WO2021169512A1 - 多肽化合物及其在预防或治疗糖尿病或糖尿病并发症中的应用 - Google Patents

多肽化合物及其在预防或治疗糖尿病或糖尿病并发症中的应用 Download PDF

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WO2021169512A1
WO2021169512A1 PCT/CN2020/137071 CN2020137071W WO2021169512A1 WO 2021169512 A1 WO2021169512 A1 WO 2021169512A1 CN 2020137071 W CN2020137071 W CN 2020137071W WO 2021169512 A1 WO2021169512 A1 WO 2021169512A1
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ser
peg
aib
gly
compound
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PCT/CN2020/137071
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English (en)
French (fr)
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蒋先兴
赵倩
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中山大学
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Priority to CN202080088462.7A priority Critical patent/CN114945589B/zh
Priority to US17/801,465 priority patent/US20230183310A1/en
Priority to EP20922203.3A priority patent/EP4112637A4/en
Priority to AU2020431973A priority patent/AU2020431973A1/en
Publication of WO2021169512A1 publication Critical patent/WO2021169512A1/zh
Priority to AU2024203093A priority patent/AU2024203093A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/605—Glucagons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/543—Lipids, e.g. triglycerides; Polyamines, e.g. spermine or spermidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention belongs to the technical field of biochemistry. Specifically, the present invention relates to a class of polypeptide compounds and their application in preventing or treating diabetes or diabetic complications.
  • diabetes In recent years, with the rapid economic development, changes in dietary structure and lifestyle, and the acceleration of population aging, the prevalence of diabetes in the world has also shown a trend of rapid increase. According to statistics, nearly 9% of adults worldwide suffer from type II diabetes. According to estimates by the International Diabetes Federation (IDF), the global prevalence of diabetes will increase from 415 million in 2015 to 642 million in 2040 (Unnikrishnan R, Pradeepa R, Joshi SR, Mohan V. Type 2Diabetes: Demystifying the Global Epidemic. Diabetes, 2017.66:1432-1442). Diabetes has become another important chronic non-communicable disease that seriously endangers human health after cardiovascular and cerebrovascular diseases and tumors.
  • IDF International Diabetes Federation
  • diabetes control is not optimistic in both developed countries in Europe and the United States or developing countries such as China.
  • diabetic vascular disease 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.
  • diabetic nephropathy Approximately 40% of patients with type I diabetes will develop diabetic nephropathy. Although there are relatively few cases of type II diabetic nephropathy developing into end-stage renal disease, the rapid increase in the number of patients with type II diabetes means that if there is a lack of Effective measures for diabetes management, the incidence of diabetic nephropathy will also increase substantially. Therefore, there is an urgent need for effective and long-acting hypoglycemic drugs 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 patients with type I diabetes develop diabetic nephropathy, which is greatly related to genetic factors.
  • GLP-1 analogs (Meier JJ.GLP-1 Receptor Agonists for Individualized Treatment of Type-2 Diabetes Mellitus. Nat. Rev. Endocrinol, 2012.8 (12): 728), which have been marketed since 2005, have a different target than before Drugs provide new treatment options for the treatment of diabetes.
  • GLP-1 analogs currently approved for the treatment of type II diabetes include exenatide, liraglutide and lixisenatide, as well as the subsequent production of abiglutide and dulaglutide.
  • Novo Nordisk's once-a-week long-acting GLP-1 analogue somaglutide has also been marketed for the treatment of type II diabetes.
  • GLP-1R/GCGR dual agonists Compared with homologous ligand single-target agonists, GLP-1R/GCGR dual agonists have synergistic effects of reducing blood sugar, lipids, and weight.
  • the GLP-1R/GCGR dual target agonist the synergistic effect of GCGR and GLP-1 can enhance the function of pancreatic ⁇ -cells, and at the same time may minimize or prevent the progression of type II diabetes.
  • These innovations increase the established effects of GLP-1, and the secondary activity may increase more effects, such as weight loss, cardiovascular benefits, and treatment of diabetic nephropathy.
  • a Chinese patent CN201711194175.0 discloses a glucagon analogue for the treatment of metabolic diseases, but studies have found that the effect of the glucagon analogue in improving glucose tolerance is equivalent to that of liraglutide, but peso Marutide has a poor effect on improving glucose tolerance, and the glucagon analogs also cannot meet the requirements of clinical use.
  • the purpose of the present invention is to provide a new class of polypeptide compounds.
  • the inventors of the present invention have conducted a large number of experimental studies to prove that this new type of polypeptide compound has a longer half-life, has insulin-promoting activity, has no adverse reactions, and can be used for the prevention or treatment of diabetic complications such as diabetes and diabetic nephropathy.
  • Another object of the present invention is to provide the application of the above-mentioned novel polypeptide compound, which can potentially be used as a new generation of drugs for preventing or treating diabetes and diabetic nephropathy and other diabetic complications, and can also be used for reducing blood sugar or reducing body weight.
  • the present invention provides a polypeptide compound, which contains a parent peptide represented by the following amino acid sequence:
  • Xaa2 Aib, Ser or D-Ser
  • Xaa15 Asp or Glu
  • Xaa16 Aib or Glu
  • Xaa17 Lys or Arg
  • Xaa21 Asp or Glu
  • Xaa24 Glu or Gln
  • Xaa27 Leu or Lys
  • Xaa28 Asp, Glu or Ala
  • Xaa35 Ala or Aib.
  • Xaa2 Aib or D-Ser.
  • Xaa21 Asp.
  • the carboxyl end of the amino acid sequence of the parent peptide is not modified or modified by amino to form a -CONH 2 group.
  • the side chain of Lys at position 10 or 12 in the amino acid sequence of the parent peptide is connected to a lipophilic substituent via a bridging group, and the bridging group is (PEG) m, (PEG) m - ⁇ Glu, (PEG) m -Asp, (Gly) x -(Gly-Ser) y -(Gly) z -, (Gly) x -(Gly-Ser) y -(Gly) z - ⁇ Glu and (Gly) One of x -(Gly-Ser) y -(Gly) z -Asp.
  • the bridging group is (PEG) m, (PEG) m - ⁇ Glu, (PEG) m -Asp, (Gly) x -(Gly-Ser) y -(Gly) z -, (Gly) x -(Gly-Ser) y -(
  • connection method is: the side chain amino group of Lys at position 10 or 12 and the carboxy group of the glycine residue at one end of the bridging group or the carboxy group modified at the end of (PEG) m form an amide bond to connect to the parent peptide ;
  • the lipophilic substituent is connected with its carboxyl group and the amino group of the bridging group at the other end by forming an amide bond.
  • the lipophilic substituent is CH 3 (CH 2 ) n C(O)- or HOOC(CH 2 ) n C(O)-, and its acyl group forms an amide bond with the amino group in the bridging group.
  • n is an integer of 14-20.
  • the 10th or 12th Lys in the amino acid sequence of the parent peptide can be replaced by HomoLys, Orn, Dap or Dab.
  • Xaa2 Aib or D-Ser
  • Xaa15 Glu
  • Xaa24 Glu
  • Xaa2 Aib or D-Ser
  • Xaa16 Glu
  • Xaa28 Asp or Glu
  • Xaa35 Ala or Aib.
  • the amino acid sequence of the parent peptide is selected from 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 shown in the amino acid sequence.
  • the 10th or 12th Lys in the amino acid sequence of the parent peptide, is connected to the lipophilic substituent via a bridging group to form a structure:
  • the present invention provides any of the following polypeptide compounds:
  • HsQGTFTSDK (GGSGSG- ⁇ Glu-CO(CH 2 ) 18 -COOH)SKYLE-Aib-KAAQDFVEWLKAGGPSSGAPPPS
  • HsQGTFTSDK (GGSGSG- ⁇ Glu-CO(CH 2 ) 18 -COOH)SKYLE-Aib-KAAQDFVEWLKAGGPSSGAPPPS-NH 2
  • H-Aib-QGTFTSDK (GGSGSG- ⁇ Glu-CO(CH 2 ) 18 -COOH) SKYLDERAAQDF-Iva-QWLLDGGPSSG-Aib-PPPS-NH 2 .
  • the present invention also provides a composition comprising the polypeptide compound of the present invention.
  • the composition is a pharmaceutical composition, which optionally further comprises a pharmaceutically acceptable carrier or adjuvant.
  • the present invention further provides an application of the polypeptide compound or the composition in the preparation of a medicine for preventing or treating diabetes and/or diabetic complications; wherein preferably, the diabetic complication is diabetic nephropathy.
  • the present invention also provides an application of the polypeptide compound or the composition in the preparation of health care products or medicines for reducing weight.
  • the first aspect of the present invention is to provide a novel agonist polypeptide compound, which contains a parent peptide represented by the following amino acid sequence:
  • R 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
  • Xaa24 Glu or Gln
  • Xaa27 Leu or Lys
  • Xaa28 Asp, Glu or Ala
  • Xaa35 Ala or Aib.
  • the side chain of Lys at position 10 or 12 is connected to a lipophilic substituent via a bridging group.
  • the bridging group includes (PEG) m, or (PEG) m - ⁇ Glu or (PEG) m -Asp; it may also include (Gly) x -(Gly-Ser) y -(Gly) z -, or (Gly) ) x -(Gly-Ser) y -(Gly) z - ⁇ Glu or (Gly) x -(Gly-Ser) y -(Gly) z -Asp.
  • connection method is: the side chain amino group of Lys at position 10 or 12 and the carboxy group of the glycine residue at one end of the bridging group or the carboxy group modified at the end of (PEG) m form an amide bond to connect to the parent peptide On; At the same time, the lipophilic substituent is connected with its carboxyl group and the amino group of the bridging group at the other end by forming an amide bond.
  • the 10th or 12th Lys to which the lipophilic substituent is attached can be replaced by HomoLys, Orn, Dap or Dab. Wherein m is an integer of 2-10; x is an integer of 0-5; y is an integer of 1-5; z is an integer of 1-5.
  • the lipophilic substituent is selected from CH 3 (CH 2 ) n CO- or HOOC(CH 2 ) n CO-, where n is an integer from 14 to 20, and the connection mode is shown in Figure 6 and Figure 7, and Figure 6 is An exemplary modification of the side chain of Lys in the parent peptide of the compound of the present invention.
  • FIG. 7 is another exemplary modification of the side chain of Lys in the parent peptide of the compound of the present invention.
  • the conventional three-letter codes are used to represent natural amino acids, and the recognized three-letter codes are used to represent other amino acids, such as Aib (aminoisobutyric acid), Orn (ornithine).
  • the compound of the present invention can stabilize the helical structure of the molecule based on the theoretical intramolecular bridge, thereby improving the efficacy and/or selectivity for the glucagon-like peptide 1 (GLP-1R) receptor and the glucagon (GCGR) receptor .
  • the compounds of the present invention are based on the theory that lipophilic substituents can bind to albumin in the blood, thereby protecting the compounds of the present invention from enzymatic degradation, thereby increasing the compound's half-life.
  • Another aspect of the present invention is to provide a pharmaceutical composition containing the novel agonist polypeptide compound of the present invention, using the novel agonist polypeptide compound as an active ingredient and adding a pharmaceutically acceptable carrier and/or adjuvant to prepare a pharmaceutical composition .
  • Another aspect of the present invention is to provide the medical use of the novel agonist polypeptide compound of the present invention.
  • Cell and animal experiments show that the novel agonist polypeptide compound of the present invention has a long-acting hypoglycemic effect and can be used as a medicine for the treatment of diabetes.
  • Another aspect of the present invention is to provide other medical uses of the novel agonist polypeptide compounds of the present invention.
  • Cell and animal experiments show that the novel agonist polypeptide compound of the present invention has a pharmacological effect on the treatment of diabetic nephropathy and other diabetic complications. Animal experiments have shown that the therapeutic effect is better than that of the marketed GLP-1 analogue somaglutide, and can be used as a medicine for the treatment of diabetic nephropathy and other diabetic complications.
  • novel agonist polypeptide compounds of the present invention can also reduce body weight and have potential applications as drugs for treating obesity.
  • the parent peptide in the novel agonist polypeptide compound mentioned in the present invention is a homologous polypeptide.
  • the homologous polypeptide in the present invention means that the polypeptide originally has oxyntomodulin (OXM), glucagon-like peptide (GLP-1), Exenatide (Exenatide) or glucagon (Glucagon) However, one or more amino acid residues have been replaced by different amino acid residues. These amino acid residues are conserved among each other, and the obtained polypeptide can be used to implement the present invention.
  • the polypeptide compounds of the present invention can also be used to prevent weight gain or promote weight loss.
  • the polypeptide compound can cause a decrease in food intake and/or an increase in energy expenditure, resulting in an observable effect on body weight. Therefore, the polypeptide compounds of the present invention can be used to directly or indirectly treat any disease caused by or characterized by overweight, such as the treatment and/or prevention of obesity, morbid obesity, obesity-related inflammation, obesity-related gallbladder Sleep apnea caused by disease and obesity.
  • the effect of the polypeptide compound of the present invention in these conditions may be caused by or related to the effect of the polypeptide compound on body weight, or may be independent of its effect on body weight.
  • the pharmaceutical composition of the present invention is suitable for various modes of administration, such as oral administration, transdermal administration, intravenous administration, intramuscular administration, topical administration, transnasal administration, and the like.
  • the pharmaceutical composition of the polypeptide compound of the present invention can be prepared into various suitable dosage forms, which contain at least one effective dose of the polypeptide compound of the present invention and at least one pharmaceutically acceptable drug.
  • suitable dosage forms are tablets, capsules, sugar-coated tablets, granules, oral solutions and syrups, ointments and patches for the surface of the skin, aerosols, nasal sprays, and sterile solutions that can be used for injection.
  • the pharmaceutical composition containing the polypeptide compound of the present invention can be made into a solution or freeze-dried powder for parenteral administration.
  • the powder can be reconstituted by adding an appropriate solvent or other pharmaceutically acceptable carrier before use.
  • the liquid formulation is generally buffered. Liquid, isotonic solution and aqueous solution.
  • the dosage of the pharmaceutical composition of the present invention can be varied within a relatively large range, and those skilled in the art can easily adjust it according to some objective factors such as the type of disease, the severity of the disease, the weight of the patient, the dosage form, the route of administration and other factors. Add to be sure.
  • the present invention has the following advantages:
  • the polypeptide compound of the present invention has better biological activity
  • the polypeptide compound of the present invention has a better pharmacodynamic effect in treating diabetic nephropathy and other diabetic complications;
  • the present invention relates to the following novel agonist polypeptide compounds, which have the following sequence and side chain modification results:
  • HsQGTFTSDK (GGSGSG- ⁇ Glu-CO(CH 2 ) 18 -COOH)SKYLE-Aib-KAAQDFVEWLKAGGPSSGAPPPS
  • HsQGTFTSDK (GGSGSG- ⁇ Glu-CO(CH 2 ) 18 -COOH)SKYLE-Aib-KAAQDFVEWLKAGGPSSGAPPPS-NH 2
  • H-Aib-QGTFTSDK (GGSGSG- ⁇ Glu-CO(CH 2 ) 18 -COOH) SKYLDERAAQDF-Iva-QWLLDGGPSSG-Aib-PPPS-NH 2 .
  • the modification of Lys can be one of the following structures:
  • Lys connected to the lipophilic substituent can be replaced with:
  • Boc is tert-butoxycarbonyl
  • Fmoc is fluorenylmethyloxycarbonyl
  • t-Bu is tert-butyl
  • ivDDe is 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methyl -Butyl
  • resin is resin
  • TFA is trifluoroacetic acid
  • EDT is 1,2-ethanedithiol
  • Phenol is phenol
  • FBS fetal bovine serum
  • BSA bovine serum albumin
  • HPLC high performance liquid phase
  • GLP -1R is the glucagon-like peptide 1 receptor
  • GCGR is the glucagon receptor
  • GLP-1 is the glucagon-like peptide
  • mPEG is monomethoxy polyethylene glycol
  • His is histidine
  • Ser is serine
  • D-Ser is D-serine
  • Gln is glutamine
  • Gly gly
  • 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.
  • Figure 1 shows the blood glucose changes in Example 2 based on the initial blood glucose level of the experimental animal 4 hours after administration and the 24-hour tracking of the oral glucose again at different time points.
  • Fig. 2A shows the electrophoresis diagrams of different groups of FN expression in different groups of FN expression gel electrophoresis detected by the in vitro renal fibrosis cell model constructed by inducing MCS mesangial cells and NRK renal epithelial cells by high glucose in Example 3, using Western blot.
  • Figure 2B is a histogram of the expression of FN in different groups detected in Example 3 by inducing MCS mesangial cells and NRK renal epithelial cells by high glucose to construct an in vitro renal fibrotic cell model, p ⁇ 0.05; *: indicates the same Compared with the control, it is within 95% confidence level (p ⁇ 0.05); **: indicates that it is within 99% confidence level (p ⁇ 0.01) compared with the control.
  • Figure 3A shows the 8-week-old db/db diabetic mice in Example 4, injected with physiological saline, compound 3 (80 and 120 ⁇ g/kg, 2 groups in total), and compound 8 (80 and 120 ⁇ g/kg) subcutaneously every day or every other day. , 2 groups), compound 13 (80 and 120 ⁇ g/kg, 2 groups), compound 15 (80 and 120 ⁇ g/kg, 2 groups) and somaglutide (80 and 120 ⁇ g/kg, 2 groups) after , The blood glucose and body weight of mice measured by fasting for 6 hours every other day after daily administration, p ⁇ 0.05.
  • Figure 3B shows the 8-week-old db/db diabetic mice in Example 4, injected with saline, compound 3 (80 and 120 ⁇ g/kg, 2 groups in total), and compound 8 (80 and 120 ⁇ g/kg) subcutaneously every day or every other day.
  • 2 groups compound 13 (80 and 120 ⁇ g/kg, 2 groups), compound 15 (80 and 120 ⁇ g/kg, 2 groups) and somaglutide (80 and 120 ⁇ g/kg, 2 groups) after ,
  • the blood glucose and body weight of the mice measured by fasting for 6 hours every other day after the administration on the next day, p ⁇ 0.05.
  • Figure 4A shows the 12-week-old db/db diabetic mice in Example 4, which were injected subcutaneously with physiological saline, polypeptide compounds 3, 8, 13, 14, 15, 16 and somaglutide, SAR425899, MED C18-acid, After continuous administration for 6 weeks, the weekly blood glucose changes of the mice.
  • Figure 4B shows the 12-week-old db/db diabetic mice in Example 4, which were injected subcutaneously with physiological saline, polypeptide compounds 3, 8, 13, 14, 15, 16 and somaglutide, SAR425899, MED C18-acid, The OGTT results and the corresponding AUC values measured in the 4th week.
  • Figure 4C shows the 12-week-old db/db diabetic mice in Example 4, which were injected subcutaneously with physiological saline, polypeptide compounds 3, 8, 13, 14, 15, 16 and somaglutide, SAR425899, MED C18-acid, The ITT result and the corresponding AUC value measured in the 5th week.
  • Figure 4D shows the 12-week-old db/db diabetic mice in Example 4, which were injected subcutaneously with physiological saline, polypeptide compounds 3, 8, 13, 14, 15, 16 and somaglutide, SAR425899, MED C18-acid, The results of ALT and AST in mouse serum, p ⁇ 0.05.
  • Figure 5A shows the urine protein content, urine sugar content, serum glycosylated hemoglobin content, urea nitrogen content, and blood creatinine content in the urine of the mice in Example 4 after 8 weeks of administration treatment from left to right.
  • Figure 5B shows the H&E staining results of the kidneys of the mice in Example 4 after 8 weeks of treatment.
  • Figure 5C shows the PAS staining results and statistics of the kidneys of the mice after 8 weeks of treatment in Example 4.
  • Figure 5D is the Sirius red staining results and statistical graphs of the kidneys of the mice after 8 weeks of treatment in Example 4.
  • Fig. 5E shows the FN staining results and statistical graphs of the kidneys of the mice after 8 weeks of treatment in Example 4. Figs.
  • Fig. 5F is the ⁇ -SMA staining results and statistical graphs of the kidneys of the mice after 8 weeks of treatment in Example 4, p ⁇ 0.05.
  • Fig. 6 is an exemplary modification of the side chain of Lys in the parent peptide of the compound of the present invention.
  • Fig. 7 is another exemplary modification of the side chain of Lys in the parent peptide of the compound of the present invention.
  • polypeptide compounds 1-16 involved in the present invention positive control polypeptide compounds 17-20 (derived from the compound in Chinese Patent Publication No. CN 104926934A), positive control polypeptide compounds 21-26 (derived from patent CN201711194175.0), SAR425899 Both MED C18-acid and MED C18-acid are synthesized by themselves, and SAR425899 and MED C18-acid are both compounds reported in the prior art.
  • amino acid sequences of the positive control polypeptide compounds 17-26, SAR425899 and MED C18-acid compounds are as follows:
  • X in compound 21 in the table is aminoisobutyric acid
  • K * in compound 21-25 indicates that this position is a lysine residue and is covalently linked to a fatty acid.
  • the structure of the fatty acid is shown in the following formula:
  • polypeptide compound 3 In order to facilitate the description of the preparation method of the above-mentioned polypeptide compound, the present invention takes polypeptide compound 3 as an example, and the specific operation steps are as follows:
  • the amount of benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate and Fmoc are used to protect the amino acid
  • the amount ratio (molar ratio) of the amount of substances used is 3:1.
  • the piperidine/N,N-dimethylformamide solution removes the Fmoc group, and then couples Fmoc- ⁇ Glu-OtBu, tBu monoprotected eicosanoic fatty acids in sequence according to conventional conditions, and obtains:
  • the column was eluted with a gradient of 30-60% acetonitrile-0.1% trifluoroacetic acid/H 2 O at 40 mL/min for 45.0 minutes, the peptide-containing fractions were collected, concentrated to remove acetonitrile, and then lyophilized.
  • the pure product with HPLC purity greater than 95% is obtained.
  • the separated products were analyzed by liquid-mass spectrometry, and it was found that the m/z value of the protonated molecular ion peak was 4860.9, and the theoretical value was 4863.5.
  • Example 2 The effect of polypeptide compounds 1-16, positive control polypeptide compounds 17-26, liraglutide and somaglutide on oral glucose tolerance (OGTT)
  • Semaglutide in this example was purchased from Zhejiang Paitide Biological Co., Ltd. (CAS No.: 910463-68-2), and Liraglutide was purchased from Shenzhen Jianyuan Pharmaceutical Technology Co., Ltd. (CAS) No.: 204656-20-2).
  • polypeptide compounds 1-16, positive control polypeptide compounds 17-26, liraglutide, somaglutide, SAR425899 and MED C18-acid are all according to 100ug/kg
  • the AUC of liraglutide and compound 17-26 in the first OGTT curve period (0-120min) can be significantly reduced (P ⁇ 0.05), and in the following three OGTT During the curve period (4-22h), its AUC began to increase. In contrast, the AUC of somaglutide was significantly reduced during the four OGTT curve periods (P ⁇ 0.05), indicating that it has a long-term hypoglycemic effect.
  • the polypeptide compounds of the present invention all have the effect of improving the glucose tolerance of mice to varying degrees. Among them, the polypeptide compounds 1-16 exhibit 4 OGTT curve periods (0-22h).
  • the peptide compound 1-16 has a more excellent and significant improvement in glucose tolerance. Compared with the peptide compound 17-26, liraglutide, SAR425899 and MED C18-acid, it has more excellent and significant long Effective hypoglycemic effect. Among them, compared with somaglutide, polypeptide compounds 1, 2, 3, 8, 13, 14 and 15 exhibited a more excellent and significant long-term hypoglycemic effect in 4 OGTT curve periods (0-22h).
  • Example 3 In the renal fibrosis model induced by high glucose, the improvement effect of polypeptide compounds 1, 2, 3, 8, 13, 14, 15 on renal fibrosis
  • the increased production of hyperglycemia and glycation end products will cause the proliferation and hypertrophy of glomerular mesangial cells, the increase of extracellular matrix (ECM), and morphological changes such as mesangial expansion, which will eventually lead to the loss of their physiological functions.
  • ECM extracellular matrix
  • morphological changes such as mesangial expansion
  • the transdifferentiation of renal tubular epithelial cells is also an important pathological basis for renal fibrosis.
  • high concentrations of glucose activate the RAS system to further induce glomerular fibrosis. Therefore, in this example, high glucose was used to induce MCS mesangial cells and NRK renal epithelial cells to construct an in vitro renal fibrotic cell model for screening drugs that can be used to treat diabetic nephropathy.
  • kidney mesangial cell line GMC and glomerular epithelial cell mTEC used in this example are cells preserved in the applicant’s laboratory (both are primary kidney cells extracted from mice), and a low-sugar medium containing 10% fetal bovine serum nourish.
  • the cells When the cells grow to 70% confluence, they are starved with serum-free culture medium for 24 hours, and the cells are divided into the following 9 groups by adjusting the glucose concentration as follows: 1Low group (5.5mmol/L glucose), 2High group (30mmol/L glucose) , 3High sugar + compound 1 group (30mmol/L glucose + NO.1 10uM), 4High sugar + compound 2 group (30mmol/L glucose + NO.2 10uM), 5High sugar + compound 3 group (30mmol/L Glucose+NO.3 10uM), 6High glucose+compound 8 group (30mmol/L glucose+NO.8 10uM), 7High glucose+compound 13 group (30mmol/L glucose+NO.13 10uM), 8High glucose+ Compound 14 group (30mmol/L glucose + NO.14 10uM) and 9 high sugar + compound 15 group (30mmol/L glucose + NO.15 10uM), cultured for 48h at 37°C under 5% CO 2 conditions, and carried out FN protein ( Fibronectin) Western
  • FIG. 2A shows the histogram of the expression of FN in different groups of the model of kidney fibrosis cells in vitro by inducing MCS mesangial cells and NRK renal epithelial cells by high glucose in Example 3, as shown in FIG. 2B.
  • db/db mouse diabetes model obtained the db/db mouse diabetes model (purchased from Nanjing University-Nanjing Institute of Biomedicine, about 8 weeks, and measured blood glucose and body weight to ensure that the subsequent experiments proceed smoothly), and randomly divided the model mice into 12 groups (including peptide compound 3, 8, 13, 15, Somaglutide and saline group), each group had 6 animals, and there was no difference in basal body weight and blood sugar.
  • Each group of rats were injected subcutaneously with compound 3 (80 and 120 ⁇ g/kg, 2 groups in total), compound 8 (80 and 120 ⁇ g/kg, 2 groups in total), and compound 13 (80 and 120 ⁇ g/kg, 2 groups in total) every day or every other day.
  • compound 15 (80 and 120 ⁇ g/kg, 2 groups in total), somaglutide (80 and 120 ⁇ g/kg, 2 groups in total) and saline (DN group: normal mice born in the same litter; DC group: db /db mouse control group).
  • mice After the mice were administered daily, the mice were fasted for 6 hours every other day to measure their blood glucose and body weight.
  • Figure 3A shows that compared with the DC group in the saline group, the somaglutide group (120 ⁇ g/kg) showed a better hypoglycemic effect, and then maintained a relatively stable normal blood sugar level, but its weight reduction effect was not significant .
  • Polypeptide compounds 3, 8, 13, and 15 in the administration group at a dose of 80ug/kg polypeptide compounds 3 and 8 have obvious hypoglycemic effects, and polypeptide compounds 13 and 15 have obvious hypoglycemic effects and have long-term effects;
  • peptide compounds 3, 8, 13, and 15 At a dose of 120ug/kg, peptide compounds 3, 8, 13, and 15 have reached a good hypoglycemic effect in 3-5 days, and blood sugar is stably maintained at a normal level, while peptide compounds 3, 8, 13, and 15 Demonstrates a more excellent weight-reducing effect, and the effect of polypeptide compounds 13 and 15 is better than that of polypeptide compounds 3 and 8. Therefore, the drug concentration is set at 120ug/kg.
  • mice were given the drug every other day, the mice were fasted for 6 hours every other day to measure their blood glucose and body weight.
  • Figure 3B shows that compared with the DC group in the saline group, after the next day administration, the somaglutide group 80 ⁇ g/kg and 120 ⁇ g/kg have the hypoglycemic effect, but the hypoglycemic effect is not obvious, and the weight reduction effect is not significant.
  • the peptide compounds 13 and 15 had a significant hypoglycemic effect on the 3rd day, but the subsequent blood glucose level changed little and tended to be stable; at the dose of 120ug/kg, the blood sugar of the somaglutide group increased on the 3rd day. Long-term effect is not enough. Polypeptide compounds 3, 8, 13, and 15 have achieved a good hypoglycemic effect in 3-5 days, and the blood sugar of polypeptide compounds 3 and 8 has little change afterwards. Polypeptide compounds 13 and 15 have always maintained a good blood sugar lowering effect, which proves that the polypeptide compounds 13 and 15 have a good long-term effect of lowering blood sugar. At the same time, polypeptide compounds 3, 8, 13, and 15 exhibited more excellent weight-reducing effects, and the effects of polypeptide compounds 13 and 15 were better than those of polypeptide compounds 3 and 8. Therefore, the drug concentration is set at 120ug/kg.
  • db/db mouse diabetes model obtained the db/db mouse diabetes model (purchased from Nanjing University-Nanjing Institute of Biomedicine, 12-week-old mice, and measured blood sugar and body weight to ensure that the subsequent experiments proceed smoothly), and randomly divided the model mice into 10 groups (polypeptide compound 3) , 8, 13, 14, 15, 16, Somaglutide, SAR425899, MED C18-acid and saline group), each group has 6 mice, and the basal body weight and blood glucose of each mouse have no difference.
  • mice Each group of mice is different every day Subcutaneous injection of peptide compounds 3, 8, 13, 14, 15, 16 (120 ⁇ g/kg), somaglutide (120 ⁇ g/kg), SAR425899 (120 ⁇ g/kg), MED C18-acid (120 ⁇ g/kg), and physiological Saline (DN group: normal mice born in the same litter; DC group: db/db mouse control group). After the mice were administered daily, the mice were fasted for 6 hours every other day to measure the blood glucose and body weight of the mice, and the water intake and food intake were measured once a week.
  • DN group normal mice born in the same litter
  • DC group db/db mouse control group
  • the OGTT was measured in the 4th week
  • the insulin tolerance (ITT) was measured in the 5th week
  • the mouse metabolic cage was used to test the metabolism of the mice in the 6-7 week
  • the samples were taken in the 8th week to detect various serological and pathological indicators.
  • the type II diabetes model is characterized by obesity, insulin resistance, hyperglycemia, dyslipidemia, and vacuolar degeneration of liver fat.
  • polypeptide compounds 3, 8, 13, 14, 15, 16 and Somaglutide, SAR425899, MED C18-acid were used to treat diabetic mice for 4 weeks and 5 weeks, oral glucose tolerance test (OGTT) and Insulin tolerance test (ITT), the results are shown in Figures 4A-4D.
  • the 20-week-old db/db diabetic mouse DC group has significantly increased proteinuria content and urine sugar content compared with the control DN group.
  • the glycosylated hemoglobin content, blood creatinine and urea nitrogen content also significantly increased. It shows that the diabetic nephropathy in mice is already very serious. After the administration, the mice's proteinuria content, urine sugar content, glycosylated hemoglobin content, blood creatinine and urea nitrogen content all improved to varying degrees.
  • the compound reported in the present invention is superior to the previously reported compound in reducing blood sugar, reducing body weight, improving and treating diabetic complications, and has more advantages in clinical application.

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Abstract

本发明公开一种多肽化合物及其在预防或治疗糖尿病或糖尿病并发症中的应用，该多肽化合物显示出了良好的长效降血糖作用和糖尿病肾病的治疗效果；同时具有高酶解稳定性、高生物活性、无不良发应发生等效果，可以用于制备治疗摄食过量、肥胖症和超重、胆固醇升高、糖尿病及糖尿病肾病药物。

Description

多肽化合物及其在预防或治疗糖尿病或糖尿病并发症中的应用 技术领域
本发明属于生物化学技术领域，具体而言，本发明涉及一类多肽化合物及其在预防或治疗糖尿病或糖尿病并发症中的应用。
背景技术
近年来，随着经济的迅速发展、膳食结构和生活方式的改变、人口老龄化速度的加快等，糖尿病在全世界的患病率也呈现快速增加的趋势。据统计，在世界范围内将近9％的成年人患有II-型糖尿病。而据国际糖尿病联盟(IDF)估计，糖尿病的全球患病率将由2015年的4.15亿人增至2040年的6.42亿人(Unnikrishnan R,Pradeepa R,Joshi SR,Mohan V.Type 2Diabetes:Demystifying the Global Epidemic.Diabetes,2017.66:1432-1442)。糖尿病已经成为继心脑血管疾病、肿瘤之后另一个严重危害人体健康的重要慢性非传染性疾病。
近年来的多项调查表明，无论是欧美发达国家还是发展中国家如中国，糖尿病控制状况均不容乐观。糖尿病最严重的后果之一是糖尿病血管病变的发展，临床上表现为微血管和大血管并发症。糖尿病肾病即是一种微血管并发症，是发达国家最常见的终末期肾病病因。
大约40％的I-型糖尿病患者会发展为糖尿病肾病，而虽然II-型糖尿病肾病发展为终末期肾病的病例相对较少，但随着II-型糖尿病患者数量的迅速增加，意味着如果缺乏糖尿病管理的有效措施，糖尿病肾病的发生率也将大幅增加。因此，急需有效且长效的降糖药物用于糖尿病肾病的治疗。大约40％的I-型糖尿病患者发展为糖尿病肾病。由于糖尿病肾病的发病率有相当大的种族变异性，只有大约三分之一的 I-型糖尿病患者发展为糖尿病肾病，且与遗传因素有极大的关系。虽然II-型糖尿病肾病发展为终末期肾病的病例相对较少，但随着II-型糖尿病患者数量的迅速增加，这意味着，如果缺乏糖尿病管理的有效措施，糖尿病肾病的发生率也将大幅增加，因此，急需有效且长效的降糖药物用于糖尿病肾病的治疗(Chen L,Magliano DJ,Zimmet PZ.The worldwide epidemiology of type 2 diabetes mellitus—present and future perspectives.Nat Rev Endocrinol,2011,8(4):228-236)。
随着糖尿病及其治疗的深入研究，人们不断发现治疗II-型糖尿病的药物作用新靶标。2005年以来上市的GLP-1类似物(Meier JJ.GLP-1 Receptor Agonists for Individualized Treatment of Type-2 Diabetes Mellitus.Nat.Rev.Endocrinol,2012.8(12):728)，其作用靶点不同于以往药物，为糖尿病的治疗提供了新的治疗选择。目前已被批准用于治疗II-型糖尿病的GLP-1类似物包括艾塞那肽、利拉鲁肽和利西拉肽，以及后续产生的阿必鲁肽及度拉糖肽。此外，诺和诺德公司的每周1次长效GLP-1类似物索玛鲁肽也已经上市销售，用于治疗II-型糖尿病。
与同源配体单靶点激动剂相比，GLP-1R/GCGR双重激动剂具有降糖、降脂、降体重的协同效应。此外，GLP-1R/GCGR双靶点激动剂，GCGR与GLP-1的协同作用可增强胰腺β-细胞的功能，同时又可能最小化或防止II-型糖尿病进展。这些创新增加了GLP-1的既定效果，且第二活性可能增加更多的功效，例如：降体重、心血管获益和糖尿病肾病的治疗等。
例如，在中国专利CN201711194175.0中公开了一种治疗代谢疾病的胰高血糖素类似物，但经研究发现，该胰高血糖素类似物改善糖耐受效果与利拉鲁肽相当，但比索玛鲁肽改善糖耐受的效果差，该胰高血糖素类似物同样不能满足临床使用要求。
综上所述，尽管已经存在许多治疗糖尿病的药物，但是II-型糖尿病以及相关并发症肾病的治疗仍然存在挑战，遏制 糖尿病肾病的全球蔓延注定是一项长期而艰巨的任务。研发新型的双靶点或多靶点激动剂多肽药物是治疗糖尿病及其并发症的主要方向。
发明内容
本发明的目的在于提供一类新型的多肽化合物。本发明的发明人经过大量的实验研究，证明该类新型的多肽化合物具有更长的半衰期，具有促胰岛素活性，没有不良反应发生，可用于糖尿病及糖尿病肾病等糖尿病并发症的预防或治疗。
本发明的另一个目的在于提供上述新型的多肽化合物的应用，该新型的多肽化合物潜在地可作为新一代预防或治疗糖尿病及糖尿病肾病等糖尿病并发症的药物，还可用于降糖或降低体重。
为达上述目的，本发明的技术方案如下：
本发明提供一种多肽化合物，其含有以下氨基酸序列表示的母体肽：
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，
其中：
Xaa2＝Aib，Ser或D-Ser；
Xaa15＝Asp或Glu；
Xaa16＝Aib或Glu；
Xaa17＝Lys或Arg；
Xaa21＝Asp或Glu；
Xaa23＝Val或Iva；
Xaa24＝Glu或Gln；
Xaa27＝Leu或Lys；
Xaa28＝Asp，Glu或Ala；
Xaa35＝Ala或Aib。
优选地，在所述母体肽的氨基酸序列中，Xaa2＝Aib或 D-Ser。
优选地，在所述母体肽的氨基酸序列中，Xaa21＝Asp。
优选地，所述母体肽的氨基酸序列的羧基端不经修饰或者经氨基修饰形成-CONH 2基团。
并且，优选地，所述母体肽的氨基酸序列中第10位或第12位Lys的侧链经由桥接基团与亲脂性取代基连接，所述桥接基团为(PEG) m、(PEG) m-γGlu、(PEG) m-Asp、(Gly) x-(Gly-Ser) y-(Gly) z-、(Gly) x-(Gly-Ser) y-(Gly) z-γGlu和(Gly) x-(Gly-Ser) y-(Gly) z-Asp中的一种。所述连接方式为：所述第10位或第12位Lys的侧链氨基与所述桥接基团一端的甘氨酸残基的羧基或(PEG) m末端修饰的羧基形成酰胺键连接到母体肽上；同时，所述亲脂性取代基以其羧基与另一端桥接基团的氨基通过形成酰胺键相连。所述亲脂性取代基为CH 3(CH 2) nC(O)-或HOOC(CH 2) nC(O)-且其酰基与所述桥接基团中的氨基形成酰胺键。
在本发明所述的桥接基团中，m为2-10的整数；x为0-5的整数；y为1-5的整数；z为1-5的整数；在本发明所述的亲脂性取代基中，n为14-20的整数。
可替代地，所述母体肽的氨基酸序列中第10位或第12位Lys可以被HomoLys、Orn、Dap或Dab代替。
进一步优选地，所述母体肽的氨基酸序列中：
Xaa2＝Aib或D-Ser；
Xaa15＝Glu；
Xaa16＝Aib；
Xaa17＝Lys；
Xaa21＝Asp；
Xaa23＝Val；
Xaa24＝Glu；
Xaa27＝Lys；
Xaa28＝Ala；
Xaa35＝Ala；
或者，
Xaa2＝Aib或D-Ser；
Xaa15＝Asp；
Xaa16＝Glu；
Xaa17＝Arg；
Xaa21＝Asp；
Xaa23＝Iva；
Xaa24＝Gln；
Xaa27＝Leu；
Xaa28＝Asp或Glu；
Xaa35＝Ala或Aib。
根据本发明的具体实施方式，所述母体肽的氨基酸序列选自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和SEQ ID NO.16所示的氨基酸序列。
根据本发明的具体实施方式，所述母体肽的氨基酸序列中第10位或第12位Lys、优选第10位Lys经由桥接基团与亲脂性取代基相连形成的结构为：
Figure PCTCN2020137071-appb-000001
Figure PCTCN2020137071-appb-000002
根据本发明的具体实施方式，本发明提供如下任一种多肽化合物：
化合物1：
H-Aib-QGTFTSDK(PEG 2-PEG 2-γGlu-CO(CH 2) 18CO 2H)SKYLE-Aib-KAAQDFVEWLKAGGPSSGAPPPS-NH 2
化合物2：
HsQGTFTSDK(PEG 2-PEG 2-CO(CH 2) 18CO 2H)SKYLE-Aib-KAAQDFVEWLKAGGPSSGAPPPS-NH 2
化合物3：
HsQGTFTSDK(PEG 2-PEG 2-γGlu-CO(CH 2) 18CO 2H)SKYLE-Aib-KAAQDFVEWLKAGGPSSGAPPPS-NH 2
化合物4：
HsQGTFTSDK(PEG 2-PEG 2-γGlu-CO(CH 2) 16CO 2H)SKYLE-Aib-KAAQDFVEWLKAGGPSSGAPPPS-NH 2
化合物5：
HsQGTFTSDK(PEG 2-PEG 2-γGlu-CO(CH 2) 18CH 3)SKYLE-Aib-KAAQDFVEWLKAGGPSSGAPPPS-NH 2
化合物6：
HsQGTFTSDK(PEG 2-PEG 2-γGlu-CO(CH 2) 18CO 2H)SKYLDERAAQDF-Iva-QWLLDGGPSSG-Aib-PPPS-NH 2
化合物7：
HsQGTFTSDK(PEG 2-PEG 2-γGlu-CO(CH 2) 18CO 2H)SKYLDERAAQDF-Iva-QWLLEGGPSSGAPPPS-NH 2
化合物8：
H-Aib-QGTFTSDK(PEG 2-PEG 2-γGlu-CO(CH 2) 18CO 2H)SKYLDERAAQDF-Iva-QWLLDGGPSSG-Aib-PPPS-NH 2
化合物9：
H-Aib-QGTFTSDK(PEG 2-PEG 2-γGlu-CO(CH 2) 16CO 2H)SKYLDERAAQDF-Iva-QWLLDGGPSSG-Aib-PPPS-NH 2
化合物10：
H-Aib-QGTFTSDK(PEG 2-PEG 2-γGlu-CO(CH 2) 18CH 3)SKYLDERAAQDF-Iva-QWLLDGGPSSG-Aib-PPPS-NH 2
化合物11：
H-Aib-QGTFTSDK(PEG 2-PEG 2-γGlu-CO(CH 2) 18CH 3)SKYLDERAAQDF-Iva-QWLLDGGPSSG-Aib-PPPS-NH 2
化合物12：
HsQGTFTSDK(PEG 2-PEG 2-γGlu-CO(CH 2) 18CO 2H)SKYLDERAAQDF-Iva-QWLLDGGPSSG-Aib-PPPS-NH 2
化合物13：
HsQGTFTSDK(GGSGSG-γGlu-CO(CH 2) 18-COOH)SKYLE-Aib-KAAQDFVEWLKAGGPSSGAPPPS
化合物14：
HsQGTFTSDK(GGSGSG-γGlu-CO(CH 2) 18-COOH)SKYLE-Aib-KAAQDFVEWLKAGGPSSGAPPPS-NH 2
化合物15：
H-Aib-QGTFTSDK(GGSGSG-γGlu-CO(CH 2) 18-COOH)SKYLDERAAQDF-Iva-QWLLDGGPSSG-Aib-PPPS
化合物16：
H-Aib-QGTFTSDK(GGSGSG-γGlu-CO(CH 2) 18-COOH)SKYLDERAAQDF-Iva-QWLLDGGPSSG-Aib-PPPS-NH 2。
本发明还提供一种组合物，其包含本发明所述的多肽化合物。
优选地，所述组合物为药物组合物，其任选地还包含药学上可接受的载体或辅料。
本发明再提供一种所述多肽化合物或所述组合物在制备预防或治疗糖尿病和/或糖尿病并发症的药物中的应用；其中优选地，所述糖尿病并发症为糖尿病肾病。
本发明又提供一种所述多肽化合物或所述组合物在制备降低体重的保健品或药物中的应用。
本发明的技术方案详细描述如下。
本发明的第一个方面是提供一种新型的激动剂多肽化合物，该新型的激动剂多肽化合物含有以下氨基酸序列表示的母体肽：
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
其中，R 1＝-NH 2或-OH；
Xaa2＝Aib，Ser或D-Ser；
Xaa15＝Asp或Glu；
Xaa16＝Aib或Glu；
Xaa17＝Lys或Arg；
Xaa21＝Asp或Glu；
Xaa23＝Val或Iva；
Xaa24＝Glu或Gln；
Xaa27＝Leu或Lys；
Xaa28＝Asp，Glu或Ala；
Xaa35＝Ala或Aib。
所述母体肽的氨基酸序列中，第10位或12位Lys的侧 链经由桥接基团与亲脂性取代基相连。
所述桥接基团包含(PEG) m，或者(PEG) m-γGlu或(PEG) m-Asp；也可以包含(Gly) x-(Gly-Ser) y-(Gly) z-，或者(Gly) x-(Gly-Ser) y-(Gly) z-γGlu或(Gly) x-(Gly-Ser) y-(Gly) z-Asp。所述连接方式为：所述第10位或第12位Lys的侧链氨基与所述桥接基团一端的甘氨酸残基的羧基或(PEG) m末端修饰的羧基形成一个酰胺键连接到母体肽上；同时，所述亲脂性的取代基以其羧基与另一端桥接基团的氨基通过形成酰胺键相连。所述亲脂性取代基连接的第10位或12位Lys能够被HomoLys、Orn、Dap或Dab代替。其中m为2-10的整数；x为0-5的整数；y为1-5的整数；z为1-5的整数。所述亲脂性取代基为选自CH 3(CH 2) nCO-或HOOC(CH 2) nCO-，其中n是14-20的整数，连接方式详见图6和图7，图6为本发明的化合物的母体肽中Lys侧链的一示例性修饰方式，图7为本发明的化合物的母体肽中Lys侧链的另一示例性修饰方式。
与本申请的说明书全文中，采用常规的三字母代码代表天然氨基酸，并采用公认的三字母代码代表其他氨基酸，如Aib(氨基异丁酸)，Orn(鸟氨酸)。本发明化合物基于理论分子内桥可以稳定分子的螺旋结构，从而提高了针对胰高血糖素样肽1(GLP-1R)受体和胰高血糖素(GCGR)受体的效力和/或选择性。
本发明化合物基于理论亲脂性取代基可以结合血液中的白蛋白，从而保护本发明化合物免受酶降解，从而提高化合物的半衰期。
本发明的另一方面是提供含有本发明的新型的激动剂多肽化合物的药物组合物，以所述新型的激动剂多肽化合物作为活性成分添加药学上可接受载体和/或辅料制成药物组合物。
本发明的再一方面是提供本发明的新型的激动剂多肽化合物的医药用途。细胞和动物实验显示，本发明的新型的激动剂多肽化合物具有长效降糖作用，可用作治疗糖尿病的药物。
本发明的还有一个方面是提供本发明的新型的激动剂多肽化合物的其他医药用。细胞和动物实验显示，本发明的新型的激动剂多肽化合物具有治疗糖尿病肾病等糖尿病并发症的药效作用。动物实验显示治疗效果优于上市的GLP-1类似物索玛鲁肽，可用作治疗糖尿病肾病等糖尿病并发症的药物。
本发明的新型的激动剂多肽化合物还可以降低体重，具有作为治疗肥胖症药物的潜在用途。
本发明中提到的新型的激动剂多肽化合物中的母体肽为同源性多肽。本发明中的同源性多肽是指，多肽本来具有胃泌酸调节素(OXM)、胰高血糖素样肽(GLP-1)、艾塞那肽(Exenatide)或胰高血糖素(Glucagon)的氨基酸序列，但其中一个或多个氨基酸残基己被不同的氨基酸残基取代，这些氨基酸残基彼此之间是保守的，并且所得到的多肽可用于实施本发明。
本发明多肽化合物还可用于防止体重增长或促进体重减轻。所述多肽化合物可引起摄食量降低和/或能量消耗升高，使得对体重产生可观察到的作用。因此，本发明多肽化合物可用于直接或间接治疗由体重超重所引起的或者以其为特征的任何病症，例如治疗和/或预防肥胖症、病态肥胖症、肥胖症相关炎症、肥胖症相关的胆囊疾病、肥胖症引起的睡眠呼吸暂停。本发明多肽化合物在这些病症中的作用可以是由多肽化合物对体重的作用所致或与之相关，或者可以独立于其对体重的作用。
本领域技术人员可以理解，本发明的药物组合物适用于各种给药方式，例如口服给药、经皮给药、静脉给药、肌肉内给药、局部给药、经鼻给药等。根据所采用的给药方式，可将本发明的多肽化合物的药物组合物制成各种合适的剂型，其中包含至少一种有效剂量的本发明的多肽化合物和至少一种药学上可接受的药用载体。适当剂型的实例为片剂、胶囊、糖衣片剂、粒剂、口服溶液和糖浆，用于皮肤表面的油膏和药贴，气雾剂、鼻喷剂，以及可用于注射的无菌溶液。
含有本发明多肽化合物的药物组合物可以制成溶液或者冻干粉末以用于胃肠外给药，在使用前可加入适当溶剂或其他可药用的载体将粉末重新配置，液体配方一般是缓冲液、等渗溶液和水溶液。
本发明的药物组合物的用量可以在一个较大范围内变动，本领域技术人员可以根据一些客观的因素如根据疾病的种类，病情严重程度，病人体重，剂型，给药途径等因素很容易的加以确定。
相较于现有技术，本发明具有以下优点：
1)在质量数相同时与天然OXM相比，本发明的多肽化合物具有更好的生物学活性；
2)在质量数相同时与GLP-1类似物相比，本发明的多肽化合物具有更好的治疗糖尿病肾病等糖尿病并发症的药效作用；
3)在药物的药代实验中显示出显著延长的半衰期和稳定性；
4)合成产率高，稳定性好，易于放大生产，成本低。
在具体的实施方案中，本发明涉及下述新型的激动剂多肽化合物，其具有的序列与侧链修饰结果如下：
化合物1(SEQ ID NO.1)：
His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(PEG 2-PEG 2-γGlu-CO(CH 2) 18CO 2H)-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) 18CO 2H)SKYLE-Aib-KAAQDFVEWLKAGGPSSGAPPPS-NH 2
化合物2(SEQ ID NO.2)：
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(PEG 2-PEG 2-CO(CH 2) 18CO 2H)-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) 18CO 2H)SKYLE-Aib-KA AQDFVEWLKAGGPSSGAPPPS-NH 2
化合物3(SEQ ID NO.3)：
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(PEG 2-PEG 2-γGlu-CO(CH 2) 18CO 2H)-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) 18CO 2H)SKYLE-Aib-KAAQDFVEWLKAGGPSSGAPPPS-NH 2
化合物4(SEQ ID NO.4)：
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(PEG 2-PEG 2-γGlu-CO(CH 2) 16CO 2H)-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) 16CO 2H)SKYLE-Aib-KAAQDFVEWLKAGGPSSGAPPPS-NH 2
化合物5(SEQ ID NO.5)：
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(PEG 2-PEG 2-γGlu-CO(CH 2) 18CH 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) 18CH 3)SKYLE-Aib-KAAQDFVEWLKAGGPSSGAPPPS-NH 2
化合物6(SEQ ID NO.6)：
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(PEG 2-PEG 2-γGlu-CO(CH 2) 18CO 2H)-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) 18CO 2H)SKYLDERAAQDF-Iva-QWLLDGGPSSG-Aib-PPPS-NH 2
化合物7(SEQ ID NO.7)：
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(PEG 2-PEG 2-γGlu-CO(CH 2) 18CO 2H)-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) 18CO 2H)SKYLDERAAQDF-Iva-QWLLEGGPSSGAPPPS-NH 2
化合物8(SEQ ID NO.8)：
His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(PEG 2-PEG 2-γGlu-CO(CH 2) 18CO 2H)-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) 18CO 2H)SKYLDERAAQDF-Iva-QWLLDGGPSSG-Aib-PPPS-NH 2
化合物9(SEQ ID NO.9)：
His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(PEG 2-PEG 2-γGlu-CO(CH 2) 16CO 2H)-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) 16CO 2H)SKYLDERAAQDF-Iva-QWLLDGGPSSG-Aib-PPPS-NH 2
化合物10(SEQ ID NO.10)：
His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(PEG 2-PEG 2-γGlu-CO(CH 2) 18CH 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) 18CH 3)SKYLDERAAQDF-Iva-QWLLDGGPSSG-Aib-PPPS-NH 2
化合物11(SEQ ID NO.11)：
His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(PEG 2-PEG 2-CO(CH 2) 18CH 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) 18CH 3)SKYLDERAAQDF-Iva-QWLLDGGPSSG-Aib-PPPS-NH 2
化合物12(SEQ ID NO.12)：
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(PEG 2-PEG 2-γGlu-CO(CH 2) 18CO 2H)-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) 18CO 2H)SKYLDERAAQDF-Iva-QWLLDGGPSSG-Aib-PPPS-NH 2
化合物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
化合物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
化合物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
化合物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-Gl y-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。
上述序列中，Lys的修饰可以为下述结构之一：
Figure PCTCN2020137071-appb-000003
Figure PCTCN2020137071-appb-000004
上述与亲脂性取代基连接的Lys可替换为：
Figure PCTCN2020137071-appb-000005
本发明中所用缩写具体含义如下：
Boc为叔丁氧羰基，Fmoc为芴甲氧羰基，t-Bu为叔丁基，ivDDe为1-(4,4-二甲基-2,6-二氧代亚环己基)-3-甲基-丁基，resin为树脂，TFA为三氟乙酸，EDT为1,2-乙二硫醇，Phenol为苯酚，FBS为胎牛血清，BSA为牛血清白蛋白，HPLC为高效液相，GLP-1R为胰高血糖素样肽1受体，GCGR为胰高血糖素受体，GLP-1为胰高血糖素样肽，mPEG为单甲氧基聚乙烯二醇，His为组氨酸，Ser为丝氨酸，D-Ser为D-型丝氨酸，Gln为谷氨酰胺，Gly为甘氨酸，Glu为谷氨酸，Ala为丙氨酸，Thr为苏氨酸，Lys为赖氨酸，Arg为精氨酸，Tyr为酪氨酸，Asp为天冬氨酸，Trp为色氨酸，Phe为苯丙氨酸，IIe为异亮氨酸，Leu为亮氨酸，Cys为半胱氨酸，Pro为脯氨酸，Val为缬氨酸，Met为蛋氨酸，Asn为天冬酰胺。HomoLys为高赖氨酸，Orn为鸟氨酸，Dap为二氨基庚二酸，Dab为2,4-二氨基丁酸，Aib为2-氨基异丁酸，Iva为异缬氨酸。
附图说明
以下，结合附图来详细说明本发明的实施方案，其中：
图1为实施例2中基于实验动物给药后4小时的初始血糖水平与不同时间点再次口服葡萄糖的24小时追踪的血糖变化，计算的曲线下面积(AUC)，p<0.05。
图2A为实施例3中通过高糖诱导MCS系膜细胞和NRK肾上皮细胞，构建体外肾纤维化细胞模型，使用Western blot检测到的不同组的FN表达凝胶电泳图。
图2B为实施例3中通过高糖诱导MCS系膜细胞和NRK肾上皮细胞，构建体外肾纤维化细胞模型，检测到的不同组的FN表达情况柱状统计图，p<0.05；*：表示与对照相比在95％置信度内(p<0.05)；**：表示与对照相比在99％置信度内(p<0.01)。
图3A为实施例4中8周龄的db/db糖尿病小鼠，分别每天或者隔天皮下注射生理盐水、化合物3(80和120μg/kg，共2组)，化合物8(80和120μg/kg，共2组)，化合物13(80和120μg/kg，共2组)，化合物15(80和120μg/kg，共2组)和索玛鲁肽(80和120μg/kg，共2组)之后，每天给药隔天禁食6h测得的小鼠血糖和体重，p<0.05。
图3B为实施例4中8周龄的db/db糖尿病小鼠，分别每天或者隔天皮下注射生理盐水、化合物3(80和120μg/kg，共2组)，化合物8(80和120μg/kg，共2组)，化合物13(80和120μg/kg，共2组)，化合物15(80和120μg/kg，共2组)和索玛鲁肽(80和120μg/kg，共2组)之后，隔天给药隔天禁食6h测得的小鼠血糖和体重，p<0.05。
图4A为实施例4中12周龄的db/db糖尿病小鼠，分别皮下注射生理盐水，多肽化合物3，8，13，14，15，16和索玛鲁肽，SAR425899，MED C18-acid，连续给药6周，小鼠每周的血糖变化值。
图4B为实施例4中12周龄的db/db糖尿病小鼠，分别皮下注射生理盐水，多肽化合物3，8，13，14，15，16和索玛鲁肽，SAR425899，MED C18-acid，第4周测得的OGTT结果及对应的AUC值。
图4C为实施例4中12周龄的db/db糖尿病小鼠，分别皮下注射生理盐水，多肽化合物3，8，13，14，15，16和 索玛鲁肽，SAR425899，MED C18-acid，第5周测得的ITT结果及对应的AUC值。
图4D为实施例4中12周龄的db/db糖尿病小鼠，分别皮下注射生理盐水，多肽化合物3，8，13，14，15，16和索玛鲁肽，SAR425899，MED C18-acid，小鼠血清中ALT和AST结果，p<0.05。
图5A从左至右依次为实施例4中小鼠给药治疗8周之后尿液中的尿蛋白含量、尿糖含量以及血清中的糖化血红蛋白含量、尿素氮含量以及血肌酐含量。
图5B为实施例4中小鼠给药治疗8周之后肾脏的H&E染色结果。
图5C为实施例4中小鼠给药治疗8周之后肾脏的PAS染色结果及统计图。
图5D为实施例4中小鼠给药治疗8周之后肾脏的天狼星红染色结果及统计图。
图5E为实施例4中小鼠给药治疗8周之后肾脏的FN染色结果及统计图。
图5F为实施例4中小鼠给药治疗8周之后肾脏的α-SMA染色结果及统计图，p<0.05。
图6为本发明的化合物的母体肽中Lys侧链的一示例性修饰方式。
图7为本发明的化合物的母体肽中Lys侧链的另一示例性修饰方式。
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例1多肽化合物的合成
本发明中涉及到的多肽化合物1-16、阳性对照多肽化合物17-20(来源于中国专利公开号CN 104926934A中的化合物)、阳性对照多肽化合物21-26(来源于专利CN201711194175.0)、SAR425899和MED C18-acid均自行合成，其中SAR425899和MED C18-acid均为现有技术报道的化合物。
上述阳性对照多肽化合物17-26、SAR425899和MED C18-acid化合物的氨基酸序列如下：
化合物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) 14CH 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) 14CH 3)RRAQDFVQWLMNTGGPSSGAPPPS-NH 2
化合物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) 14CH 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) 14CH 3)RRAQDFVQWLLNTGGPSSGAPPPS-NH 2
化合物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) 16CO 2H)-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) 16CO 2H)KLD-Aib-RRAQDFVQWLMNTGGPSSGAPPPS-NH 2
化合物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) 14CH 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) 14CH 3)RRAQDFVQWLMNTGGPSSGAPPPS-NH 2
化合物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
化合物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
化合物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
化合物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
化合物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-OH
HSQGTFTSDK *SKYLDERAAQDFVQWLMNTGPSSGAPPPS-OH
化合物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-OH
HSQGTFTSDK *SKYLDSRAAQDFVQWLMNTGPSSGAPPPS-OH
注：表中化合物21中的X为氨基异丁酸，化合物21-25中的K *表示此位置为赖氨酸残基并共价连接至脂肪酸，该脂肪酸结构如下式所示：
Figure PCTCN2020137071-appb-000006
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) 17COOH)-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) 16CO 2H)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) 14CH 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) 14CH 3)ESKAAQDFIEWLKAGGPSSGAPPPS-NH 2
为方便阐述上述多肽化合物的制备方法，本发明以多肽 化合物3为例，具体操作步骤如下：
(一)材料：
所有的氨基酸均购自NovaBiochem公司。如果没有特别说明，其他所有试剂均为分析纯，购自Sigma公司。CS336X多肽合成仪(美国C S Bio公司)。Phenomenex Luna C18制备柱(46mm x 250mm)用来纯化多肽。高效液相色谱仪为Waters公司产品。质谱分析采用Agilent质谱仪进行测定。
(二)方法：
1.多肽化合物3的合成：
结构序列：
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(PEG 2-PEG 2-γGlu-CO(CH 2) 18CO 2H)-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)主肽链组装：
按照Fmoc/t-Bu策略在CS336X多肽合成仪(美国C S Bio公司)上合成0.25mol规模的如下多肽：
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) 18CO 2H)-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-Pro-Ser(t-Bu)-rink amide树脂
(1)第一步：将0.75克Rink amide树脂在二氯甲烷中溶胀，用N,N-二甲基甲酰胺洗涤树脂三次；
(2)第二步：以Rink amide树脂为载体，以由苯并三氮唑-N,N,N',N'-四甲基脲六氟磷酸盐、1-羟基苯并三唑和N,N-二异丙基乙胺三者按物质的量比(摩尔比)1:1:1配制的混合液为偶联剂，以N,N-二甲基甲酰胺为溶剂，进行程序反应，依次进行缩合反应连接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，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) 18CO 2H)-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，得：
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) 18CO 2H)-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-Pro-Ser(t-Bu)-rink amide树脂，其中每次缩合反应中Fmoc保护氨基酸的投料量与树脂用量的物质的量比(摩尔比)为1:1～6:1，每次缩合反应中以由苯并三氮唑-N,N,N',N'-四甲基脲六氟磷酸盐用量与Fmoc保护氨基酸用量的物质的量比(摩尔比)为3:1。
1b)Lys(PEG 2-PEG 2-γGlu-CO(CH 2) 18CO 2H)的脱除与亲脂性取代基的引入：
在N-甲基吡咯烷酮:二氯甲烷＝1:1(体积比)的混合溶液中，将1a)中合成的保护的肽基树脂洗涤两次，加入新鲜制备的2.0％的肼水合物N-甲基吡咯烷酮溶液，将该反应混合物在室温下振摇12.0分钟，然后过滤。将肼处理步骤重复两次，得：
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-Gly- Pro-Ser(t-Bu)-Ser(t-Bu)-Gly-Ala-Pro-Pro-Pro-Ser(t-Bu)-rinkamide树脂。此后用二氯甲烷和N-甲基吡咯烷酮充分洗涤树脂。向其中加入FmocNH-PEG 2-OH、苯并三氮唑-N,N,N',N'-四甲基脲六氟磷酸盐、1-羟基苯并三唑、二异丙基乙基胺的N-甲基吡咯烷酮混合偶联液，振摇3.0小时后，过滤，洗涤将肼处理步骤重复两次，得：
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-Pro-Ser(t-Bu)-rink amide树脂，哌啶/N,N-二甲基甲酰胺溶液脱除Fmoc基团，重复一次Fmoc-PEG 2-OH偶连反应，得：
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-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-Pro-Ser(t-Bu)-rink amide树脂。哌啶/N,N-二甲基甲酰胺溶液脱除Fmoc基团，然后按照常规条件依次偶连Fmoc-γGlu-OtBu,tBu单保护的二十碳脂肪酸，得：
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) 18CO 2H)-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-Pro-Ser(t-Bu)-rink amide树脂。
1c)多肽全保护的脱除：
将
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) 18CO 2H)-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-Pro-Ser(t-Bu)-rink amide树脂加入至圆底烧瓶中，在冰浴条件下，加入切割液TFA/EDT/Phenol/H 2O(88/2/5/5，体积比)升温，控制裂解液温度25℃，反应120分钟。过滤，滤饼用少量三氟乙酸洗涤3次，合并滤液。滤液在搅拌下缓慢倒入冰乙醚中。静置1.0小时以上，待沉淀完全。倾去上清液，沉淀离心，用冰乙醚洗涤6次，得到粗品化合物：
His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(PEG 2-PEG 2-γGlu-CO(CH 2) 18CO 2H)-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)多肽化合物的精制纯化：
将1c中所得粗品溶于5.0％乙酸在乙腈:H 2O＝1:1(体积比)的溶液中，通过反相C18的填充的50mm x 250mm柱上进行2次半制备型HPLC而纯化。用30-60％乙腈-0.1％三氟乙酸/H 2O梯度以40mL/min将该柱洗脱45.0分钟，收集含有肽的组分，浓缩除去乙腈后冻干。得到HPLC纯度大于95％的纯品。用液质联用分析分离出的产物，发现质子化分子离子峰的m/z值为：4860.9，理论值为4863.5。
表1.本发明的多肽化合物
Figure PCTCN2020137071-appb-000007
Figure PCTCN2020137071-appb-000008
Figure PCTCN2020137071-appb-000009
Figure PCTCN2020137071-appb-000010
实施例2多肽化合物1-16、阳性对照多肽化合物17-26、利拉鲁肽和索玛鲁肽对口服葡萄糖耐量(OGTT)的作用
本实施例的索玛鲁肽(Semaglutide)购自浙江湃肽生物有限公司(CAS No.：910463-68-2)，利拉鲁肽(Liraglutide)购自深圳市健元医药科技有限公司(CAS No.：204656-20-2)。
将8周龄的雄性C57BL/6J小鼠(南京大学模式动物研究中心)按照相似的血糖(从尾尖获取的血液样品评估)随机分组，每组6只。
禁食(6小时)后对动物给药，本发明中多肽化合物1-16、阳性对照多肽化合物17-26、利拉鲁肽、索玛鲁肽、SAR425899和MED C18-acid均按照100ug/kg的剂量，皮下进行给药，对照组给予PBS。约4小时后，获取初始血液样品(禁食血液葡萄糖水平)。随后，给予口服剂量的葡萄糖 (2g/kg)，并将动物放回其饲养笼中(t＝0)。在t＝15分钟、t＝30分钟、t＝60分钟和t＝120分钟时测量血糖。然后在给药8h，给药12h以及给药24h后，重复给予口服剂量的葡萄糖，并检测血糖变化，跟踪血糖直到24个小时。使用软件GraphPadPrism处理数据作出血糖变化折线图，并计算曲线下面积得到AUC图，结果如图1所示。
与载剂(对照组PBS)相比，利拉鲁肽和化合物17-26在第一个OGTT曲线时段(0-120min)其AUC可显著降低(P<0.05)，而在随后的三个OGTT曲线时段(4-22h)，其AUC开始提升。相比之下，索玛鲁肽在四个OGTT曲线时段其AUC均显著降低(P<0.05)，说明其具有长效降糖效果。而与载剂(对照组PBS)相比，本发明的多肽化合物均有不同程度地改善小鼠糖耐受的效果，其中多肽化合物1-16在4个OGTT曲线时段(0-22h)，展现了更为优秀和显著地改善葡萄糖耐受效果，同时结果也说明多肽化合物1-16，相比多肽化合物17-26，利拉鲁肽，SAR425899和MED C18-acid具有更为优秀和显著的长效降糖效果。其中与索玛鲁肽相比，多肽化合物1、2、3、8、13、14和15在4个OGTT曲线时段(0-22h)，展现了更为优秀和显著地长效降糖效果。
实施例3在高糖诱导的肾纤维化模型中，多肽化合物1、2、3、8、13、14、15对肾纤维化的改善作用
高血糖及糖基化终产物生成增多后将引起肾小球系膜细胞增生、肥大，细胞外基质(extracellular matrix，ECM)增多，系膜扩张等形态学改变，最终导致其生理功能的丧失。此外，肾小管上皮细胞转分化也是肾脏纤维化的重要病理基础，有研究发现高浓度葡萄糖通过激活RAS系统，进一步诱导肾小球纤维化。因此，本实施例采用高糖诱导MCS系膜细胞和NRK肾上皮细胞，构建了体外肾纤维化细胞模型，用来筛选可用于治疗糖尿病肾病的药物。
本实施例使用的肾脏系膜细胞株GMC和肾小球上皮细胞mTEC均为申请人实验室内保存的细胞(均是提取老鼠的 肾原代细胞)，用含10％胎牛血清低糖培养基培养。细胞生长到汇合度70％时，用无血清的培养液饥饿24h，并通过如下调整葡萄糖浓度将细胞分为以下9组：①Low组(5.5mmol/L葡萄糖)、②High组(30mmol/L葡萄糖)、③高糖+化合物1组(30mmol/L葡萄糖+NO.1 10uM)、④高糖+化合物2组(30mmol/L葡萄糖+NO.2 10uM)、⑤高糖+化合物3组(30mmol/L葡萄糖+NO.3 10uM)、⑥高糖+化合物8组(30mmol/L葡萄糖+NO.8 10uM)、⑦高糖+化合物13组(30mmol/L葡萄糖+NO.13 10uM)、⑧高糖+化合物14组(30mmol/L葡萄糖+NO.14 10uM)和⑨高糖+化合物15组(30mmol/L葡萄糖+NO.15 10uM)，于37℃5％CO 2条件下培养48h，进行FN蛋白(纤连蛋白)的Western blot检测。如图2A所示，为本实施例中通过高糖诱导MCS系膜细胞和NRK肾上皮细胞，构建体外肾纤维化细胞模型，使用Western blot检测到的不同组的FN表达凝胶电泳图；如图2B所示，为本实施例3中通过高糖诱导MCS系膜细胞和NRK肾上皮细胞，构建体外肾纤维化细胞模型，检测到的不同组的FN表达情况柱状统计图。
根据前期的实验研究及以往文献提示，30mmol/L葡萄糖即可诱导肾小球系膜细胞增生肥大，以及肾上皮细胞转分化。Western blot检测结果显示，在高糖刺激下，High组FN蛋白的表达水平比对照Low组高；而给药组均能下调FN蛋白的表达，提示本发明的多肽化合物能够显著减少高糖导致的ECM的增多。其中，多肽化合物3、8、13、14和15抑制FN表达的作用更为显著。
实施例4多肽化合物3、8、13、14、15和16，索玛鲁肽，SAR425899，MED C18-acid对糖尿病小鼠的治疗作用
(一)多肽化合物3、8、13和15的药物不同浓度测定实验
获得db/db小鼠糖尿病模型(购自南京大学-南京生物医药研究院，8周左右，并测定血糖和体重保证后续实验顺利 进行)，对模型小鼠随机分成12组(包括多肽化合物3、8、13、15、索玛鲁肽和生理盐水组)，每组6只，基础体重和血糖无差异。每组老鼠每天或隔天分别皮下注射化合物3(80和120μg/kg，共2组)，化合物8(80和120μg/kg，共2组)，化合物13(80和120μg/kg，共2组)，化合物15(80和120μg/kg，共2组)，索玛鲁肽(80和120μg/kg，共2组)和生理盐水(DN组：同窝出生的正常小鼠；DC组：db/db小鼠对照组)。
在小鼠每天给药之后，隔天禁食6h测量小鼠的血糖和体重。
图3A显示，与生理盐水组DC组比较，索玛鲁肽组(120μg/kg)展现出了更好的降糖效果，之后维持在一个比较稳定的正常血糖水平，但其降体重效果不显著。给药组中多肽化合物3、8、13和15在80ug/kg的剂量下，多肽化合物3和8有明显的降糖效果，多肽化合物13和15降糖效果明显，并具有长效性；在120ug/kg的剂量下，多肽化合物3、8、13和15在3-5天已经达到了很好的降糖效果，且血糖稳定的维持在正常水平，同时多肽化合物3、8、13和15展现了更为优秀的降体重效果，且多肽化合物13和15的效果优于多肽化合物3和8。故药物浓度定为120ug/kg。
在小鼠隔天给药之后，隔天禁食6h测小鼠的血糖和体重。
图3B显示，与生理盐水组DC组比较，隔天给药后，索玛鲁肽组80μg/kg和120μg/kg均有降糖效果，但是降糖效果不明显，其降体重效果不显著。给药组中多肽化合物3和8，在80ug/kg的剂量下，多肽化合物3和8的血糖出现先降后升的现象，总体降糖效果明显，并且多肽化合物3和8具有降体重的效果。而多肽化合物13和15在第3天具有显著地降糖效果，但后续血糖水平变化不大，趋于稳定；在120ug/kg的剂量下，索玛鲁肽组在第3天血糖回升，其长效性不够。多肽化合物3、8、13和15在3-5天已经达到了很好的降糖效果，多肽化合物3和8之后血糖变化不大。多肽 化合物13和15一直保持着良好的降血糖效果，证明了多肽化合物13和15具有良好的降血糖长效性。同时多肽化合物3、8、13和15展现了更为优秀的降体重效果，且多肽化合物13和15的效果优于多肽化合物3和8。故药物浓度定为120ug/kg。
(二)多肽化合物3、8、13、14、15、16、索玛鲁肽、SAR425899和MED C18-acid对糖尿病小鼠的糖尿病改善作用
获得db/db小鼠糖尿病模型(购于南京大学-南京生物医药研究院，12周左右小鼠，并测定血糖和体重保证后续实验顺利进行)，对模型小鼠随机分成10组(多肽化合物3、8、13、14、15、16、索玛鲁肽、SAR425899、MED C18-acid和生理盐水组)，每组6只，每只小鼠的基础体重和血糖无差异，每组老鼠每天分别皮下注射多肽化合物3、8、13、14、15、16(120μg/kg)，索玛鲁肽(120μg/kg)，SAR425899(120μg/kg)，MED C18-acid(120μg/kg)，和生理盐水(DN组：同窝出生的正常小鼠；DC组：db/db小鼠对照组)。在小鼠每天给药之后，隔天禁食6h测小鼠的血糖和体重，一周测一次进水量和进食量。在第4周测OGTT，第5周测胰岛素耐量(ITT)，第6-7周使用小鼠代谢笼检测小鼠代谢情况，第8周取材，检测各种血清学指标和病理学指标。
1、药物对糖尿病小鼠的降糖降体重及胰岛素抵抗改善作用
II-型糖尿病模型，其表现特征为：肥胖、胰岛素抵抗、高糖血症、血脂异常和肝脏脂肪空泡样变性等。在多肽化合物3、8、13、14、15、16和索玛鲁肽，SAR425899，MED C18-acid治疗糖尿病小鼠4周和5周的时候，分别进行了口服葡萄糖耐受实验(OGTT)和胰岛素耐受测试(ITT)，结果如图4A-4D所示。
图4A至4C中的结果显示，与DN组相比，多肽化合物3、8、13、14、15、16、索玛鲁肽、SAR425899和MED C18-acid均有显著的降糖效果，在第1周的时候血糖已经降到正常水 平，且此后血糖相对稳定。但是，在改善糖尿病小鼠口服葡萄糖耐受能力(OGTT)及胰岛素抵抗(ITT)方面多肽化合物3、8、13、14、15、16表现更为优秀。
此外，图4D的结果显示，多肽化合物3、8、13、14、15、16具有更为优秀的肝脏保护功能。
2、药物对糖尿病小鼠的糖尿病肾病治疗作用
在药物治疗6周之后，使用代谢笼收集小鼠尿液，使用相关试剂盒检测尿液中的蛋白含量。在给药7周之后，取材，血液用于检测血肌酐、尿素氮等血清学指标。结果如图5A至5F所示。
由图5A可知，20周龄的db/db糖尿病小鼠DC组相比对照DN组，蛋白尿含量以及尿糖含量均明显增加，此外，糖化血红蛋白含量、血肌酐以及尿素氮含量也明显增加，说明小鼠的糖尿病肾病已经很严重。在给药之后，小鼠的蛋白尿含量、尿糖含量、糖化血红蛋白含量、血肌酐以及尿素氮含量均有不同程度的改善。
取小鼠肾脏进行石蜡包埋，用于后续的染色及免疫组化研究。根据图5B小鼠肾脏H&E染色结果可知，小鼠肾脏组织出现局部的炎症，肾小球黏连现象等。给药之后，肾小球黏连现象得到缓解，相比于阳性对照药物索玛鲁肽、SAR425899和MED C18-acid组，多肽化合物3、8、13、14、15和16的改善和治疗作用更为显著。一般来说，肾脏病变的观察多集中在皮质，主要包括肾小球和肾间质，小球的观察集中在系膜区。由PAS染色(图5C)可以观察到，相比于DN组，DC组肾小球系膜区增宽，系膜细胞和系膜基质增生，基底膜增厚，给药之后，肾小球血管袢薄而清晰，内皮细胞和系膜细胞数目正常，周围的肾小管也正常，多肽化合物3、8、13、14、15和16的改善作用更为显著。通过图5D至5F中天狼新红以及FN、α-SMA染色可知，相比于DN组，DC组胶原沉积明显增加，药物组可显著降低胶原含量，其中多肽化合物3、8、13、14、15和16改善作用更为显著，其治疗效果优于阳性对照索玛鲁肽，SAR425899和MED  C18-acid组。
总结，通过对氨基酸序列的进一步优化和设计，同时侧链技术的修饰。本发明报道的化合物在降糖，降体重，改善和治疗糖尿病并发症方面优于之前报道的化合物，在临床应用方面更具有优势。
以上对本发明具体实施方式的描述并不限制本发明，本领域技术人员可以根据本发明作出各种改变或变形，只要不脱离本发明的精神，均应属于本发明所附权利要求的范围。

Claims (15)

  1. 一种多肽化合物，其含有以下氨基酸序列表示的母体肽：
    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，
    其中：
    Xaa2＝Aib，Ser或D-Ser；
    Xaa15＝Asp或Glu；
    Xaa16＝Aib或Glu；
    Xaa17＝Lys或Arg；
    Xaa21＝Asp或Glu；
    Xaa23＝Val或Iva；
    Xaa24＝Glu或Gln；
    Xaa27＝Leu或Lys；
    Xaa28＝Asp，Glu或Ala；
    Xaa35＝Ala或Aib。
  2. 根据权利要求1所述的多肽化合物，其特征在于，所述Xaa2＝Aib或D-Ser。
  3. 根据权利要求2所述的多肽化合物，其特征在于，所述Xaa21＝Asp。
  4. 根据权利要求3所述的多肽化合物，其特征在于，所述母体肽的氨基酸序列的羧基端不经修饰，或者经氨基修饰形成-CONH 2基团。
  5. 根据权利要求1至4任一项所述的多肽化合物，其特征在于，所述母体肽的氨基酸序列中第10位或第12位Lys的侧链经由桥接基团与亲脂性取代基连接；所述桥接基团为(PEG) m、(PEG) m-γGlu、(PEG) m-Asp、(Gly) x-(Gly-Ser) y-(Gly) z-、(Gly) x-(Gly-Ser) y-(Gly) z-γGlu和(Gly) x-(Gly-Ser) y-(Gly) z-Asp中的一种；所述连接方式为所述第10位或第12位Lys的侧链氨基与所述桥接基团一端的甘氨酸残基的羧基或(PEG) m末端修饰的羧基形成酰胺键，且所述亲脂性取代基以其羧基与另一端桥接基团的氨基通过形成酰胺键相连；所述亲脂性取代基为CH 3(CH 2) nC(O)-或HOOC(CH 2) nC(O)-且其酰基与所述桥接基团中的氨基形成酰胺键；其中，m 为2-10的整数；n为14-20的整数；x为0-5的整数；y为1-5的整数；z为1-5的整数。
  6. 根据权利要求1所述的多肽化合物，其特征在于，所述母体肽的氨基酸序列中第10位或第12位Lys被HomoLys、Orn、Dap或Dab代替。
  7. 根据权利要求1所述的多肽化合物，其特征在于，所述母体肽的氨基酸序列中：
    Xaa2＝Aib或D-Ser；
    Xaa15＝Glu；
    Xaa16＝Aib；
    Xaa17＝Lys；
    Xaa21＝Asp；
    Xaa23＝Val；
    Xaa24＝Glu；
    Xaa27＝Lys；
    Xaa28＝Ala；
    Xaa35＝Ala。
  8. 根据权利要求1所述的多肽化合物，其特征在于，所述母体肽的氨基酸序列中：
    Xaa2＝Aib或D-Ser；
    Xaa15＝Asp；
    Xaa16＝Glu；
    Xaa17＝Arg；
    Xaa21＝Asp；
    Xaa23＝Iva；
    Xaa24＝Gln；
    Xaa27＝Leu；
    Xaa28＝Asp或Glu；
    Xaa35＝Ala或Aib。
  9. 根据权利要求1所述的多肽化合物，其特征在于，所述母体肽的氨基酸序列选自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和SEQ ID NO.12、SEQ ID NO.13、SEQ ID  NO.14、SEQ ID NO.15和SEQ ID NO.16所示的氨基酸序列。
  10. 根据权利要求5所述的多肽化合物，其特征在于，所述母体肽的氨基酸序列中第10位或第12位Lys经由桥接基团与亲脂性取代基相连形成的结构为：
    Figure PCTCN2020137071-appb-100001
    Figure PCTCN2020137071-appb-100002
  11. 根据权利要求1所述的多肽化合物，其特征在于，所述多肽化合物为如下化合物中的任一种：
    化合物1：
    H-Aib-QGTFTSDK(PEG 2-PEG 2-γGlu-CO(CH 2) 18CO 2H)SKYLE-Aib-KAAQDFVEWLKAGGPSSGAPPPS-NH 2
    化合物2：
    HsQGTFTSDK(PEG 2-PEG 2-CO(CH 2) 18CO 2H)SKYLE-Aib-KAAQDFVEWLKAGGPSSGAPPPS-NH 2
    化合物3：
    HsQGTFTSDK(PEG 2-PEG 2-γGlu-CO(CH 2) 18CO 2H)SKYLE-Aib-KAAQDFVEWLKAGGPSSGAPPPS-NH 2
    化合物4：
    HsQGTFTSDK(PEG 2-PEG 2-γGlu-CO(CH 2) 16CO 2H)SKYLE-Aib-KAAQDFVEWLKAGGPSSGAPPPS-NH 2
    化合物5：
    HsQGTFTSDK(PEG 2-PEG 2-γGlu-CO(CH 2) 18CH 3)SKYLE-Aib-KAAQDFVEWLKAGGPSSGAPPPS-NH 2
    化合物6：
    HsQGTFTSDK(PEG 2-PEG 2-γGlu-CO(CH 2) 18CO 2H)SKYLDERAAQDF-Iva-QWLLDGGPSSG-Aib-PPPS-NH 2
    化合物7：
    HsQGTFTSDK(PEG 2-PEG 2-γGlu-CO(CH 2) 18CO 2H)SKYLDERAAQDF-Iva-QWLLEGGPSSGAPPPS-NH 2
    化合物8：
    H-Aib-QGTFTSDK(PEG 2-PEG 2-γGlu-CO(CH 2) 18CO 2H)SKYLDERAAQDF-Iva-QWLLDGGPSSG-Aib-PPPS-NH 2
    化合物9：
    H-Aib-QGTFTSDK(PEG 2-PEG 2-γGlu-CO(CH 2) 16CO 2H)SKYLDERAAQDF-Iva-QWLLDGGPSSG-Aib-PPPS-NH 2
    化合物10：
    H-Aib-QGTFTSDK(PEG 2-PEG 2-γGlu-CO(CH 2) 18CH 3)SKYLDERAAQDF-Iva-QWLLDGGPSSG-Aib-PPPS-NH 2
    化合物11：
    H-Aib-QGTFTSDK(PEG 2-PEG 2-γGlu-CO(CH 2) 18CH 3)SKYLDERAAQDF-Iva-QWLLDGGPSSG-Aib-PPPS-NH 2
    化合物12：
    HsQGTFTSDK(PEG 2-PEG 2-γGlu-CO(CH 2) 18CO 2H)SKYLDERAAQDF-Iva-QWLLDGGPSSG-Aib-PPPS-NH 2
    化合物13：
    HsQGTFTSDK(GGSGSG-γGlu-CO(CH 2) 18-COOH)SKYLE-Aib-KAAQDFVEWLKAGGPSSGAPPPS
    化合物14：
    HsQGTFTSDK(GGSGSG-γGlu-CO(CH 2) 18-COOH)SKYLE-Aib-KAAQDFVEWLKAGGPSSGAPPPS-NH 2
    化合物15：
    H-Aib-QGTFTSDK(GGSGSG-γGlu-CO(CH 2) 18-COOH)SKYLDERAAQDF-Iva-QWLLDGGPSSG-Aib-PPPS
    化合物16：
    H-Aib-QGTFTSDK(GGSGSG-γGlu-CO(CH 2) 18-COOH)SKYLDERAAQDF-Iva-QWLLDGGPSSG-Aib-PPPS-NH 2。
  12. 一种组合物，其包含权利要求1至11任一项所述的多肽化合物。
  13. 根据权利要求12所述的组合物，其特征在于，所述组合物为药物组合物，还包含药学上可接受的载体或辅料。
  14. 一种权利要求1至11任一项所述的多肽化合物或者权利要求12至13任一项所述的组合物在制备预防或治疗糖尿病和/或糖尿病并发症的药物中的应用；所述糖尿病并发症为糖尿病肾病。
  15. 一种权利要求1至11任一项所述的多肽化合物或者权利要求12至13任一项所述的组合物在制备降低体重的保健品或药物中的应用。
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