h with serum-free medium and divided into the following 9 groups by adjusting the glucose concentration as follows: low group (5.5mmol/L glucose), High group (30mmol/L glucose), High sugar + compound 1 group (30mmol/L glucose + NO. 110uM), High sugar + compound 2 group (30mmol/L glucose + NO. 210uM), High sugar + compound 3 group (30mmol/L glucose + NO. 310uM), High sugar + compound 8 group (30mmol/L glucose + NO. 810uM), High sugar + compound 13 group (30mmol/L glucose + NO. 131uM), High sugar + compound 14 group (30mmol/L glucose + NO. 1410uM) and High sugar + -%, respectivelyCompound 15 (30mmol/L glucose + NO. 1510uM), 5% CO at 37 deg.C 2 Culturing for 48h under the condition, and carrying out Western blot detection on FN protein (fibronectin). As shown in fig. 2A, different groups of FN expression gel electrophoresis patterns detected using Western blot were constructed for the in vitro renal fibrotic cell model by high-sugar induction of MCS lineage membrane cells and NRK renal epithelial cells in this example; as shown in fig. 2B, a histogram of FN expression in different groups was constructed by inducing MCS lineage membrane cells and NRK renal epithelial cells with high glucose in example 3, and then constructing an in vitro renal fibrotic cell model.
According to the previous experimental study and the previous literature, 30mmol/L glucose can induce the proliferation and hypertrophy of mesangial cells and the transdifferentiation of renal epithelial cells. Western blot detection results show that the expression level of the FN protein in the High group is higher than that in the Low group under the stimulation of High sugar; the administration group can down-regulate the expression of FN protein, and the polypeptide compound of the invention can obviously reduce the increase of ECM caused by high sugar. Among them, the polypeptide compounds 3, 8, 13, 14 and 15 showed more significant effects in inhibiting FN expression.
Example 4Therapeutic effects of polypeptide compounds 3, 8, 13, 14, 15 and 16, somaglutide, SAR425899, MED C18-acid on diabetic mice
Measurement of drug-induced concentrations of polypeptide Compounds 3, 8, 13 and 15
Obtaining a db/db mouse diabetes model (purchased from Nanjing university-Nanjing biomedical research institute, about 8 weeks, and measuring blood sugar and body weight to ensure that subsequent experiments are smoothly carried out), randomly dividing model mice into 12 groups (comprising polypeptide compounds 3, 8, 13, 15, somaltulin and physiological saline), wherein 6 mice in each group have no difference in basic body weight and blood sugar. Each group of mice was subcutaneously injected daily or every other day with Compound 3(80 and 120. mu.g/kg, in total of 2 groups), Compound 8(80 and 120. mu.g/kg, in total of 2 groups), Compound 13(80 and 120. mu.g/kg, in total of 2 groups), Compound 15(80 and 120. mu.g/kg, in total of 2 groups), Somaltulin (80 and 120. mu.g/kg, in total of 2 groups) and physiological saline (DN group: littermate normal mice; DC group: db/db mouse control group).
After the mice were dosed daily, they were fasted for 6h every other day to measure blood glucose and body weight.
Fig. 3A shows that the somaglutide group (120 μ g/kg) exhibited better hypoglycemic effect compared to the saline group DC group, and then maintained at a more stable normal blood glucose level, but its weight-reducing effect was insignificant. In the administration group, under the dosage of 80ug/kg of polypeptide compounds 3, 8, 13 and 15, the polypeptide compounds 3 and 8 have obvious blood sugar reducing effect, and the polypeptide compounds 13 and 15 have obvious blood sugar reducing effect and long-acting property; at a dose of 120ug/kg, the peptide compounds 3, 8, 13 and 15 achieved good blood sugar lowering effect in 3-5 days, and blood sugar was stably maintained at a normal level, while the peptide compounds 3, 8, 13 and 15 showed more excellent weight lowering effect, and the peptide compounds 13 and 15 showed better effect than the peptide compounds 3 and 8. Therefore, the concentration of the drug is determined to be 120 ug/kg.
After the mice were dosed every other day, the mice were fasted for 6h every other day for blood glucose and body weight measurements.
Fig. 3B shows that after the administration of the normal saline group DC, 80 μ g/kg and 120 μ g/kg of the somaglutide groups both had the effect of reducing blood glucose, but the effect of reducing blood glucose was not significant, and the effect of reducing body weight was not significant. In the administration group, the polypeptide compounds 3 and 8 have the phenomenon that the blood sugar of the polypeptide compounds 3 and 8 is reduced firstly and then increased under the dosage of 80ug/kg, the total blood sugar reducing effect is obvious, and the polypeptide compounds 3 and 8 have the effect of reducing the weight. The polypeptide compounds 13 and 15 have obvious blood sugar reducing effect on day 3, but the subsequent blood sugar level changes little and tends to be stable; at a dose of 120ug/kg, the sumalutamide group had an insufficient long-lasting effect with a blood glucose rise at day 3. The polypeptide compounds 3, 8, 13 and 15 achieve good blood sugar reducing effect in 3-5 days, and the blood sugar of the polypeptide compounds 3 and 8 does not change greatly. The polypeptide compounds 13 and 15 always maintain good blood sugar reducing effect, and the polypeptide compounds 13 and 15 are proved to have good long-acting blood sugar reducing effect. Meanwhile, the polypeptide compounds 3, 8, 13 and 15 show more excellent weight-reducing effects, and the effect of the polypeptide compounds 13 and 15 is better than that of the polypeptide compounds 3 and 8. Therefore, the concentration of the drug is determined to be 120 ug/kg.
Diabetes improving effect of (di) polypeptide compounds 3, 8, 13, 14, 15, 16, somaglutide, SAR425899 and MED C18-acid on diabetic mice
Db/db mouse diabetes models (purchased from Nanjing university-Nanjing biomedical institute, mice for about 12 weeks, and blood glucose and body weight were measured to ensure smooth progress of subsequent experiments) were obtained, and the model mice were randomly divided into 10 groups (groups of polypeptide compounds 3, 8, 13, 14, 15, 16, somaglutide, SAR425899, MED C18-acid and physiological saline), 6 mice each having no difference in basal body weight and blood glucose, and mice each having subcutaneous injections of polypeptide compounds 3, 8, 13, 14, 15, 16 (120. mu.g/kg), somagluteline (120. mu.g/kg), SAR425899 (120. mu.g/kg), MED C18-acid (120. mu.g/kg), and physiological saline (DN group: normal mice of the same litter; DC group: db/db mouse control group). After the mice were dosed daily, they were fasted every other day for 6h to measure blood glucose and body weight, and once a week to measure water intake and food intake. OGTT is measured at week 4, insulin tolerance (ITT) is measured at week 5, mouse metabolism conditions are measured by using mouse metabolism cages at weeks 6-7, materials are taken at week 8, and various serological indexes and pathological indexes are measured.
1. The medicine has effects of lowering blood sugar, reducing body weight and improving insulin resistance of diabetic mice
Type II diabetes mellitus model characterized by: obesity, insulin resistance, hyperglycemia, dyslipidemia, liver steatorvacuole-like degeneration, and the like. Oral Glucose Tolerance Test (OGTT) and insulin resistance test (ITT) were performed at 4 weeks and 5 weeks of treatment of diabetic mice with polypeptide compounds 3, 8, 13, 14, 15, 16 and somaglutide, SAR425899, MED C18-acid, respectively, and the results are shown in fig. 4A-4D.
The results in fig. 4A to 4C show that the polypeptide compounds 3, 8, 13, 14, 15, 16, somaglutide, SAR425899 and MED C18-acid all had significant hypoglycemic effects compared to the DN group, blood glucose had decreased to normal levels by week 1 and thereafter blood glucose was relatively stable. However, the polypeptide compounds 3, 8, 13, 14, 15 and 16 are more excellent in improving oral glucose tolerance (OGTT) and insulin resistance (ITT) in diabetic mice.
In addition, the results of fig. 4D show that the polypeptide compounds 3, 8, 13, 14, 15, and 16 have more excellent liver protection functions.
2. Therapeutic effect of drug on diabetic nephropathy of diabetic mice
After 6 weeks of drug treatment, mouse urine was collected using a metabolic cage and the protein content in the urine was determined using a relevant kit. After 7 weeks of administration, the material was taken and the blood was used to test serological indicators such as serum creatinine, urea nitrogen, etc. The results are shown in FIGS. 5A to 5F.
As can be seen from FIG. 5A, the 20-week-old db/db diabetic mice in the DC group had significantly increased proteinuria and urine glucose levels as compared to the control DN group, and also had significantly increased glycated hemoglobin, serum creatinine and urea nitrogen levels, indicating that diabetic nephropathy in the mice was already severe. After administration, the mice were improved to varying degrees in proteinuria content, urine glucose content, glycated hemoglobin content, blood creatinine, and urea nitrogen content.
The mouse kidneys were paraffin embedded for subsequent staining and immunohistochemical studies. As shown in FIG. 5B, H & E staining results of mouse kidney tissue showed local inflammation and glomerular adhesion. After administration, glomerular adhesion was alleviated, and the improvement and therapeutic effects of polypeptide compounds 3, 8, 13, 14, 15 and 16 were more significant compared to the positive control drugs of somaglutide, SAR425899 and MED C18-acid group. In general, the observation of renal lesions is mostly focused on the cortex, mainly including glomeruli and renal interstitium, and the observation of glomeruli is focused on the mesangial region. As can be observed by PAS staining (fig. 5C), the glomerular mesangial region of DC group was broadened, mesangial cells and mesangial matrix were proliferated, basement membrane was thickened, and after administration, glomerular vascular loops were thin and clear, the number of endothelial cells and mesangial cells was normal, the peripheral renal tubules were also normal, and the improving effects of the polypeptide compounds 3, 8, 13, 14, 15 and 16 were more significant, compared to DN group. As can be seen from fig. 5D to 5F, sirius new red and FN, α -SMA staining, compared to the DN group, the DC group collagen deposition was significantly increased, and the drug group could significantly reduce the collagen content, wherein the polypeptide compounds 3, 8, 13, 14, 15 and 16 had more significant improvement effects, and the therapeutic effects thereof were superior to those of the positive control somaglutide, SAR425899 and MED C18-acid groups.
In conclusion, the modification of the side chain technology is simultaneously carried out through further optimization and design of the amino acid sequence. The compound reported by the invention is superior to the previously reported compounds in terms of reducing blood sugar, reducing weight and improving and treating diabetic complications, and has more advantages in terms of clinical application.
The above description of the specific embodiments of the present invention is not intended to limit the present invention, and those skilled in the art may make various changes and modifications according to the present invention without departing from the spirit of the present invention, which is defined by the scope of the appended claims.
Sequence listing
<110> Zhongshan university
<120> polypeptide compound and use thereof for preventing or treating diabetes or diabetic complications
<130> GD1899-21P125434
<160> 28
<170> PatentIn version 3.5
<210> 1
<211> 39
<212> PRT
<213> Artificial sequence
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> X=Aib
<220>
<221> MISC_FEATURE
<222> (10)..(10)
<223> side chain attachment of Lys-PEG 2-PEG 2-gamma Glu-CO (CH2)18CO2H
<220>
<221> MISC_FEATURE
<222> (16)..(16)
<223> X=Aib
<400> 1
His Xaa Gln Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Glu Xaa
1 5 10 15
Lys Ala Ala Gln Asp Phe Val Glu Trp Leu Lys Ala Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 2
<211> 39
<212> PRT
<213> Artificial sequence
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> X=D-Ser
<220>
<221> MISC_FEATURE
<222> (10)..(10)
<223> side chain attachment of Lys PEG2-PEG2-CO (CH2)18CO2H
<220>
<221> MISC_FEATURE
<222> (16)..(16)
<223> X=Aib
<400> 2
His Xaa Gln Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Glu Xaa
1 5 10 15
Lys Ala Ala Gln Asp Phe Val Glu Trp Leu Lys Ala Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 3
<211> 39
<212> PRT
<213> Artificial sequence
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> X=D-Ser
<220>
<221> MISC_FEATURE
<222> (10)..(10)
<223> side chain attachment of Lys-PEG 2-PEG 2-gamma Glu-CO (CH2)18CO2H
<220>
<221> MISC_FEATURE
<222> (16)..(16)
<223> X=Aib
<400> 3
His Xaa Gln Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Glu Xaa
1 5 10 15
Lys Ala Ala Gln Asp Phe Val Glu Trp Leu Lys Ala Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 4
<211> 39
<212> PRT
<213> Artificial sequence
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> X=D-Ser
<220>
<221> MISC_FEATURE
<222> (10)..(10)
<223> side chain attachment of Lys-PEG 2-PEG 2-gamma Glu-CO (CH2)16CO2H
<220>
<221> MISC_FEATURE
<222> (16)..(16)
<223> X=Aib
<400> 4
His Xaa Gln Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Glu Xaa
1 5 10 15
Lys Ala Ala Gln Asp Phe Val Glu Trp Leu Lys Ala Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 5
<211> 39
<212> PRT
<213> Artificial sequence
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> X=D-Ser
<220>
<221> MISC_FEATURE
<222> (10)..(10)
<223> side chain attachment of Lys-PEG 2-PEG 2-gamma Glu-CO (CH2)18CH3
<220>
<221> MISC_FEATURE
<222> (16)..(16)
<223> X=Aib
<400> 5
His Xaa Gln Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Glu Xaa
1 5 10 15
Lys Ala Ala Gln Asp Phe Val Glu Trp Leu Lys Ala Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 6
<211> 39
<212> PRT
<213> Artificial sequence
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> X=D-Ser
<220>
<221> MISC_FEATURE
<222> (10)..(10)
<223> side chain attachment of Lys-PEG 2-PEG 2-gamma Glu-CO (CH2)18CO2H
<220>
<221> MISC_FEATURE
<222> (23)..(23)
<223> X=Iva
<220>
<221> MISC_FEATURE
<222> (35)..(35)
<223> X=Aib
<400> 6
His Xaa Gln Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Asp Glu
1 5 10 15
Arg Ala Ala Gln Asp Phe Xaa Gln Trp Leu Leu Asp Gly Gly Pro Ser
20 25 30
Ser Gly Xaa Pro Pro Pro Ser
35
<210> 7
<211> 39
<212> PRT
<213> Artificial sequence
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> X=D-Ser
<220>
<221> MISC_FEATURE
<222> (10)..(10)
<223> side chain attachment of Lys-PEG 2-PEG 2-gamma Glu-CO (CH2)18CO2H
<220>
<221> MISC_FEATURE
<222> (23)..(23)
<223> X=Iva
<400> 7
His Xaa Gln Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Asp Glu
1 5 10 15
Arg Ala Ala Gln Asp Phe Xaa Gln Trp Leu Leu Glu Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 8
<211> 39
<212> PRT
<213> Artificial sequence
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> X=Aib
<220>
<221> MISC_FEATURE
<222> (10)..(10)
<223> side chain attachment of Lys-PEG 2-PEG 2-gamma Glu-CO (CH2)18CO2H
<220>
<221> MISC_FEATURE
<222> (23)..(23)
<223> X=Iva
<220>
<221> MISC_FEATURE
<222> (35)..(35)
<223> X=Aib
<400> 8
His Xaa Gln Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Asp Glu
1 5 10 15
Arg Ala Ala Gln Asp Phe Xaa Gln Trp Leu Leu Asp Gly Gly Pro Ser
20 25 30
Ser Gly Xaa Pro Pro Pro Ser
35
<210> 9
<211> 39
<212> PRT
<213> Artificial sequence
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> X=Aib
<220>
<221> MISC_FEATURE
<222> (10)..(10)
<223> side chain attachment of Lys-PEG 2-PEG 2-gamma Glu-CO (CH2)16CO2H
<220>
<221> MISC_FEATURE
<222> (23)..(23)
<223> X=Iva
<220>
<221> MISC_FEATURE
<222> (35)..(35)
<223> X=Aib
<400> 9
His Xaa Gln Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Asp Glu
1 5 10 15
Arg Ala Ala Gln Asp Phe Xaa Gln Trp Leu Leu Asp Gly Gly Pro Ser
20 25 30
Ser Gly Xaa Pro Pro Pro Ser
35
<210> 10
<211> 39
<212> PRT
<213> Artificial sequence
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> X=Aib
<220>
<221> MISC_FEATURE
<222> (10)..(10)
<223> side chain attachment of Lys-PEG 2-PEG 2-gamma Glu-CO (CH2)18CH3
<220>
<221> MISC_FEATURE
<222> (23)..(23)
<223> X=Iva
<220>
<221> MISC_FEATURE
<222> (35)..(35)
<223> X=Aib
<400> 10
His Xaa Gln Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Asp Glu
1 5 10 15
Arg Ala Ala Gln Asp Phe Xaa Gln Trp Leu Leu Asp Gly Gly Pro Ser
20 25 30
Ser Gly Xaa Pro Pro Pro Ser
35
<210> 11
<211> 39
<212> PRT
<213> Artificial sequence
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> X=Aib
<220>
<221> MISC_FEATURE
<222> (10)..(10)
<223> side chain attachment of Lys-PEG 2-PEG2-CO (CH2)18CH3
<220>
<221> MISC_FEATURE
<222> (23)..(23)
<223> X=Iva
<220>
<221> MISC_FEATURE
<222> (35)..(35)
<223> X=Aib
<400> 11
His Xaa Gln Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Asp Glu
1 5 10 15
Arg Ala Ala Gln Asp Phe Xaa Gln Trp Leu Leu Asp Gly Gly Pro Ser
20 25 30
Ser Gly Xaa Pro Pro Pro Ser
35
<210> 12
<211> 39
<212> PRT
<213> Artificial sequence
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> X=D-Ser
<220>
<221> MISC_FEATURE
<222> (10)..(10)
<223> side chain attachment of Lys-PEG 2-PEG 2-gamma Glu-CO (CH2)18CO2H
<220>
<221> MISC_FEATURE
<222> (23)..(23)
<223> X=Iva
<220>
<221> MISC_FEATURE
<222> (35)..(35)
<223> X=Aib
<400> 12
His Xaa Gln Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Asp Glu
1 5 10 15
Arg Ala Ala Gln Asp Phe Xaa Gln Trp Leu Leu Asp Gly Gly Pro Ser
20 25 30
Ser Gly Xaa Pro Pro Pro Ser
35
<210> 13
<211> 39
<212> PRT
<213> Artificial sequence
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> X=D-Ser
<220>
<221> MISC_FEATURE
<222> (10)..(10)
<223> side chain attachment of Lys-Gly-Gly-Ser-Gly-Ser-Gly-gamma Glu-CO (CH2)18-COOH
<220>
<221> MISC_FEATURE
<222> (16)..(16)
<223> X=Aib
<400> 13
His Xaa Gln Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Glu Xaa
1 5 10 15
Lys Ala Ala Gln Asp Phe Val Glu Trp Leu Lys Ala Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 14
<211> 39
<212> PRT
<213> Artificial sequence
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> X=D-Ser
<220>
<221> MISC_FEATURE
<222> (10)..(10)
<223> side chain attachment of Lys-Gly-Gly-Ser-Gly-Ser-Gly-gamma Glu-CO (CH2)18-COOH
<220>
<221> MISC_FEATURE
<222> (16)..(16)
<223> X=Aib
<400> 14
His Xaa Gln Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Glu Xaa
1 5 10 15
Lys Ala Ala Gln Asp Phe Val Glu Trp Leu Lys Ala Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 15
<211> 39
<212> PRT
<213> Artificial sequence
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> X=Aib
<220>
<221> MISC_FEATURE
<222> (10)..(10)
<223> side chain attachment of Lys-Gly-Gly-Ser-Gly-Ser-Gly-gamma Glu-CO (CH2)18-COOH
<220>
<221> MISC_FEATURE
<222> (23)..(23)
<223> X=Iva
<220>
<221> MISC_FEATURE
<222> (35)..(35)
<223> X=Aib
<400> 15
His Xaa Gln Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Asp Glu
1 5 10 15
Arg Ala Ala Gln Asp Phe Xaa Gln Trp Leu Leu Asp Gly Gly Pro Ser
20 25 30
Ser Gly Xaa Pro Pro Pro Ser
35
<210> 16
<211> 39
<212> PRT
<213> Artificial sequence
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> X=Aib
<220>
<221> MISC_FEATURE
<222> (10)..(10)
<223> side chain attachment of Lys-Gly-Gly-Ser-Gly-Ser-Gly-gamma Glu-CO (CH2)18-COOH
<220>
<221> MISC_FEATURE
<222> (23)..(23)
<223> X=Iva
<220>
<221> MISC_FEATURE
<222> (35)..(35)
<223> X=Aib
<400> 16
His Xaa Gln Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Asp Glu
1 5 10 15
Arg Ala Ala Gln Asp Phe Xaa Gln Trp Leu Leu Asp Gly Gly Pro Ser
20 25 30
Ser Gly Xaa Pro Pro Pro Ser
35
<210> 17
<211> 40
<212> PRT
<213> Artificial sequence
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> X=D-Ser
<220>
<221> MISC_FEATURE
<222> (16)..(16)
<223> side chain attachment of Lys-PEG 2-PEG 2-gamma Glu-CO (CH2)14CH3
<400> 17
His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Lys
1 5 10 15
Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Gly Gly Pro
20 25 30
Ser Ser Gly Ala Pro Pro Pro Ser
35 40
<210> 18
<211> 40
<212> PRT
<213> Artificial sequence
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> X=D-Ser
<220>
<221> MISC_FEATURE
<222> (16)..(16)
<223> side chain attachment of Lys-PEG 2-PEG 2-gamma Glu-CO (CH2)14CH3
<400> 18
His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Lys
1 5 10 15
Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Leu Asn Thr Gly Gly Pro
20 25 30
Ser Ser Gly Ala Pro Pro Pro Ser
35 40
<210> 19
<211> 40
<212> PRT
<213> Artificial sequence
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> X=D-Ser
<220>
<221> MISC_FEATURE
<222> (12)..(12)
<223> side chain attachment of Lys-PEG 2-PEG2-CO (CH2)16CO2H
<220>
<221> MISC_FEATURE
<222> (16)..(16)
<223> X=Aib
<400> 19
His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Lys Leu Asp Xaa
1 5 10 15
Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Gly Gly Pro
20 25 30
Ser Ser Gly Ala Pro Pro Pro Ser
35 40
<210> 20
<211> 40
<212> PRT
<213> Artificial sequence
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> X=D-Ser
<220>
<221> MISC_FEATURE
<222> (16)..(16)
<223> side chain attachment of Lys- (PEG2-PEG 2-gamma Glu-CO (CH2)14CH3
<400> 20
His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Lys
1 5 10 15
Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Gly Gly Pro
20 25 30
Ser Ser Gly Ala Pro Pro Pro Ser
35 40
<210> 21
<211> 38
<212> PRT
<213> Artificial sequence
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> Xaa = aminoisobutyric acid
<400> 21
His Xaa Gln Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Asp Glu
1 5 10 15
Gln Ala Ala Gln Asp Phe Val Gln Trp Leu Leu Asp Gly Pro Ser Ser
20 25 30
Gly Ala Pro Pro Pro Ser
35
<210> 22
<211> 39
<212> PRT
<213> Artificial sequence
<400> 22
His Ser Gln Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Asp Ser
1 5 10 15
Gln Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 23
<211> 39
<212> PRT
<213> Artificial sequence
<400> 23
His Ser Gln Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Asp Glu
1 5 10 15
Glu Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 24
<211> 39
<212> PRT
<213> Artificial sequence
<400> 24
His Ser Gln Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Asp Glu
1 5 10 15
Gln Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 25
<211> 39
<212> PRT
<213> Artificial sequence
<400> 25
His Ser Gln Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Asp Glu
1 5 10 15
Arg Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 26
<211> 39
<212> PRT
<213> Artificial sequence
<400> 26
His Ser Gln Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Asp Ser
1 5 10 15
Arg Ala Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 27
<211> 30
<212> PRT
<213> Artificial sequence
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> X=D-Ser
<220>
<221> MISC_FEATURE
<222> (13)..(13)
<223> side chain attachment of Lys-PEG 2-PEG 2-gamma Glu-CO (CH2)17COOH
<400> 27
His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Lys Leu Asp Ser
1 5 10 15
Glu Arg Ala Arg Asp Phe Val Ala Trp Leu Val Ala Gly Gly
20 25 30
<210> 28
<211> 39
<212> PRT
<213> Artificial sequence
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> X=D-Ser
<220>
<221> MISC_FEATURE
<222> (14)..(14)
<223> side chain attachment of Lys-CO (CH2)14CH3
<400> 28
His Xaa Gln Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Lys Glu Ser
1 5 10 15
Lys Ala Ala Gln Asp Phe Ile Glu Trp Leu Lys Ala Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35

Claims (15)

1. A polypeptide compound comprising a parent peptide represented by the amino acid sequence:
His-Xaa2-Gln-Gly-Thr 5 -Phe-Thr-Ser-Asp-Lys 10 -Ser-Lys-Tyr-Leu-Xaa15 15 -Xaa16-Xaa17-Ala-Ala-Gln 20 -Xaa21-Phe-Xaa23-Xaa24-Trp 25 -Leu-Xaa27-Xaa28-Gly-Gly 30 -Pro-Ser-Ser-Gly-Xaa35 35 -Pro-Pro-Pro-Ser，
wherein:
xaa2 ═ Aib, Ser or D-Ser;
xaa15 ═ Asp or Glu;
xaa16 ═ Aib or Glu;
xaa17 ═ Lys or Arg;
xaa21 ═ Asp or Glu;
xaa23 Val or Iva;
xaa24 ═ Glu or Gln;
xaa27 ═ Leu or Lys;
xaa28 ═ Asp, Glu, or Ala;
xaa35 ═ Ala or Aib.
2. The polypeptide compound of claim 1 wherein Xaa2 ═ Aib or D-Ser.
3. The polypeptide compound of claim 2 wherein Xaa21 is Asp.
4. The polypeptide compound of claim 3, wherein the parent peptide has an amino acid sequence which is carboxy-terminal unmodified or amino-modified to form-CONH 2 A group.
5. The polypeptide compound of any one of claims 1 to 4 wherein the side chain of Lys at position 10 or position 12 of the amino acid sequence of the parent peptide is attached to a lipophilic substituent via a bridging group; the bridging group is (PEG) m 、(PEG) m -γGlu、(PEG) m -Asp、(Gly) x -(Gly-Ser) y -(Gly) z -、(Gly) x -(Gly-Ser) y -(Gly) z -gamma Glu and (Gly) x -(Gly-Ser) y -(Gly) z -one of Asp; the connection mode is the side chain amino of the 10 th or 12 th Lys and the carboxyl of the glycine residue at one end of the bridging group or (PEG) m The carboxyl modified at the tail end forms an amido bond, and the lipophilic substituent is connected with the amino of the bridging group at the other end through the formation of the amido bond by the carboxyl; the lipophilic substituent is CH 3 (CH 2 ) n C (O) -or HOOC (CH) 2 ) n C (O) -and the acyl group thereof forms an amide bond with the amino group in the bridging group; wherein m is an integer of 2 to 10; n is an integer of 14 to 20; x is an integer of 0 to 5; y is an integer of 1 to 5; z is an integer of 1 to 5.
6. The polypeptide compound of claim 1, wherein Lys at position 10 or 12 in the amino acid sequence of the parent peptide is replaced by HomoLys, Orn, Dap, or Dab.
7. The polypeptide compound of claim 1, wherein the amino acid sequence of the parent peptide:
xaa2 ═ Aib or D-Ser;
Xaa15＝Glu；
Xaa16＝Aib；
Xaa17＝Lys；
Xaa21＝Asp；
Xaa23＝Val；
Xaa24＝Glu；
Xaa27＝Lys；
Xaa28＝Ala；
Xaa35＝Ala。
8. the polypeptide compound of claim 1, wherein the amino acid sequence of the parent peptide:
xaa2 ═ Aib or D-Ser;
Xaa15＝Asp；
Xaa16＝Glu；
Xaa17＝Arg；
Xaa21＝Asp；
Xaa23＝Iva；
Xaa24＝Gln；
Xaa27＝Leu；
xaa28 ═ Asp or Glu;
xaa35 ═ Ala or Aib.
9. The polypeptide compound of claim 1, wherein the amino acid sequence of the parent peptide is selected from the group consisting of the amino acid sequences shown in SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6, SEQ ID No.7, SEQ ID No.8, SEQ ID No.9, SEQ ID No.10, SEQ ID No.11 and SEQ ID No.12, SEQ ID No.13, SEQ ID No.14, SEQ ID No.15 and SEQ ID No. 16.
10. A polypeptide compound according to claim 5 wherein the Lys position 10 or 12 of the amino acid sequence of the parent peptide is linked to a lipophilic substituent via a bridging group forming the structure:
Figure FDA0002977865580000031
Figure FDA0002977865580000041
11. the polypeptide compound of claim 1, wherein the polypeptide compound is any one of the following compounds:
compound 1:
H-Aib-QGTFTSDK(PEG 2 -PEG 2 -γGlu-CO(CH 2 ) 18 CO 2 H)SKYLE-Aib-KAAQDFVEWLKAGGPSSGAPPPS-NH 2
compound 2:
HsQGTFTSDK(PEG 2 -PEG 2 -CO(CH 2 ) 18 CO 2 H)SKYLE-Aib-KAAQDFVEWLKAGGPSSGAPPPS-NH 2
compound 3:
HsQGTFTSDK(PEG 2 -PEG 2 -γGlu-CO(CH 2 ) 18 CO 2 H)SKYLE-Aib-KAAQDFVEWLKAGGPSSGAPPPS-NH 2
compound 4:
HsQGTFTSDK(PEG 2 -PEG 2 -γGlu-CO(CH 2 ) 16 CO 2 H)SKYLE-Aib-KAAQDFVEWLKAGGPSSGAPPPS-NH 2
compound 5:
HsQGTFTSDK(PEG 2 -PEG 2 -γGlu-CO(CH 2 ) 18 CH 3 )SKYLE-Aib-KAAQDFVEWLKAGGPSSGAPPPS-NH 2
compound 6:
HsQGTFTSDK(PEG 2 -PEG 2 -γGlu-CO(CH 2 ) 18 CO 2 H)SKYLDERAAQDF-Iva-QWLLDGGPSSG-Aib-PPPS-NH 2
compound 7:
HsQGTFTSDK(PEG 2 -PEG 2 -γGlu-CO(CH 2 ) 18 CO 2 H)SKYLDERAAQDF-Iva-QWLLEGGPSSGAPPPS-NH 2
compound 8:
H-Aib-QGTFTSDK(PEG 2 -PEG 2 -γGlu-CO(CH 2 ) 18 CO 2 H)SKYLDERAAQDF-Iva-QWLLDGGPSSG-Aib-PPPS-NH 2
compound 9:
H-Aib-QGTFTSDK(PEG 2 -PEG 2 -γGlu-CO(CH 2 ) 16 CO 2 H)SKYLDERAAQDF-Iva-QWLLDGGPSSG-Aib-PPPS-NH 2
compound 10:
H-Aib-QGTFTSDK(PEG 2 -PEG 2 -γGlu-CO(CH 2 ) 18 CH 3 )SKYLDERAAQDF-Iva-QWLLDGGPSSG-Aib-PPPS-NH 2
compound 11:
H-Aib-QGTFTSDK(PEG 2 -PEG 2 -γGlu-CO(CH 2 ) 18 CH 3 )SKYLDERAAQDF-Iva-QWLLDGGPSSG-Aib-PPPS-NH 2
compound 12:
HsQGTFTSDK(PEG 2 -PEG 2 -γGlu-CO(CH 2 ) 18 CO 2 H)SKYLDERAAQDF-Iva-QWLLDGGPSSG-Aib-PPPS-NH 2
compound 13:
HsQGTFTSDK(GGSGSG-γGlu-CO(CH 2 ) 18 -COOH)SKYLE-Aib-KAAQDFVEWLKAGGPSSGAPPPS
compound 14:
HsQGTFTSDK(GGSGSG-γGlu-CO(CH 2 ) 18 -COOH)SKYLE-Aib-KAAQDFVEWLKAGGPSSGAPPPS-NH 2
compound 15:
H-Aib-QGTFTSDK(GGSGSG-γGlu-CO(CH 2 ) 18 -COOH)SKYLDERAAQDF-Iva-QWLLDGGPSSG-Aib-PPPS
compound 16:
H-Aib-QGTFTSDK(GGSGSG-γGlu-CO(CH 2 ) 18 -COOH)SKYLDERAAQDF-Iva-QWLLDGGPSSG-Aib-PPPS-NH 2。
12. a composition comprising the polypeptide compound of any one of claims 1 to 11.
13. The composition of claim 12, wherein the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier or excipient.
14. Use of a polypeptide compound according to any one of claims 1 to 11 or a composition according to any one of claims 12 to 13 for the manufacture of a medicament for the prevention or treatment of diabetes and/or diabetic complications; the diabetic complication is diabetic nephropathy.
15. Use of a polypeptide compound according to any one of claims 1 to 11 or a composition according to any one of claims 12 to 13 for the preparation of a weight-reducing nutraceutical or pharmaceutical product.
CN202110280107.6A 2021-03-16 2021-03-16 Polypeptide compound and application thereof in preventing or treating diabetes or diabetic complications Pending CN115073582A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202110280107.6A CN115073582A (en) 2021-03-16 2021-03-16 Polypeptide compound and application thereof in preventing or treating diabetes or diabetic complications

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202110280107.6A CN115073582A (en) 2021-03-16 2021-03-16 Polypeptide compound and application thereof in preventing or treating diabetes or diabetic complications

Publications (1)

Publication Number Publication Date
CN115073582A true CN115073582A (en) 2022-09-20

Family

ID=83246126

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202110280107.6A Pending CN115073582A (en) 2021-03-16 2021-03-16 Polypeptide compound and application thereof in preventing or treating diabetes or diabetic complications

Country Status (1)

Country Link
CN (1) CN115073582A (en)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106046145A (en) * 2016-04-22 2016-10-26 深圳市图微安创科技开发有限公司 Application of GLP-1R/GCGR double-target agonist polypeptide to treatment of fatty liver diseases, hyperlipidemia and arteriosclerosis
CN106519015A (en) * 2014-09-23 2017-03-22 蒋先兴 Oxyntomodulin analogue

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106519015A (en) * 2014-09-23 2017-03-22 蒋先兴 Oxyntomodulin analogue
CN106046145A (en) * 2016-04-22 2016-10-26 深圳市图微安创科技开发有限公司 Application of GLP-1R/GCGR double-target agonist polypeptide to treatment of fatty liver diseases, hyperlipidemia and arteriosclerosis

Similar Documents

Publication Publication Date Title
AU2015321161B2 (en) Oxyntomodulin analogue
CN111278853B (en) Treatment of biliary cirrhosis based on oxyntomodulin analog GLP-1R/GCGR dual-target agonist polypeptide
EP3447067B1 (en) Glp-1r/gcgr dual target agonist polypeptide for treating non-alcoholic fatty liver diseases
JP2022023029A (en) Glucagon analogues
CN104395338B (en) People&#39;s amylin analog
CN113429471B (en) Long-acting GLP-1 polypeptide analogue, and preparation method and application thereof
AU2024203093A1 (en) Polypeptide compounds and use thereof in the prevention or treatment of diabetes or diagnostic compounds
JP2022551233A (en) Use of GLP-1 Analogue Peptide Modified Dimers of Different Structures and Methods for Their Preparation in the Treatment of Type II Diabetes
CN112898405B (en) Polypeptide compound and application thereof in preventing or treating diabetes or diabetic complications
CN115073582A (en) Polypeptide compound and application thereof in preventing or treating diabetes or diabetic complications
JP7483040B2 (en) Incretin analogs and methods for their preparation and use
CN115572326A (en) Triplex agonists of GLP-1, GCG and GIP receptors
CN114585640B (en) GLP-1R/GCGR double-target agonist polypeptide derivative for treating liver fibrosis related to viral hepatitis
EP4289860A1 (en) Use of polypeptide compound in prevention or treatment of inflammatory bowel disease and intestinal fibrosis related thereto
CN116970064A (en) GLP-1/GCG receptor co-agonists, pharmaceutical compositions comprising same and uses thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination