WO2022111370A1 - Glp-1/glucagon receptor dual-agonist and application thereof - Google Patents

Glp-1/glucagon receptor dual-agonist and application thereof Download PDF

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WO2022111370A1
WO2022111370A1 PCT/CN2021/131369 CN2021131369W WO2022111370A1 WO 2022111370 A1 WO2022111370 A1 WO 2022111370A1 CN 2021131369 W CN2021131369 W CN 2021131369W WO 2022111370 A1 WO2022111370 A1 WO 2022111370A1
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gly
pro
lys
asp
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韩京
孟庆华
杨启萌
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江苏师范大学
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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates to biomedicine, in particular to a class of GLP-1/glucagon receptor dual agonists and applications thereof.
  • T2DM type 2 diabetes mellitus
  • NAFLD nonalcoholic fatty liver disease
  • NASH nonalcoholic steatohepatitis
  • dyslipidemia The incidence and course of the disease are closely related to obesity. Studies have shown that 80-90% of T2DM patients are clinically overweight or obese.
  • the use of weight loss therapy is beneficial to prevent and control the disease, including controlling blood sugar, reducing morbidity and disability (death) rates. It is generally difficult to achieve ideal weight loss through exercise and diet control alone.
  • the body's energy and blood sugar regulation signaling system includes a variety of different polypeptides.
  • Endogenous gastrointestinal hormones proglucagon (proglucagon) is a 160 amino acid precursor polypeptide, which is cleaved in different tissues. Converted into different products, such as GLP-1, glucagon-like peptide-2 (GLP-2), glucagon (Glucagon, GCG) and oxyntomodulin (Oxyntomodulin, OXM) and other endogenous gastric gut hormones.
  • GLP-1 glucagon-like peptide-2
  • Glucagon Glucagon
  • GCG glucagon
  • Oxyntomodulin oxyntomodulin
  • GLP-1 is a glucose-dependent hypoglycemic polypeptide hormone secreted by L cells in the terminal jejunum, ileum and colon, and exerts a hypoglycemic effect after specifically binding to the GLP-1 receptor.
  • the main advantage of GLP-1 is its glucose-dependent incretin secretion, which avoids the risk of hypoglycemia that is often present in the treatment of diabetes.
  • GLP-1 can also prevent pancreatic ⁇ -cell degeneration, stimulate ⁇ -cell proliferation and differentiation, and improve diabetes progression at the source.
  • GLP-1 also has the effects of inhibiting gastric acid secretion, delaying gastric emptying, and suppressing appetite, and has partial weight loss effects.
  • GLP-1 drugs such as liraglutide, semaglutide and dulaglutide.
  • GLP-1 drugs have a safe hypoglycemic effect, if you need to achieve better weight loss effect, it is generally necessary to increase the dosage, and large doses of GLP-1 drugs are prone to gastrointestinal side effects, which are tolerated The poor performance results in a narrow therapeutic window. Therefore, there remains a need for safer and more tolerated therapeutic agents that are effective for weight loss and glycemic control.
  • GCG is a hormone produced in the alpha cells of the pancreas. It acts on the liver under stress conditions such as cold and starvation, and decomposes glycogen in the liver to increase blood sugar.
  • GCG In addition to its blood sugar-raising effect, GCG also has the effect of promoting lipolysis, fat oxidation, and fever in the body (Diabetologia, 2017, 60, 1851–1861). However, these beneficial effects of GCG on energy metabolism have not been applied due to its inherent hypoglycemic effect.
  • OXM is an endogenous GLP-1 receptor and GCG receptor dual agonist in the human body, and its agonistic activity on GLP-1 receptor and GCG receptor is weaker than the natural ligand of each receptor (natural GLP). -1 or GCG).
  • the acute physiological effects of OXM include inhibition of gastric emptying, food intake and exocrine secretion of the stomach and pancreas, enhancement of resting energy expenditure, etc., resulting in weight loss.
  • intraperipheral administration of OXM in animals and humans reduces body weight and food intake, and in obese subjects increases metabolic rate and activity-related energy expenditure.
  • large doses of OXM are less prone to common gastrointestinal side effects such as nausea and vomiting while reducing body weight.
  • the reported polypeptide GLP-1/GCG receptor dual agonists can be divided into four categories based on GCG, OXM, GLP-1 or exendin-4 (exendin-4) according to their sequence structure. ⁇ :CN201911103118.6 ⁇ CN201780013643.1 ⁇ CN201680021972.6 ⁇ CN201580030150.X ⁇ CN201380048137.8 ⁇ WO2008/071972 ⁇ WO 2008/101017 ⁇ WO 2009/155258 ⁇ WO 2010/096052 ⁇ WO 2010/096142 ⁇ WO2011/ 075393, WO 2008/152403, WO 2010/070251, WO 2010/070252, WO 2010/070253, WO2010/070255, WO 2011/160630, WO 2011/006497, WO 2011/0876171, WO2111/08 WO2011/117416, WO 2012/177443, WO 2012/177444, WO 2012/150503, WO2013/004983, WO 2013/092703, WO 2014/041195 and WO 2014/041375
  • GLP-1/GCG/GIP receptor triple agonists that activate not only the GLP-1 receptor and GCG receptor, but also the glucose-dependent insulinotropic polypeptide (GIP) receptor.
  • GIP glucose-dependent insulinotropic polypeptide
  • XenGLP-1 is a class of animal-derived GLP-1 analogs found in Xenopus laevis. Compared with natural GLP-1, XenGLP-1 has better hypoglycemic activity and stability. Furthermore, in addition to being more resistant to degradation by dipeptidyl peptidase (DPP-IV) compared to GLP-1, OXM and GCG, XenGLP-1 also showed stable degradation against neutral endopeptidase (NEP). many.
  • DPP-IV dipeptidyl peptidase
  • NEP neutral endopeptidase
  • XenGLP-1 is a potent agonist of the GLP-1 receptor, however it does not activate the GCG receptor.
  • XenGLP-1 has many of the glucose-regulating effects observed with native GLP-1, and many preclinical studies have shown that XenGLP-1 has several beneficial antidiabetic properties, including glucose-dependent enhancement of insulin synthesis and secretion, slowed gastric emptying , food intake and weight loss, as well as promoting ⁇ -cell proliferation and restoring islet function, etc. (Biochem. Pharmacol., 2017, 142, 155-167; FASEB J., 2019, 33, 7113-7125). These effects are beneficial not only for diabetics, but also for patients suffering from obesity. Patients with obesity have a higher risk of developing hypertension, hyperlipidemia, diabetes, NAFLD, NASH, musculoskeletal and cardiovascular disease.
  • the object of the present invention is to provide a novel polypeptide compound with dual agonistic effect of GLP-1/GCG receptor, the polypeptide is a variant designed based on the sequence of XenGLP-1, which retains the therapeutic effect of XenGLP-1 on diabetes while having The beneficial effects of GCG on lipid metabolism and energy metabolism, resulting in synergistic effects on sugar, lipid, and energy metabolism, are more useful than single receptor agonists in the preparation of drugs for the treatment of metabolic syndrome, such as obesity, diabetes, NAFLD, NASH and other diseases more potential.
  • a class of GLP-1/GCG receptor dual agonist polypeptide compounds the general amino acid sequence formula of this class of GLP-1 receptor/GCG receptor dual agonist polypeptide compounds is:
  • Xaa 1 is taken from Ser, D-Ser or Aib;
  • Xaa 2 is taken from Glu or Gln;
  • Xaa 3 is taken from Thr or Ser
  • Xaa 4 is taken from Glu, Lys or Lys whose side chain is modified;
  • Xaa 5 is taken from Leu, Lys or Lys whose side chain is modified;
  • Xaa 6 is taken from Glu or Asp
  • Xaa 7 is taken from Glu or Ser
  • Xaa 8 is taken from Glu or Arg
  • Xaa 9 is taken from Ala or Arg
  • Xaa 11 is taken from Glu or Asp
  • Xaa 12 taken from Ile or Lys
  • Xaa 15 is taken from -NH 2 or Lys whose side chain is modified;
  • the Lys in which the side chain is modified is selected from Lys( ⁇ -Glu-CO-(CH 2 ) n -CH 3 ) or Lys(AEEA-AEEA- ⁇ -Glu-CO-(CH 2 ) n -COOH),
  • Lys( ⁇ -Glu-CO-(CH 2 ) n -CH 3 ) The structural formula of Lys( ⁇ -Glu-CO-(CH 2 ) n -CH 3 ) is shown below:
  • n is a natural number, and 12 ⁇ n ⁇ 20.
  • the n is 14, 16, 18 or 20.
  • amino acid sequence of the polypeptide compound is one of the following sequences:
  • the present invention also provides pharmaceutically acceptable salts of a class of GLP-1/GCG receptor dual agonist polypeptide compounds.
  • the salt is a salt formed by a GLP-1/GCG receptor dual agonist polypeptide compound and one of the following compounds: hydrobromic acid, hydrochloric acid, methanesulfonic acid, phosphoric acid, ethanesulfonic acid, formic acid, p-toluenesulfonic acid, acetic acid, acetoacetic acid, pyruvic acid, pectic acid, butyric acid, caproic acid, benzenesulfonic acid, heptanoic acid, undecanoic acid, benzoic acid, salicylic acid, lauric acid, 2-(4 -Hydroxybenzoyl)benzoic acid, cinnamic acid, camphoric acid, cyclopentanepropionic acid, 3-hydroxy-2-naphthoic acid, camphorsulfonic acid, digluconic acid, niacin, pamoic acid, propionic acid, persulfuric acid ,,,
  • the present invention also provides a pharmaceutical composition of the GLP-1/GCG receptor dual agonist polypeptide compound, the pharmaceutical composition comprising: using any of the above GLP-1/GCG receptor dual agonist polypeptide compounds or a pharmaceutically acceptable salt thereof It is composed of effective raw materials, together with a pharmaceutically acceptable carrier or diluent.
  • the present invention also provides a medicament containing the above-mentioned GLP-1/GCG receptor dual agonist polypeptide compound, the medicament is any of the capsules, tablets, sprays, inhalants, injections, patches mentioned in pharmacy , emulsion, film, powder or compound preparation, the medicament is composed of GLP-1/GCG receptor dual agonist polypeptide compound and pharmaceutically acceptable pharmaceutical excipients, carriers or diluents.
  • the present invention also provides the GLP-1/GCG receptor dual agonist polypeptide compound of the present invention, its pharmaceutically acceptable salt, its pharmaceutical composition or its medicament in the preparation of a medicament for the treatment of metabolic diseases or disorders Applications.
  • the metabolic disease or disorder is diabetes, NAFLD, NASH, hyperlipidemia or obesity.
  • the diabetes is type 1 diabetes, T2DM or gestational diabetes.
  • the medicament is for the treatment of more than one metabolic disease or disorder, eg, diabetes and NAFLD, NASH or obesity; obesity and NASH or NAFLD; diabetes, NASH and obesity; diabetes, NAFLD and obesity; or diabetes and obesity .
  • the GLP-1/GCG receptor dual agonist polypeptide compound of the present invention has the functions of promoting weight loss and preventing weight gain while lowering blood sugar more effectively, and reversing insulin resistance. , modulates lipid metabolism with unexpected beneficial effects compared with existing drugs.
  • the agonistic activity of the polypeptide compound of the present invention on GLP-1 receptor and GCG receptor is higher than that of the natural ligands of each receptor, while the agonistic activity on GIP receptor is lower.
  • the polypeptide compound provided by the invention has stable chemical properties, is not easily degraded by DPP-IV and NEP in the body, and is not easily filtered by the glomerulus. Generation dynamics characteristics.
  • the polypeptide compound provided by the present invention has improved biophysical properties, and the solubility at neutral pH and pH 4.5 is higher than that of natural GLP-1 and GCG, and has properties that are beneficial to formulations.
  • the polypeptide compound provided by the invention has low immunogenicity, and the therapeutic effect on metabolic diseases such as T2DM, obesity, NAFLD, NASH and hyperlipidemia is better than that of the existing marketed drugs. Therefore, the polypeptide compound provided by the present invention is suitable as an active ingredient of medicines for the treatment of metabolic diseases, such as diabetes, obesity, hyperlipidemia, NAFLD, NASH and the like.
  • Figure 1 shows the long-acting hypoglycemic effect of a single dose of each test substance in db/db mice in a non-fasted state
  • Figure 2 shows the hypoglycemic effect of each test substance in the oral glucose tolerance test after long-term administration of DIO mice for 21 days;
  • Figure 3 shows the in vitro immunogenicity of each test substance.
  • the 12-position Lys can be Fmoc-Lys(Alloc)-OH, Fmoc-Lys(Dde)-OH, Fmoc-Lys(Mtt)-OH or Fmoc-Lys(ivDde)-OH, etc.
  • the Fmoc-Lys(Dde)-OH protection strategy was adopted, and the N-terminal His was Boc-His(Boc)-OH.
  • the crude target polypeptide was dissolved in water, filtered with a 0.25 ⁇ m microporous membrane, and then purified by a Shimadzu preparative reverse-phase HPLC system. Chromatographic conditions were C18 reversed-phase preparative column (250mm ⁇ 20mm, 12 ⁇ m); mobile phase A: 0.1% TFA/water (V/V), mobile phase B: methanol (V/V); flow rate was 8mL/min; detection wavelength is 214nm. A linear gradient (20%B ⁇ 70%B/30min) was used for elution, the target peak was collected, and 0.14g of pure product was obtained by lyophilization after removing methanol. The purity was greater than 98%. The molecular weight of the target polypeptide was confirmed by LC-MS.
  • the synthetic method was the same as that of Example 1, and the target peak was collected and lyophilized to obtain 0.16 g of pure product.
  • the synthesis method was the same as that in Example 1, and the target peak was collected and lyophilized to obtain 0.13 g of pure product.
  • the 12-position Lys can be Fmoc-Lys(Alloc)-OH, Fmoc-Lys(Dde)-OH, Fmoc-Lys(Mtt)-OH or Fmoc-Lys(ivDde)-OH, etc.
  • the Fmoc-Lys(Dde)-OH protection strategy was adopted, and the N-terminal His was Boc-His(Boc)-OH.
  • the crude target polypeptide was dissolved in water, filtered through a 0.25 ⁇ m microporous membrane, and then purified by a Shimadzu preparative reverse-phase HPLC system. Chromatographic conditions were C18 reversed-phase preparative column (250mm ⁇ 20mm, 12 ⁇ m); mobile phase A: 0.1% TFA/water (V/V), mobile phase B: methanol (V/V); flow rate was 8mL/min; detection wavelength is 214nm. A linear gradient (20%B ⁇ 80%B/30min) was used for elution, the target peak was collected, and 0.18g of pure product was obtained by lyophilization after removing methanol. The purity was greater than 98%. The molecular weight of the target polypeptide was confirmed by LC-MS.
  • the synthetic method was the same as that in Example 4, and the target peak was collected and lyophilized to obtain 0.17g of pure product.
  • the synthetic method was the same as that in Example 4, and the target peak was collected and lyophilized to obtain 0.15 g of pure product.
  • the synthesis method was the same as that of Example 1, and the target peak was collected and lyophilized to obtain 0.15 g of pure product.
  • the synthetic method was the same as that of Example 1, and the target peak was collected and lyophilized to obtain 0.16 g of pure product.
  • the synthesis method was the same as that in Example 1, and the target peak was collected and lyophilized to obtain 0.14 g of pure product.
  • the synthetic method was the same as that in Example 4, and the target peak was collected and lyophilized to obtain 0.17g of pure product.
  • the synthetic method was the same as that in Example 4, and the target peak was collected and lyophilized to obtain 0.16 g of pure product.
  • the synthetic method was the same as that of Example 4, and the target peak was collected and lyophilized to obtain 0.14g of pure product.
  • the synthetic method was the same as that of Example 1, and the target peak was collected and lyophilized to obtain 0.13 g of pure product.
  • the synthetic method was the same as that of Example 1, and the target peak was collected and lyophilized to obtain 0.15 g of pure product.
  • the synthetic method was the same as that of Example 1, and the target peak was collected and lyophilized to obtain 0.16 g of pure product.
  • the synthesis method was the same as that in Example 4, and the target peak was collected and lyophilized to obtain 0.15 g of pure product.
  • the synthetic method was the same as that in Example 4, and the target peak was collected and lyophilized to obtain 0.16 g of pure product.
  • the synthesis method was the same as that in Example 4, and the target peak was collected and lyophilized to obtain 0.15 g of pure product.
  • the agonistic effect of polypeptide compounds on the receptor is determined by functional assays that measure the cAMP response of HEK-293 cell lines stably expressing the human GLP-1 receptor, GCG receptor or GIP receptor.
  • Cells stably expressing the above three receptors were split into T175 flasks and grown overnight in medium (DMEM/10% FBS) to near confluence, then the medium was removed and cells were washed with PBS without calcium and magnesium, then Protease treatment with Accutase enzyme.
  • Detached cells were washed and resuspended in assay buffer (20 mM HEPES, 0.1% BSA, 2 mM IBMX, 1 x HBSS) and cell density was determined and aliquots of 25 ⁇ L were aliquoted into 96-well plates. in the hole. For measurements, 25 [mu]L of a solution of the test polypeptide compound in assay buffer was added to the wells, followed by incubation at room temperature for 30 minutes. The cAMP content of cells was determined based on homogeneous time-resolved fluorescence (HTRF) using a kit from Cisbio.
  • assay buffer 20 mM HEPES, 0.1% BSA, 2 mM IBMX, 1 x HBSS
  • HTRF reagent diluted in lysis buffer (kit component)
  • the plate was incubated for 1 hour and then the fluorescence ratio at 665/620 nm was measured.
  • the in vitro potency of agonists is quantified by measuring the concentration that elicits 50% activation of the maximal response ( EC50 ).
  • the detection data (nM) in the examples of the present patent application are shown in Table 1 below. Although the detection data is stated with a certain number of significant figures, it should not be considered that the data has been determined to be exactly the number of significant figures.
  • Table 1 EC 50 values of polypeptide compounds for human GLP-1 receptor, GCG receptor and GIP receptor (expressed in nM)
  • SEQ ID NO: 13 0.049 0.059 2869.9
  • SEQ ID NO: 14 0.035 0.062 3215.6
  • SEQ ID NO: 15 0.061 0.052 3015.3
  • SEQ ID NO: 16 0.077 0.060 3225.6
  • SEQ ID NO: 17 0.075 0.056 3568.6
  • SEQ ID NO: 18 0.072 0.061 3348.5
  • the agonistic activity of all polypeptide compounds on GLP-1 receptor is higher than that of natural GLP-1, and the agonistic activity of most polypeptide compounds on GCG receptor is also higher than that of natural GCG. Shows weaker agonistic activity of GIP receptors.
  • SEQ ID NO: 6 94 100 >8 >8 SEQ ID NO: 7 93 100 >8 >8 SEQ ID NO: 8 95 98 >8 >8 SEQ ID NO: 9 90 99 >8 >8 SEQ ID NO: 10 89 99 >8 >8 SEQ ID NO: 11 88 100 >8 >8 SEQ ID NO: 12 93 98 >8 >8 SEQ ID NO: 13 95 97 >8 >8 SEQ ID NO: 14 94 100 >8 >8 SEQ ID NO: 15 92 100 >8 >8 SEQ ID NO: 16 94 99 >8 >8 SEQ ID NO: 17 90 98 >8 >8 SEQ ID NO: 18 88 100 >8 >8 >8
  • the solubility of the polypeptide compound of the present invention is greatly improved under the pH condition of the injection solution acceptable to the body, and has the characteristics that are beneficial to the formulation.
  • the polypeptide compounds of the present invention also have high solubility at pH 4.5, a property that may allow co-formulation for combination therapy with insulin or insulin derivatives.
  • the polypeptide compounds of the present invention also have high stability under pH 4.5 and neutral pH conditions.
  • test sample was incubated with purified human DPP-IV or NEP enzyme at 37°C for 0, 2, 4, and 8 hours.
  • the peak area of the residual sample in the solution at each time point was measured by HPLC, and the half-life of the sample was calculated. The results are shown in the table. 3 shown.
  • SEQ ID NO: 7 >8 >8 SEQ ID NO: 8 >8 >8 SEQ ID NO: 9 >8 >8 SEQ ID NO: 11 >8 >8 SEQ ID NO: 12 >8 >8 SEQ ID NO: 13 >8 >8 SEQ ID NO: 14 >8 >8 SEQ ID NO: 15 >8 >8 SEQ ID NO: 17 >8 >8 SEQ ID NO: 18 >8 >8
  • Rats were administered a subcutaneous (s.c.) injection of 50 nmol/kg and blood samples were collected at 0.25, 0.5, 1, 2, 4, 8, 16, 24, 36 and 48 hours post-dose. Plasma samples were analyzed by LC-MS after protein precipitation using acetonitrile. Pharmacokinetic parameters and half-life were calculated using WinonLin 5.2.1 (non-compartmental model) (Table 4).
  • SEQ ID NO: 8 3.9 539 SEQ ID NO: 9 4.6 551 SEQ ID NO: 11 11.5 541 SEQ ID NO: 12 12.6 561
  • the in vivo half-life of the polypeptide compound of the present invention is significantly prolonged, and has the pharmacokinetic characteristics supporting once-daily or once-weekly administration.
  • mice Male db/db mice were randomly divided into groups of 6 mice. The blank group was subcutaneously injected with normal saline (10 mg/kg), and the administration group was divided into 6 groups. During the experiment, the mice were free to eat and drink, and the mice were subcutaneously injected with 25 nmol/kg of liraglutide, semaglutide, SEQ in a non-fasting state. ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12. Blood glucose levels of mice in each group were measured with a blood glucose meter at 0 h before administration and at 4, 6, 24 and 48 h after administration.
  • the results of the hypoglycemic experiments in db/db mice show that the polypeptide compounds of the present invention show a long-acting hypoglycemic activity superior to that of the positive control drugs liraglutide and semaglutide.
  • mice Male C57BL/6J mice, weighing about 22 g, with a total of 42 models in the model group, were fed with D12492 high-fat diet from Research Diets for 18 weeks to establish the DIO mouse model.
  • the blank control group was fed with standard rat chow (control standard diet group).
  • the DIO mice in each group were randomly divided into 7 groups according to their body weight, with 6 mice in each group, namely the normal saline group (control high-fat diet group), the positive control group (liraglutide and semaglutide) and the control group.
  • Test sample group SEQ ID NO: 8, 9, 11, 12).
  • control standard diet group and the control high-fat diet group were subcutaneously injected twice a day with normal saline (10mg/kg), liraglutide, SEQ ID NO: 8, and SEQ ID NO: 9 groups were subcutaneously injected twice a day (25nmol/kg), semaglutide,
  • SEQ ID NO: 11 and SEQ ID NO: 12 groups were subcutaneously injected (25 nmol/kg) once a day, and the dosing cycle was 21 days.
  • the body weight changes of the mice were recorded every day, and nuclear magnetic resonance (NMR) was used to measure body fat before and at the end of the experiment.
  • NMR nuclear magnetic resonance
  • mice in each group were fasted for 12 h and then orally administered glucose (1.5 g/kg) and The blood glucose values of each group of mice were measured at 15, 30, 60 and 120 min after glucose administration using a blood glucose meter (Table 5).
  • the results of the hypoglycemic experiment show that the polypeptide compound of the present invention exhibits hypoglycemic activity equivalent to that of the positive control drugs liraglutide and semaglutide.
  • mice Male db/db mice were randomly divided into groups of 6 mice. After one week of adaptive feeding, tail blood was taken to measure the initial HbA1c value and fasting blood glucose value before the start of treatment.
  • the blank group was subcutaneously injected with normal saline (10 mg/kg) twice a day, and the administration group was divided into 6 groups. twice daily), SEQ ID NO: 9 (twice daily), SEQ ID NO: 11 (once daily), SEQ ID NO: 12 (once daily).
  • the treatment period was 5 weeks. After the treatment, the mice were fasted overnight to measure the fasting blood glucose value, and at the same time, blood was taken to measure the HbA1c (%) value (Tables 6 and 7).
  • PBMCs peripheral blood mononuclear cells
  • Stimulation index was calculated by dividing the proliferative response (cpm) for each donor's test wells by the proliferative response to the medium treatment (cpm), with an SI greater than 2.0 considered positive.
  • Donor response percentages were calculated by dividing the number of positive donors over the entire time course (5-8 days) as a percentage of the total number of donors tested.
  • the donor response ratio of the polypeptide compound of the present invention is lower than that of liraglutide and semaglutide, indicating that the polypeptide compound of the present invention has low immunogenicity.

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Abstract

Provided are a GLP-1/glucagon (GCG) receptor dual-agonist polypeptide compound. The GLP-1/GCG receptor dual-agonist polypeptide compound has the functions of promoting weight reduction and preventing weight gain while lowering blood glucose, reversing insulin resistance, and regulating lipid metabolism. The polypeptide compound has higher agonist activity on a GLP-1 receptor and a GCG receptor than natural ligands for the receptors, and has lower agonist activity on a GIP receptor. The polypeptide compound provided in the present invention has stable chemical properties and low immunogenicity, and is suitable for being used as an active ingredient of drugs for treating metabolic diseases, such as diabetes, obesity, hyperlipidemia, NAFLD, and NASH.

Description

一类GLP-1/胰高血糖素受体双重激动剂及其应用A class of GLP-1/glucagon receptor dual agonists and their applications 技术领域technical field
本发明涉及生物医药，具体涉及一类GLP-1/胰高血糖素受体双重激动剂及其应用。The invention relates to biomedicine, in particular to a class of GLP-1/glucagon receptor dual agonists and applications thereof.
背景技术Background technique
肥胖及其相关代谢综合征已成为全球性的公众健康问题，许多代谢综合征如2型糖尿病(T2DM)、非酒精性脂肪肝病(NAFLD)、非酒精性脂肪肝炎(NASH)、血脂代谢异常的发病率与病程发展都与肥胖密切相关。研究表明，临床上80-90％的T2DM患者伴有超重或肥胖，使用减重疗法有利于预防和控制病情，包括控制血糖、减少患病率和致残(死)率等。仅靠锻炼和饮食控制来减轻体重，一般很难达到理想的减重效果。目前治疗肥胖的药物疗效较为有限，许多治疗肥胖的药物还具有较显著的副作用，如作用于中枢神经引起的精神症状和严重的心血管影响等副作用。目前仅有少数药物单独使用能实现5-10％的体重降幅，在治疗T2DM的药物中，仅有胰高血糖素样肽(GLP-1)受体激动剂和钠-葡萄糖共转运蛋白2(SGLT2)抑制剂具有较好的体重控制效果(J.Med.Chem.,2018,61,5580-5593)。减肥手术对肥胖的治疗效果显著，但是患者所遭受的手术风险较大，并且手术的长期效应仍不明确。因此，用于体重控制的药物目前仍存在巨大的临床需求，能够安全有效地控制体重兼具原发病症治疗作用的药物是理想的选择。Obesity and its associated metabolic syndrome have become a global public health problem, and many metabolic syndromes such as type 2 diabetes mellitus (T2DM), nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), dyslipidemia The incidence and course of the disease are closely related to obesity. Studies have shown that 80-90% of T2DM patients are clinically overweight or obese. The use of weight loss therapy is beneficial to prevent and control the disease, including controlling blood sugar, reducing morbidity and disability (death) rates. It is generally difficult to achieve ideal weight loss through exercise and diet control alone. At present, the efficacy of drugs for the treatment of obesity is relatively limited, and many drugs for the treatment of obesity also have significant side effects, such as psychiatric symptoms and severe cardiovascular effects caused by acting on the central nervous system. Currently only a few drugs alone can achieve a 5-10% weight loss. Among the drugs for the treatment of T2DM, only glucagon-like peptide (GLP-1) receptor agonists and sodium-glucose cotransporter 2 ( SGLT2) inhibitors have better weight control effect (J. Med. Chem., 2018, 61, 5580-5593). Bariatric surgery is effective in treating obesity, but patients suffer from greater surgical risks, and the long-term effects of surgery remain unclear. Therefore, there is still a huge clinical demand for drugs for weight control, and drugs that can safely and effectively control body weight and treat the primary disease are an ideal choice.
机体的能量和血糖调节信号系统包括多种不同的多肽类内源性胃肠道激素，胰高血糖素原(proglucagon)是一种具有160个氨基酸的前体多肽，其在不同组织中裂解后转化为不同的产物，诸如GLP-1、胰高血糖素样肽-2(GLP-2)、胰高血糖素(Glucagon，GCG)及胃泌酸调节素(Oxyntomodulin，OXM)等内源性胃肠道激素。这些内源性胃肠道激素参与胰岛素分泌、食物摄取、胃排空以及葡萄糖体内平衡等多种生理功能的调节。因此，基于内源性胃肠道激素的疗法已成为代谢综合征研究领域深受关注的研究方向。The body's energy and blood sugar regulation signaling system includes a variety of different polypeptides. Endogenous gastrointestinal hormones, proglucagon (proglucagon) is a 160 amino acid precursor polypeptide, which is cleaved in different tissues. Converted into different products, such as GLP-1, glucagon-like peptide-2 (GLP-2), glucagon (Glucagon, GCG) and oxyntomodulin (Oxyntomodulin, OXM) and other endogenous gastric gut hormones. These endogenous gastrointestinal hormones are involved in the regulation of various physiological functions such as insulin secretion, food intake, gastric emptying, and glucose homeostasis. Therefore, therapy based on endogenous gastrointestinal hormones has become a research direction of great concern in the field of metabolic syndrome research.
GLP-1是由末端空肠、回肠和结肠的L细胞所分泌的一种葡萄糖依赖性降血糖多肽激素，与GLP-1受体特异性结合后发挥降糖作用。GLP-1的主要优点是具有血糖依赖性的肠促胰岛素分泌作用，避免了糖尿病治疗中常存在的产生低血糖症的危险。除了调节血糖，GLP-1也可以阻止胰腺β细胞退化，刺激β细胞的增殖和分化，能从源头上改善糖尿病进程。此外，GLP-1还具有抑制胃酸分泌、延迟胃排空、抑制食欲等作用，具有部分减重效果。目前已上市多个长效GLP-1类药物，诸如liraglutide、semaglutide和dulaglutide等。虽然GLP-1类药物具有安全的降血糖作用，但是如果需要实现较好的体重减轻作用，一般需要加大给药剂量，而大剂量给予GLP-1类药物容易产生胃肠道副作用，耐受性差而导致治疗窗较窄。因此，仍然需要更为安全耐受的，可有效减轻体重和控制血糖的治疗剂。GCG是在胰脏的α细胞中生成的激素，在机体寒冷、饥饿等应激状态下作用于肝脏，将肝脏中的糖原进行分解而提高血糖。除了其升血糖作用，GCG在体内还具有促进脂解、脂肪氧化、发热等作用(Diabetologia,2017,60,1851–1861)，长期给药可以通过增加能量代谢量而呈现出体重减轻药效，但GCG这些对能量代谢的有益作用因其固有的升血糖作用而未能得以应用。GLP-1 is a glucose-dependent hypoglycemic polypeptide hormone secreted by L cells in the terminal jejunum, ileum and colon, and exerts a hypoglycemic effect after specifically binding to the GLP-1 receptor. The main advantage of GLP-1 is its glucose-dependent incretin secretion, which avoids the risk of hypoglycemia that is often present in the treatment of diabetes. In addition to regulating blood sugar, GLP-1 can also prevent pancreatic β-cell degeneration, stimulate β-cell proliferation and differentiation, and improve diabetes progression at the source. In addition, GLP-1 also has the effects of inhibiting gastric acid secretion, delaying gastric emptying, and suppressing appetite, and has partial weight loss effects. A number of long-acting GLP-1 drugs, such as liraglutide, semaglutide and dulaglutide, have been marketed. Although GLP-1 drugs have a safe hypoglycemic effect, if you need to achieve better weight loss effect, it is generally necessary to increase the dosage, and large doses of GLP-1 drugs are prone to gastrointestinal side effects, which are tolerated The poor performance results in a narrow therapeutic window. Therefore, there remains a need for safer and more tolerated therapeutic agents that are effective for weight loss and glycemic control. GCG is a hormone produced in the alpha cells of the pancreas. It acts on the liver under stress conditions such as cold and starvation, and decomposes glycogen in the liver to increase blood sugar. In addition to its blood sugar-raising effect, GCG also has the effect of promoting lipolysis, fat oxidation, and fever in the body (Diabetologia, 2017, 60, 1851–1861). However, these beneficial effects of GCG on energy metabolism have not been applied due to its inherent hypoglycemic effect.
OXM是人体内的一种内源性GLP-1受体和GCG受体双重激动剂，其对GLP-1受体和GCG受体的激动活性效力弱于各受体的天然配体(天然GLP-1或GCG)。OXM的急性生理作用包括抑制胃排空、摄食以及胃和胰腺的外分泌、提升静息能量消耗等，可产生体重减轻作用。实验表明，在动物和人体内外周给予OXM可减轻体重和降低摄食量，在肥胖对象中可提高代谢率以及和活动相关的能量消耗。此外，在临床中OXM大剂量给药在减轻体重的同时不易发生恶心、呕吐等常见胃肠道副作用。上述实验证实了基于OXM或GLP-1/GCG受体双重激动剂的疗法对代谢综合征的治疗显示了潜在的应用价值。OXM is an endogenous GLP-1 receptor and GCG receptor dual agonist in the human body, and its agonistic activity on GLP-1 receptor and GCG receptor is weaker than the natural ligand of each receptor (natural GLP). -1 or GCG). The acute physiological effects of OXM include inhibition of gastric emptying, food intake and exocrine secretion of the stomach and pancreas, enhancement of resting energy expenditure, etc., resulting in weight loss. Experiments have shown that intraperipheral administration of OXM in animals and humans reduces body weight and food intake, and in obese subjects increases metabolic rate and activity-related energy expenditure. In addition, in clinical practice, large doses of OXM are less prone to common gastrointestinal side effects such as nausea and vomiting while reducing body weight. The above experiments confirmed that the therapy based on OXM or GLP-1/GCG receptor dual agonist showed potential application value for the treatment of metabolic syndrome.
目前已报道的多肽类GLP-1/GCG受体双重激动剂，按序列结构可以分为基于GCG、OXM、GLP-1或毒蜥外泌肽-4(exendin-4)四类，已公开的专利文件有：CN201911103118.6、CN201780013643.1、CN201680021972.6、CN201580030150.X、CN201380048137.8、WO2008/071972、WO 2008/101017、WO 2009/155258、WO 2010/096052、WO 2010/096142、WO2011/075393、WO 2008/152403、WO 2010/070251、WO 2010/070252、WO 2010/070253、WO2010/070255、WO 2011/160630、WO 2011/006497、WO 2011/087671、WO 2011/087672、WO2011/117415、WO2011/117416、WO 2012/177443、WO 2012/177444、WO 2012/150503、WO2013/004983、WO 2013/092703、WO 2014/041195和WO 2014/041375等。The reported polypeptide GLP-1/GCG receptor dual agonists can be divided into four categories based on GCG, OXM, GLP-1 or exendin-4 (exendin-4) according to their sequence structure.专利文件有：CN201911103118.6、CN201780013643.1、CN201680021972.6、CN201580030150.X、CN201380048137.8、WO2008/071972、WO 2008/101017、WO 2009/155258、WO 2010/096052、WO 2010/096142、WO2011/ 075393, WO 2008/152403, WO 2010/070251, WO 2010/070252, WO 2010/070253, WO2010/070255, WO 2011/160630, WO 2011/006497, WO 2011/0876171, WO2111/08 WO2011/117416, WO 2012/177443, WO 2012/177444, WO 2012/150503, WO2013/004983, WO 2013/092703, WO 2014/041195 and WO 2014/041375 etc.
此外，还有部分研究描述了不仅能激活GLP-1受体和GCG受体，还激活葡萄糖依赖性促胰岛素多肽(GIP)受体的GLP-1/GCG/GIP受体三重激动剂描述于Brian Finan等(Nat.Med.,2015,21,27-36)，Victor A.Gault等(Biochem.Pharmacol.,2013,85,1655–1662；Diabetologia,2013,56,1417–1424)以及描述于CN104902919B、WO 2012/088116等。In addition, several studies have described GLP-1/GCG/GIP receptor triple agonists that activate not only the GLP-1 receptor and GCG receptor, but also the glucose-dependent insulinotropic polypeptide (GIP) receptor. Finan et al. (Nat. Med., 2015, 21, 27-36), Victor A. Gault et al. (Biochem. Pharmacol., 2013, 85, 1655-1662; Diabetologia, 2013, 56, 1417-1424) and described in CN104902919B , WO 2012/088116, etc.
两栖动物体内的GLP-1作用效果与人GLP-1类似，所以针对两栖动物GLP-1进行结构修饰，有望发现具有更高效和长效降糖作用的新型GLP-1类药物。XenGLP-1是从非洲爪蟾体内发现的一类动物源属的GLP-1类似物，与天然GLP-1相比，XenGLP-1的降糖活性和稳定性更优。此外，与GLP-1、OXM和GCG相比，除了更为耐受二肽基肽酶(DPP-IV)的降解，XenGLP-1还显示出对于中性内肽酶(NEP)的降解稳定得多。XenGLP-1是GLP-1受体的高效激动剂，然而其不会激活GCG受体。XenGLP-1具有许多用天然GLP-1观察到的葡萄糖调控作用，许多临床前研究都显示XenGLP-1具有若干有益的抗糖尿病特性，包括血糖依赖性的胰岛素合成和分泌增强、胃排空放慢、食物摄入和体重减少，以及促进β细胞增殖和恢复胰岛功能等(Biochem.Pharmacol.,2017,142,155–167；FASEB J.,2019,33,7113-7125)。这些效果不仅对于糖尿病人是有益的，并且对罹患肥胖症的患者也是有益的。患有肥胖者的患者具有更高的患上高血压、高血脂、糖尿病、NAFLD、NASH、肌肉骨骼和心血管疾病的风险。The effect of GLP-1 in amphibians is similar to that of human GLP-1, so structural modification of amphibian GLP-1 is expected to discover new GLP-1 drugs with more efficient and long-acting hypoglycemic effects. XenGLP-1 is a class of animal-derived GLP-1 analogs found in Xenopus laevis. Compared with natural GLP-1, XenGLP-1 has better hypoglycemic activity and stability. Furthermore, in addition to being more resistant to degradation by dipeptidyl peptidase (DPP-IV) compared to GLP-1, OXM and GCG, XenGLP-1 also showed stable degradation against neutral endopeptidase (NEP). many. XenGLP-1 is a potent agonist of the GLP-1 receptor, however it does not activate the GCG receptor. XenGLP-1 has many of the glucose-regulating effects observed with native GLP-1, and many preclinical studies have shown that XenGLP-1 has several beneficial antidiabetic properties, including glucose-dependent enhancement of insulin synthesis and secretion, slowed gastric emptying , food intake and weight loss, as well as promoting β-cell proliferation and restoring islet function, etc. (Biochem. Pharmacol., 2017, 142, 155-167; FASEB J., 2019, 33, 7113-7125). These effects are beneficial not only for diabetics, but also for patients suffering from obesity. Patients with obesity have a higher risk of developing hypertension, hyperlipidemia, diabetes, NAFLD, NASH, musculoskeletal and cardiovascular disease.
发明内容SUMMARY OF THE INVENTION
本发明的目的是提供一种新的具有GLP-1/GCG受体双重激动作用的多肽化合物，所述多肽是基于XenGLP-1序列设计的变体，保留XenGLP-1对糖尿病的治疗作用同时具有GCG对脂代谢和能量代谢的有益作用，从而对糖、脂、能量代谢产生协同影响，比单一受体激动剂在制备用于治疗代谢综合征，诸如肥胖、糖尿病、NAFLD、NASH等疾病的药物方面更具潜力。The object of the present invention is to provide a novel polypeptide compound with dual agonistic effect of GLP-1/GCG receptor, the polypeptide is a variant designed based on the sequence of XenGLP-1, which retains the therapeutic effect of XenGLP-1 on diabetes while having The beneficial effects of GCG on lipid metabolism and energy metabolism, resulting in synergistic effects on sugar, lipid, and energy metabolism, are more useful than single receptor agonists in the preparation of drugs for the treatment of metabolic syndrome, such as obesity, diabetes, NAFLD, NASH and other diseases more potential.
为实现上述发明目的，本发明的技术方案具体如下：In order to realize the above-mentioned purpose of the invention, the technical scheme of the present invention is as follows:
一类GLP-1/GCG受体双重激动多肽化合物，该类GLP-1受体/GCG受体双重激动多肽 化合物的氨基酸序列通式为：A class of GLP-1/GCG receptor dual agonist polypeptide compounds, the general amino acid sequence formula of this class of GLP-1 receptor/GCG receptor dual agonist polypeptide compounds is:
His-Xaa 1-Xaa 2-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Xaa 3-Xaa 4-Tyr-Xaa 5-Xaa 6-Xaa 7-Xaa 8-Xaa 9-Ala-Xaa 10-Xaa 11-Phe-Ile-Glu-Trp-Leu-Xaa 12-Xaa 13-Gly-Xaa 14-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Xaa 15 His-Xaa 1 -Xaa 2 -Gly-Thr-Tyr-Thr-Asn-Asp-Val-Xaa 3 -Xaa 4 -Tyr-Xaa 5 -Xaa 6 -Xaa 7 -Xaa 8 -Xaa 9 -Ala-Xaa 10 - Xaa 11 -Phe-Ile-Glu-Trp-Leu-Xaa 12 -Xaa 13 -Gly-Xaa 14 -Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Xaa 15
其中：in:
Xaa 1取自Ser，D-Ser或Aib； Xaa 1 is taken from Ser, D-Ser or Aib;
Xaa 2取自Glu或Gln； Xaa 2 is taken from Glu or Gln;
Xaa 3取自Thr或Ser； Xaa 3 is taken from Thr or Ser;
Xaa 4取自Glu、Lys或侧链被修饰的Lys； Xaa 4 is taken from Glu, Lys or Lys whose side chain is modified;
Xaa 5取自Leu、Lys或侧链被修饰的Lys； Xaa 5 is taken from Leu, Lys or Lys whose side chain is modified;
Xaa 6取自Glu或Asp； Xaa 6 is taken from Glu or Asp;
Xaa 7取自Glu或Ser； Xaa 7 is taken from Glu or Ser;
Xaa 8取自Glu或Arg； Xaa 8 is taken from Glu or Arg;
Xaa 9取自Ala或Arg； Xaa 9 is taken from Ala or Arg;
Xaa 10取自Lys或Gln； Xaa 10 from Lys or Gln;
Xaa 11取自Glu或Asp； Xaa 11 is taken from Glu or Asp;
Xaa 12取自Ile或Lys； Xaa 12 taken from Ile or Lys;
Xaa 13取自Lys或Asn； Xaa 13 from Lys or Asn;
Xaa 14取自Lys或Gly； Xaa 14 from Lys or Gly;
Xaa 15取自-NH 2或侧链被修饰的Lys； Xaa 15 is taken from -NH 2 or Lys whose side chain is modified;
其中侧链被修饰的Lys选自Lys(γ-Glu-CO-(CH 2) n-CH 3)或Lys(AEEA-AEEA-γ-Glu-CO-(CH 2) n-COOH)， The Lys in which the side chain is modified is selected from Lys(γ-Glu-CO-(CH 2 ) n -CH 3 ) or Lys(AEEA-AEEA-γ-Glu-CO-(CH 2 ) n -COOH),
Lys(γ-Glu-CO-(CH 2) n-CH 3)的结构式如下式所示： The structural formula of Lys(γ-Glu-CO-(CH 2 ) n -CH 3 ) is shown below:
Figure PCTCN2021131369-appb-000001
Figure PCTCN2021131369-appb-000001
Lys(AEEA-AEEA-γ-Glu-CO-(CH 2) n-COOH)的结构式如下式所示： The structural formula of Lys(AEEA-AEEA-γ-Glu-CO-(CH 2 ) n -COOH) is shown below:
Figure PCTCN2021131369-appb-000002
Figure PCTCN2021131369-appb-000002
其中，n为自然数，且12≤n≤20。Among them, n is a natural number, and 12≤n≤20.
优选的，所述n是14、16、18或20。Preferably, the n is 14, 16, 18 or 20.
优选的，所述多肽化合物的氨基酸序列是下列序列之一：Preferably, the amino acid sequence of the polypeptide compound is one of the following sequences:
(1)SEQ ID NO:1(1) SEQ ID NO: 1
His-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys(γ-Glu-CO-(CH 2) 14-CH 3)-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2 His-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys(γ-Glu-CO-(CH 2 ) 14 -CH 3 )-Tyr-Leu-Asp-Ser-Arg- Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2
(2)SEQ ID NO:2(2) SEQ ID NO: 2
His-D-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys(γ-Glu-CO-(CH 2) 14- CH 3)-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2 His-D-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys(γ-Glu-CO-(CH 2 ) 14 -CH 3 )-Tyr-Leu-Asp-Ser- Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2
(3)SEQ ID NO:3(3) SEQ ID NO: 3
His-Aib-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys(γ-Glu-CO-(CH 2) 14-CH 3)-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2 His-Aib-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys(γ-Glu-CO-(CH 2 ) 14 -CH 3 )-Tyr-Leu-Asp-Ser-Arg- Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2
(4)SEQ ID NO:4(4) SEQ ID NO: 4
His-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys(AEEA-AEEA-γ-Glu-CO-(CH 2) 16-COOH)-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2 His-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys(AEEA-AEEA-γ-Glu-CO-(CH 2 ) 16 -COOH)-Tyr-Leu-Asp-Ser -Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2
(5)SEQ ID NO:5(5) SEQ ID NO: 5
His-D-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys(AEEA-AEEA-γ-Glu-CO-(CH 2) 16-COOH)-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2 His-D-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys(AEEA-AEEA-γ-Glu-CO-(CH 2 ) 16 -COOH)-Tyr-Leu-Asp -Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2
(6)SEQ ID NO:6(6) SEQ ID NO: 6
His-Aib-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys(AEEA-AEEA-γ-Glu-CO-(CH 2) 16-COOH)-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2 His-Aib-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys(AEEA-AEEA-γ-Glu-CO-(CH 2 ) 16 -COOH)-Tyr-Leu-Asp-Ser -Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2
(7)SEQ ID NO:7(7) SEQ ID NO: 7
His-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Lys(γ-Glu-CO-(CH 2) 14-CH 3)-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2 His-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Lys(γ-Glu-CO-(CH 2 ) 14 -CH 3 )-Asp-Ser-Arg- Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2
(8)SEQ ID NO:8(8) SEQ ID NO: 8
His-D-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Lys(γ-Glu-CO-(CH 2) 14-CH 3)-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2 His-D-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Lys(γ-Glu-CO-(CH 2 ) 14 -CH 3 )-Asp-Ser- Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2
(9)SEQ ID NO:9(9) SEQ ID NO: 9
His-Aib-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Lys(γ-Glu-CO-(CH 2) 14-CH 3)-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2 His-Aib-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Lys(γ-Glu-CO-(CH 2 ) 14 -CH 3 )-Asp-Ser-Arg- Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2
(10)SEQ ID NO:10(10) SEQ ID NO: 10
His-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Lys(AEEA-AEEA-γ-Glu-CO-(CH 2) 16-COOH)-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2 His-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Lys(AEEA-AEEA-γ-Glu-CO-(CH 2 ) 16 -COOH)-Asp-Ser -Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2
(11)SEQ ID NO:11(11) SEQ ID NO: 11
His-D-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Lys(AEEA-AEEA-γ-Glu-CO-(CH 2) 16-COOH)-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2 His-D-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Lys(AEEA-AEEA-γ-Glu-CO-(CH 2 ) 16 -COOH)-Asp -Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2
(12)SEQ ID NO:12(12) SEQ ID NO: 12
His-Aib-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Lys(AEEA-AEEA-γ-Glu-CO-(CH 2) 16-COOH)-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2 His-Aib-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Lys(AEEA-AEEA-γ-Glu-CO-(CH 2 ) 16 -COOH)-Asp-Ser -Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2
(13)SEQ ID NO:13(13) SEQ ID NO: 13
His-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Lys(γ-Glu-CO-(CH 2) 14-CH 3)-NH 2 His-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp- Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Lys(γ-Glu-CO-(CH 2 ) 14 -CH 3 )-NH 2
(14)SEQ ID NO:14(14) SEQ ID NO: 14
His-D-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Lys(γ-Glu-CO-(CH 2) 14-CH 3)-NH 2 His-D-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu- Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Lys(γ-Glu-CO-(CH 2 ) 14 -CH 3 )-NH 2
(15)SEQ ID NO:15(15) SEQ ID NO: 15
His-Aib-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Lys(γ-Glu-CO-(CH 2) 14-CH 3)-NH 2 His-Aib-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp- Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Lys(γ-Glu-CO-(CH 2 ) 14 -CH 3 )-NH 2
(16)SEQ ID NO:16(16) SEQ ID NO: 16
His-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Lys(AEEA-AEEA-γ-Glu-CO-(CH 2) 16-COOH)-NH 2 His-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp- Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Lys(AEEA-AEEA-γ-Glu-CO-(CH 2 ) 16 -COOH)-NH 2
(17)SEQ ID NO:17(17) SEQ ID NO: 17
His-D-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Lys(AEEA-AEEA-γ-Glu-CO-(CH 2) 16-COOH)-NH 2 His-D-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu- Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Lys(AEEA-AEEA-γ-Glu-CO-(CH 2 ) 16 -COOH) -NH2
(18)SEQ ID NO:18(18) SEQ ID NO: 18
His-Aib-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Lys(AEEA-AEEA-γ-Glu-CO-(CH 2) 16-COOH)-NH 2 His-Aib-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp- Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Lys(AEEA-AEEA-γ-Glu-CO-(CH 2 ) 16 -COOH)-NH 2
本发明还提供了一类GLP-1/GCG受体双重激动多肽化合物药学上可接受的盐。The present invention also provides pharmaceutically acceptable salts of a class of GLP-1/GCG receptor dual agonist polypeptide compounds.
优选的，所述的盐为GLP-1/GCG受体双重激动多肽化合物与下述化合物中的一种所形成的盐：氢溴酸、盐酸、甲磺酸、磷酸、乙磺酸、甲酸、对甲苯磺酸、乙酸、乙酰乙酸、丙酮酸、果胶酯酸、丁酸、己酸、苯磺酸、庚酸、十一烷酸、苯甲酸、水杨酸、月桂酸、2-(4-羟基苯甲酰基)苯甲酸、肉桂酸、樟脑酸、环戊烷丙酸、3-羟基-2-萘甲酸、樟脑磺酸、二葡糖酸、烟酸、扑酸、丙酸、过硫酸、、苦味酸、3-苯基丙酸、特戊酸、衣康酸、2-羟基乙磺酸、氨基磺酸、十二烷基硫酸、三氟甲磺酸、萘二磺酸、2-萘磺酸、柠檬酸、扁桃酸、抗坏血酸、酒硬脂酸、石酸、草酸、乳酸、琥珀酸、丙二酸、半硫酸、苹果酸、马来酸、藻酸、富马酸、D-葡糖酸、甘油磷酸、葡庚酸、天冬氨酸、硫氰酸或者磺基水杨酸。Preferably, the salt is a salt formed by a GLP-1/GCG receptor dual agonist polypeptide compound and one of the following compounds: hydrobromic acid, hydrochloric acid, methanesulfonic acid, phosphoric acid, ethanesulfonic acid, formic acid, p-toluenesulfonic acid, acetic acid, acetoacetic acid, pyruvic acid, pectic acid, butyric acid, caproic acid, benzenesulfonic acid, heptanoic acid, undecanoic acid, benzoic acid, salicylic acid, lauric acid, 2-(4 -Hydroxybenzoyl)benzoic acid, cinnamic acid, camphoric acid, cyclopentanepropionic acid, 3-hydroxy-2-naphthoic acid, camphorsulfonic acid, digluconic acid, niacin, pamoic acid, propionic acid, persulfuric acid ,, picric acid, 3-phenylpropionic acid, pivalic acid, itaconic acid, 2-hydroxyethanesulfonic acid, sulfamic acid, dodecyl sulfuric acid, trifluoromethanesulfonic acid, naphthalenedisulfonic acid, 2- Naphthalenesulfonic acid, citric acid, mandelic acid, ascorbic acid, wine stearic acid, tartaric acid, oxalic acid, lactic acid, succinic acid, malonic acid, hemisulfuric acid, malic acid, maleic acid, alginic acid, fumaric acid, D- Gluconic acid, glycerophosphoric acid, glucoheptanoic acid, aspartic acid, thiocyanic acid or sulfosalicylic acid.
本发明还提供了GLP-1/GCG受体双重激动多肽化合物的药物组合物，该药物组合物包括：以上述任一GLP-1/GCG受体双重激动多肽化合物或其药学上可接受的盐为有效原料，再加上药学上可接受的载体或稀释剂组成。The present invention also provides a pharmaceutical composition of the GLP-1/GCG receptor dual agonist polypeptide compound, the pharmaceutical composition comprising: using any of the above GLP-1/GCG receptor dual agonist polypeptide compounds or a pharmaceutically acceptable salt thereof It is composed of effective raw materials, together with a pharmaceutically acceptable carrier or diluent.
本发明还提供了含有上述GLP-1/GCG受体双重激动多肽化合物的药剂，所述的药剂是任何一种药剂学上所说的胶囊、片剂、喷雾剂、吸入剂、注射剂、贴剂、乳剂、膜剂、散剂或者复方制剂，药剂由GLP-1/GCG受体双重激动多肽化合物和药学上可接受的药用辅料、载体或稀释剂组成。The present invention also provides a medicament containing the above-mentioned GLP-1/GCG receptor dual agonist polypeptide compound, the medicament is any of the capsules, tablets, sprays, inhalants, injections, patches mentioned in pharmacy , emulsion, film, powder or compound preparation, the medicament is composed of GLP-1/GCG receptor dual agonist polypeptide compound and pharmaceutically acceptable pharmaceutical excipients, carriers or diluents.
本发明还提供了本发明所述的GLP-1/GCG受体双重激动多肽化合物、其药学上可接受的盐、其药物组合物或其药剂在制备用于治疗代谢性疾病或病症的药物中的应用。在特定方面，代谢性疾病或病症为糖尿病、NAFLD、NASH、高血脂或肥胖。在特定方面，糖尿病为1型糖尿病、T2DM或妊娠糖尿病。在特定方面，所述药物用于治疗超过一种代谢疾病或病症，例如，糖尿病和NAFLD、NASH或肥胖；肥胖和NASH或NAFLD；糖尿病、NASH和肥胖；糖尿病、NAFLD和肥胖；或糖尿病和肥胖。The present invention also provides the GLP-1/GCG receptor dual agonist polypeptide compound of the present invention, its pharmaceutically acceptable salt, its pharmaceutical composition or its medicament in the preparation of a medicament for the treatment of metabolic diseases or disorders Applications. In particular aspects, the metabolic disease or disorder is diabetes, NAFLD, NASH, hyperlipidemia or obesity. In certain aspects, the diabetes is type 1 diabetes, T2DM or gestational diabetes. In certain aspects, the medicament is for the treatment of more than one metabolic disease or disorder, eg, diabetes and NAFLD, NASH or obesity; obesity and NASH or NAFLD; diabetes, NASH and obesity; diabetes, NAFLD and obesity; or diabetes and obesity .
与现有技术相比，本发明的有益效果：Compared with the prior art, the beneficial effects of the present invention:
与现有的GLP-1受体激动剂相比，本发明的GLP-1/GCG受体双重激动多肽化合物在更为有效的降低血糖的同时具有促进减重和防止增重作用，逆转胰岛素抵抗，调节脂质代谢，与现有药物相比具有意想不到的有益作用。本发明的多肽化合物对GLP-1受体和GCG受体的激动活性高于各受体的天然配体，同时对GIP受体具有更低的激动活性。本发明提供的多肽化合物化学性质稳定，不易被体内的DPP-IV和NEP降解，不易被肾小球滤过，化合物的稳定性显著提高，具有支持每天一次给药或每周一次给药的药代动力学特征。本发明提供的多肽化合物具有提高的生物物理特性，在中性pH和pH 4.5的溶解性都高于天然GLP-1和GCG，具备有利于制剂的特性。本发明提供的多肽化合物具有低的免疫原性特性，对T2DM、肥胖、NAFLD、NASH和高血脂症等代谢性疾病的治疗作用优于现有上市药物。因此，本发明提供的多肽化合物，适合作为治疗代谢性疾病，如糖尿病、肥胖、高血脂症、NAFLD、NASH等药物的活性成分。Compared with the existing GLP-1 receptor agonists, the GLP-1/GCG receptor dual agonist polypeptide compound of the present invention has the functions of promoting weight loss and preventing weight gain while lowering blood sugar more effectively, and reversing insulin resistance. , modulates lipid metabolism with unexpected beneficial effects compared with existing drugs. The agonistic activity of the polypeptide compound of the present invention on GLP-1 receptor and GCG receptor is higher than that of the natural ligands of each receptor, while the agonistic activity on GIP receptor is lower. The polypeptide compound provided by the invention has stable chemical properties, is not easily degraded by DPP-IV and NEP in the body, and is not easily filtered by the glomerulus. Generation dynamics characteristics. The polypeptide compound provided by the present invention has improved biophysical properties, and the solubility at neutral pH and pH 4.5 is higher than that of natural GLP-1 and GCG, and has properties that are beneficial to formulations. The polypeptide compound provided by the invention has low immunogenicity, and the therapeutic effect on metabolic diseases such as T2DM, obesity, NAFLD, NASH and hyperlipidemia is better than that of the existing marketed drugs. Therefore, the polypeptide compound provided by the present invention is suitable as an active ingredient of medicines for the treatment of metabolic diseases, such as diabetes, obesity, hyperlipidemia, NAFLD, NASH and the like.
附图说明Description of drawings
图1显示的是各受试物单次给药在db/db小鼠非禁食状态下的长效降血糖作用；Figure 1 shows the long-acting hypoglycemic effect of a single dose of each test substance in db/db mice in a non-fasted state;
图2显示的是各受试物在DIO小鼠长期给药21天后的口服糖耐量实验中的降血糖作用；Figure 2 shows the hypoglycemic effect of each test substance in the oral glucose tolerance test after long-term administration of DIO mice for 21 days;
图3显示的是各受试物在体外的免疫原性。Figure 3 shows the in vitro immunogenicity of each test substance.
具体实施方式Detailed ways
在本说明书全文中采用以下缩写：The following abbreviations are used throughout this specification:
英文缩写  中文English abbreviation Chinese
Gly       甘氨酸Gly Glycine
Ser       丝氨酸Ser Serine
Ala       丙氨酸Ala Alanine
Thr       苏氨酸Thr Threonine
Val       缬氨酸Val valine
Ile       异亮氨酸Ile Isoleucine
Leu       亮氨酸Leu Leucine
Tyr    酪氨酸Tyr Tyrosine
Phe    苯丙氨酸Phe Phenylalanine
His    组氨酸His Histidine
Pro    脯氨酸Pro Proline
Asp    天冬氨酸Asp Aspartate
Met    蛋氨酸Met methionine
Glu    谷氨酸Glu Glutamate
Trp    色氨酸Trp Tryptophan
Lys    赖氨酸Lys Lysine
Arg    精氨酸Arg Arginine
Asn    天冬酰胺Asn Asparagine
Gln    谷氨酰胺Gln Glutamine
Cys    半胱氨酸Cys cysteine
Aib    α-氨基异丁酸Aib α-aminoisobutyric acid
AEEA   8-氨基-3,6二氧杂辛酸AEEA 8-amino-3,6 dioxa octanoic acid
DCM    二氯甲烷DCM Dichloromethane
DMF    二甲基甲酰胺DMF Dimethylformamide
Fmoc   9-芴基甲氧基羰基Fmoc 9-Fluorenylmethoxycarbonyl
Boc    叔丁氧羰基Boc tert-Butoxycarbonyl
DMSO   二甲基亚砜DMSO Dimethyl Sulfoxide
DIC    N,N’-二异丙基碳二亚胺DIC N,N’-diisopropylcarbodiimide
HOBT   1-羟基-苯并三氮唑HOBT 1-Hydroxy-benzotriazole
Alloc  烯丙氧羰基Alloc Allyloxycarbonyl
Dde    1-(4,4-二甲基-2,6-二氧代亚环己基)-乙基Dde 1-(4,4-Dimethyl-2,6-dioxocyclohexylene)-ethyl
Mtt    4-甲基三苯甲基Mtt 4-methyltrityl
ivDde  1-(4,4-二甲基-2,6-二氧代亚环己基)3-甲基-丁基ivDde 1-(4,4-dimethyl-2,6-dioxocyclohexylene)3-methyl-butyl
TFA    三氟乙酸TFA trifluoroacetic acid
EDT    二巯基乙烷EDT dimercaptoethane
HPLC   高效液相色谱HPLC High Performance Liquid Chromatography
LC-MS  液质联用质谱LC-MS mass spectrometry
DMEM   杜贝可氏修饰伊格氏培养基DMEM Dulbecco's Modified Eagle's Medium
FBS    胎牛血清FBS Fetal Bovine Serum
PBS    磷酸盐缓冲盐水PBS Phosphate Buffered Saline
HEPES  2-[4-(2-羟乙基)哌嗪-1-基]乙磺酸HEPES 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid
BSA    牛血清白蛋白BSA bovine serum albumin
IBMX   3-异丁基-1-甲基黄嘌呤IBMX 3-isobutyl-1-methylxanthine
HBSS   Hanks’平衡盐溶液HBSS Hanks' Balanced Salt Solution
AIMV   无血清细胞培养基AIMV serum-free cell culture medium
实施例1Example 1
SEQ ID NO:1多肽化合物的合成Synthesis of SEQ ID NO:1 polypeptide compound
His-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys(γ-Glu-CO-(CH 2) 14-CH 3)-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2 His-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys(γ-Glu-CO-(CH 2 ) 14 -CH 3 )-Tyr-Leu-Asp-Ser-Arg- Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2
(1)树脂的溶胀(1) Swelling of resin
称取担载量为0.382mmol/g的Rink Amide MBHA树脂0.262g(0.1mmol当量)，放入25mL的反应器中，用7mL的DCM和甲醇交替清洗树脂1次，7mL的DCM清洗树脂2次，然后用7mL的DCM溶胀树脂1h，最后用7mL DMF清洗树脂3次。Weigh 0.262 g (0.1 mmol equivalent) of Rink Amide MBHA resin with a loading of 0.382 mmol/g, put it into a 25 mL reactor, wash the resin alternately with 7 mL of DCM and methanol once, and wash the resin twice with 7 mL of DCM , then swelled the resin with 7 mL of DCM for 1 h, and finally washed the resin 3 times with 7 mL of DMF.
(2)树脂Fmoc保护基的脱除(2) Removal of resin Fmoc protecting group
将溶胀后的树脂转入PSI200多肽合成仪，加入7mL 20％哌啶/DMF(v/v)室温反应5min，滤去脱保护溶液，7mL DMF清洗树脂一次，再加入7mL 20％哌啶/DMF(v/v)脱保护溶剂与树脂反应15min，最后7mL DMF清洗树脂4次，每次1.5min，得到脱除Fmoc保护基的Rink树脂。Transfer the swollen resin to a PSI200 peptide synthesizer, add 7 mL of 20% piperidine/DMF (v/v) to react at room temperature for 5 min, filter off the deprotection solution, wash the resin once with 7 mL of DMF, and then add 7 mL of 20% piperidine/DMF (v/v) The deprotection solvent was reacted with the resin for 15 min, and finally 7 mL of DMF was used to wash the resin 4 times, each 1.5 min, to obtain the Rink resin with the Fmoc protecting group removed.
(3)Fmoc-Ser-Rink amide-MBHA Resin的合成(3) Synthesis of Fmoc-Ser-Rink amide-MBHA Resin
称Fmoc-Ser(Boc)-OH(0.4mmol)，用3mL 10％DMF/DMSO(v/v)溶解，加入2mL DIC/HOBt(0.4mmol/0.44mmol)缩合剂，预活化30min后，将活化好的氨基酸加入反应器中，室温震荡反应2h，滤去反应液后用7mL DMF清洗树脂4次，使用Kaiser试剂检测反应耦合是否完全，如不完全则2次耦合。Weigh Fmoc-Ser(Boc)-OH (0.4mmol), dissolve it with 3mL 10% DMF/DMSO (v/v), add 2mL DIC/HOBt (0.4mmol/0.44mmol) condensing agent, pre-activate for 30min, then activate The good amino acid was added to the reactor, and the reaction was shaken at room temperature for 2 h. After filtering off the reaction solution, the resin was washed with 7 mL of DMF for 4 times. Kaiser reagent was used to check whether the reaction coupling was complete. If not, the coupling was performed twice.
(4)肽链的延长(4) Extension of the peptide chain
按照肽链的序列，重复上述脱保护和耦合的步骤依次连接上相应的氨基酸，直至肽链合成完毕。其中12位Lys可以采用Fmoc-Lys(Alloc)-OH、Fmoc-Lys(Dde)-OH、Fmoc-Lys(Mtt)-OH或Fmoc-Lys(ivDde)-OH等。本实例中采用Fmoc-Lys(Dde)-OH保护策略，同时N末端的His使用的是Boc-His(Boc)-OH。According to the sequence of the peptide chain, the above steps of deprotection and coupling are repeated to connect the corresponding amino acids in turn, until the synthesis of the peptide chain is completed. The 12-position Lys can be Fmoc-Lys(Alloc)-OH, Fmoc-Lys(Dde)-OH, Fmoc-Lys(Mtt)-OH or Fmoc-Lys(ivDde)-OH, etc. In this example, the Fmoc-Lys(Dde)-OH protection strategy was adopted, and the N-terminal His was Boc-His(Boc)-OH.
(5)Lys侧链的修饰(5) Modification of Lys side chain
肽链合成完毕后，加入7mL 2％水合肼/DMF(v/v)选择性脱除12位Lys的Dde保护基，Dde保护基脱除后加入0.4mmol的Fmoc-Glu-OtBu，0.4mmol的DIC及0.44mmol的HOBt，震荡反应2h。然后使用上述相同方法脱除Fmoc保护基后，加入0.4mmol的棕榈酸，0.4mmol的DIC及0.44mmol的HOBt缩合反应2h，反应完全后用7mL DMF清洗树脂4次。After the synthesis of the peptide chain, 7 mL of 2% hydrazine hydrate/DMF (v/v) was added to selectively remove the Dde protecting group of the 12-position Lys. After the Dde protecting group was removed, 0.4 mmol of Fmoc-Glu-OtBu was added, and 0.4 mmol of DIC and 0.44mmol of HOBt were shaken for 2h. Then use the same method as above to remove the Fmoc protecting group, add 0.4 mmol of palmitic acid, 0.4 mmol of DIC and 0.44 mmol of HOBt for condensation reaction for 2 h, and wash the resin 4 times with 7 mL of DMF after the reaction is complete.
(6)多肽的裂解(6) Peptide cleavage
将上述得到的连有多肽的树脂转移至圆底瓶中，使用切割剂Reagent R(TFA/苯甲硫醚/苯酚/EDT，90:5:3:2，V/V)5mL切割树脂，在油浴中恒温30℃反应2h，切割液倾入40mL冰乙醚中，冷冻离心后粗品用15mL冰乙醚洗涤3次，最后用氮气吹干，得到粗肽。Transfer the above-obtained peptide-linked resin into a round-bottomed flask, and use 5 mL of cutting agent Reagent R (TFA/anisole/phenol/EDT, 90:5:3:2, V/V) to cut the resin. The reaction was carried out at a constant temperature of 30 °C in an oil bath for 2 h, the cutting solution was poured into 40 mL of ice ether, and the crude product was washed three times with 15 mL of ice ether after freezing and centrifugation, and finally dried with nitrogen to obtain the crude peptide.
(7)多肽的纯化(7) Purification of polypeptides
将目标多肽粗品溶于水中，用0.25μm微孔滤膜过滤后进岛津制备型反相HPLC系统纯化。色谱条件为C18反相制备柱(250mm×20mm，12μm)；流动相A：0.1％TFA/水(V/V)，流动相B：甲醇(V/V)；流速为8mL/min；检测波长为214nm。采用线性梯度(20％B～70％B/30min)洗脱，收集目标峰，除去甲醇后冻干得纯品0.14g，纯度大于98％，通过LC-MS确认目标多肽的分子量。The crude target polypeptide was dissolved in water, filtered with a 0.25 μm microporous membrane, and then purified by a Shimadzu preparative reverse-phase HPLC system. Chromatographic conditions were C18 reversed-phase preparative column (250mm×20mm, 12μm); mobile phase A: 0.1% TFA/water (V/V), mobile phase B: methanol (V/V); flow rate was 8mL/min; detection wavelength is 214nm. A linear gradient (20%B～70%B/30min) was used for elution, the target peak was collected, and 0.14g of pure product was obtained by lyophilization after removing methanol. The purity was greater than 98%. The molecular weight of the target polypeptide was confirmed by LC-MS.
实施例2Example 2
SEQ ID NO:2多肽化合物的合成Synthesis of SEQ ID NO:2 polypeptide compound
His-D-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys(γ-Glu-CO-(CH 2) 14-CH 3)-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2 His-D-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys(γ-Glu-CO-(CH 2 ) 14 -CH 3 )-Tyr-Leu-Asp-Ser- Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2
合成方法同实施例1，收集目标峰冻干得纯品0.16g。The synthetic method was the same as that of Example 1, and the target peak was collected and lyophilized to obtain 0.16 g of pure product.
实施例3Example 3
SEQ ID NO:3多肽化合物的合成Synthesis of SEQ ID NO:3 polypeptide compound
His-Aib-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys(γ-Glu-CO-(CH 2) 14-CH 3)-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2 His-Aib-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys(γ-Glu-CO-(CH 2 ) 14 -CH 3 )-Tyr-Leu-Asp-Ser-Arg- Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2
合成方法同实施例1，收集目标峰冻干得纯品0.13g。The synthesis method was the same as that in Example 1, and the target peak was collected and lyophilized to obtain 0.13 g of pure product.
实施例4Example 4
SEQ ID NO:4多肽化合物的合成Synthesis of SEQ ID NO:4 polypeptide compound
His-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys(AEEA-AEEA-γ-Glu-CO-(CH 2) 16-COOH)-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2 His-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys(AEEA-AEEA-γ-Glu-CO-(CH 2 ) 16 -COOH)-Tyr-Leu-Asp-Ser -Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2
(1)树脂的溶胀(1) Swelling of resin
称取担载量为0.382mmol/g的Rink Amide MBHA树脂0.262g(0.1mmol当量)，放入25mL的反应器中，用7mL的DCM和甲醇交替清洗树脂1次，7mL的DCM清洗树脂2次，然后用7mL的DCM溶胀树脂1h，最后用7mL DMF清洗树脂3次。Weigh 0.262 g (0.1 mmol equivalent) of Rink Amide MBHA resin with a loading of 0.382 mmol/g, put it into a 25 mL reactor, wash the resin alternately with 7 mL of DCM and methanol once, and wash the resin twice with 7 mL of DCM , then swelled the resin with 7 mL of DCM for 1 h, and finally washed the resin 3 times with 7 mL of DMF.
(2)树脂Fmoc保护基的脱除(2) Removal of resin Fmoc protecting group
将溶胀后的树脂转入PSI200多肽合成仪，加入7mL 20％哌啶/DMF(v/v)室温反应5min，滤去脱保护溶液，7mL DMF清洗树脂一次，再加入7mL 20％哌啶/DMF(v/v)脱保护溶剂与树脂反应15min，最后7mL DMF清洗树脂4次，每次1.5min，得到脱除Fmoc保护基的Rink树脂。Transfer the swollen resin to a PSI200 peptide synthesizer, add 7 mL of 20% piperidine/DMF (v/v) to react at room temperature for 5 min, filter off the deprotection solution, wash the resin once with 7 mL of DMF, and then add 7 mL of 20% piperidine/DMF (v/v) The deprotection solvent was reacted with the resin for 15 min, and finally 7 mL of DMF was used to wash the resin 4 times, each 1.5 min, to obtain the Rink resin with the Fmoc protecting group removed.
(3)Fmoc-Ser-Rink amide-MBHA Resin的合成(3) Synthesis of Fmoc-Ser-Rink amide-MBHA Resin
称Fmoc-Ser(Boc)-OH(0.4mmol)，用3mL 10％DMF/DMSO(v/v)溶解，加入2ml DIC/HOBt(0.4mmol/0.44mmol)缩合剂，预活化30min后，将活化好的氨基酸加入反应器中，室温震荡反应2h，滤去反应液后用7mL DMF清洗树脂4次，使用Kaiser试剂检测反应耦合是否完全，如不完全则2次耦合。Weigh Fmoc-Ser(Boc)-OH (0.4mmol), dissolve with 3mL 10% DMF/DMSO (v/v), add 2ml DIC/HOBt (0.4mmol/0.44mmol) condensing agent, pre-activate for 30min, then activate The good amino acid was added to the reactor, and the reaction was shaken at room temperature for 2 h. After filtering off the reaction solution, the resin was washed with 7 mL of DMF for 4 times. Kaiser reagent was used to check whether the reaction coupling was complete. If not, the coupling was performed twice.
(4)肽链的延长(4) Extension of the peptide chain
按照肽链的序列，重复上述脱保护和耦合的步骤依次连接上相应的氨基酸，直至肽链合成完毕。其中12位Lys可以采用Fmoc-Lys(Alloc)-OH、Fmoc-Lys(Dde)-OH、Fmoc-Lys(Mtt)-OH或Fmoc-Lys(ivDde)-OH等。本实例中采用Fmoc-Lys(Dde)-OH保护策略，同时N末端的His使用的是Boc-His(Boc)-OH。According to the sequence of the peptide chain, the above steps of deprotection and coupling are repeated to connect the corresponding amino acids in turn, until the synthesis of the peptide chain is completed. The 12-position Lys can be Fmoc-Lys(Alloc)-OH, Fmoc-Lys(Dde)-OH, Fmoc-Lys(Mtt)-OH or Fmoc-Lys(ivDde)-OH, etc. In this example, the Fmoc-Lys(Dde)-OH protection strategy was adopted, and the N-terminal His was Boc-His(Boc)-OH.
(5)Lys侧链的修饰(5) Modification of Lys side chain
肽链合成完毕后，加入7mL 2％水合肼/DMF(v/v)选择性脱除12位Lys的Dde保护基，Dde保护基脱除后加入0.4mmol的Fmoc-AEEA-OH，0.4mmol的DIC及0.44mmol的HOBt，震荡缩合反应2h。脱除Fmoc保护基后，再次加入0.4mmol的Fmoc-AEEA-OH，0.4mmol的DIC及 0.44mmol的HOBt，震荡缩合反应2h。脱除Fmoc保护基后，加入0.4mmol的Fmoc-Glu-OtBu，0.4mmol的DIC及0.44mmol的HOBt，震荡缩合反应2h。脱除Fmoc保护基后，加入0.4mmol的十八烷二酸单叔丁酯，0.4mmol的DIC及0.44mmol的HOBt缩合反应2h，反应完全后用7mL DMF清洗树脂4次。After the synthesis of the peptide chain, 7 mL of 2% hydrazine hydrate/DMF (v/v) was added to selectively remove the Dde protecting group of the 12-position Lys. After the Dde protecting group was removed, 0.4 mmol of Fmoc-AEEA-OH, 0.4 mmol of DIC and 0.44 mmol of HOBt, shaking condensation reaction for 2h. After removing the Fmoc protecting group, 0.4 mmol of Fmoc-AEEA-OH, 0.4 mmol of DIC and 0.44 mmol of HOBt were added again, and the condensation reaction was conducted for 2 h. After removing the Fmoc protecting group, 0.4 mmol of Fmoc-Glu-OtBu, 0.4 mmol of DIC and 0.44 mmol of HOBt were added, and the reaction was shaken for 2 h. After removing the Fmoc protecting group, 0.4 mmol of octadecanedioic acid mono-tert-butyl ester, 0.4 mmol of DIC and 0.44 mmol of HOBt were added for a condensation reaction for 2 h. After the reaction was complete, the resin was washed with 7 mL of DMF for 4 times.
(6)多肽的裂解(6) Peptide cleavage
将上述得到的连有多肽的树脂转移至圆底瓶中，使用切割剂Reagent R(TFA/苯甲硫醚/苯酚/EDT，90:5:3:2，V/V)5mL切割树脂，在油浴中恒温30℃反应2h，切割液倾入40mL冰乙醚中，冷冻离心后粗品用15mL冰乙醚洗涤3次，最后用氮气吹干，得到粗肽。Transfer the above-obtained peptide-linked resin into a round-bottomed flask, and use a cutting agent Reagent R (TFA/anisole/phenol/EDT, 90:5:3:2, V/V) 5mL to cut the resin, The reaction was carried out at a constant temperature of 30 °C in an oil bath for 2 h, and the cutting solution was poured into 40 mL of ice ether. After freezing and centrifugation, the crude product was washed three times with 15 mL of ice ether, and finally dried with nitrogen to obtain the crude peptide.
(7)多肽的纯化(7) Purification of polypeptides
将目标多肽粗品溶于水中，用0.25μm微孔滤膜过滤后进岛津制备型反相HPLC系统纯化。色谱条件为C18反相制备柱(250mm×20mm，12μm)；流动相A：0.1％TFA/水(V/V)，流动相B：甲醇(V/V)；流速为8mL/min；检测波长为214nm。采用线性梯度(20％B～80％B/30min)洗脱，收集目标峰，除去甲醇后冻干得纯品0.18g，纯度大于98％，通过LC-MS确认目标多肽的分子量。The crude target polypeptide was dissolved in water, filtered through a 0.25 μm microporous membrane, and then purified by a Shimadzu preparative reverse-phase HPLC system. Chromatographic conditions were C18 reversed-phase preparative column (250mm×20mm, 12μm); mobile phase A: 0.1% TFA/water (V/V), mobile phase B: methanol (V/V); flow rate was 8mL/min; detection wavelength is 214nm. A linear gradient (20%B～80%B/30min) was used for elution, the target peak was collected, and 0.18g of pure product was obtained by lyophilization after removing methanol. The purity was greater than 98%. The molecular weight of the target polypeptide was confirmed by LC-MS.
实施例5Example 5
SEQ ID NO:5多肽化合物的合成Synthesis of SEQ ID NO:5 polypeptide compound
His-D-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys(AEEA-AEEA-γ-Glu-CO-(CH 2) 16-COOH)-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2 His-D-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys(AEEA-AEEA-γ-Glu-CO-(CH 2 ) 16 -COOH)-Tyr-Leu-Asp -Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2
合成方法同实施例4，收集目标峰冻干得纯品0.17g。The synthetic method was the same as that in Example 4, and the target peak was collected and lyophilized to obtain 0.17g of pure product.
实施例6Example 6
SEQ ID NO:6多肽化合物的合成Synthesis of SEQ ID NO:6 polypeptide compound
His-Aib-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys(AEEA-AEEA-γ-Glu-CO-(CH 2) 16-COOH)-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2 His-Aib-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys(AEEA-AEEA-γ-Glu-CO-(CH 2 ) 16 -COOH)-Tyr-Leu-Asp-Ser -Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2
合成方法同实施例4，收集目标峰冻干得纯品0.15g。The synthetic method was the same as that in Example 4, and the target peak was collected and lyophilized to obtain 0.15 g of pure product.
实施例7Example 7
SEQ ID NO:7多肽化合物的合成Synthesis of SEQ ID NO:7 polypeptide compound
His-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Lys(γ-Glu-CO-(CH 2) 14-CH 3)-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2 His-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Lys(γ-Glu-CO-(CH 2 ) 14 -CH 3 )-Asp-Ser-Arg- Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2
合成方法同实施例1，收集目标峰冻干得纯品0.15g。The synthesis method was the same as that of Example 1, and the target peak was collected and lyophilized to obtain 0.15 g of pure product.
实施例8Example 8
SEQ ID NO:8多肽化合物的合成Synthesis of SEQ ID NO:8 polypeptide compound
His-D-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Lys(γ-Glu-CO-(CH 2) 14-CH 3)-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2 His-D-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Lys(γ-Glu-CO-(CH 2 ) 14 -CH 3 )-Asp-Ser- Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2
合成方法同实施例1，收集目标峰冻干得纯品0.16g。The synthetic method was the same as that of Example 1, and the target peak was collected and lyophilized to obtain 0.16 g of pure product.
实施例9Example 9
SEQ ID NO:9多肽化合物的合成Synthesis of SEQ ID NO:9 polypeptide compound
His-Aib-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Lys(γ-Glu-CO-(CH 2) 14-CH 3)-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2 His-Aib-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Lys(γ-Glu-CO-(CH 2 ) 14 -CH 3 )-Asp-Ser-Arg- Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2
合成方法同实施例1，收集目标峰冻干得纯品0.14g。The synthesis method was the same as that in Example 1, and the target peak was collected and lyophilized to obtain 0.14 g of pure product.
实施例10Example 10
SEQ ID NO:10多肽化合物的合成Synthesis of SEQ ID NO:10 polypeptide compound
His-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Lys(AEEA-AEEA-γ-Glu-CO-(CH 2) 16-COOH)-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2 His-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Lys(AEEA-AEEA-γ-Glu-CO-(CH 2 ) 16 -COOH)-Asp-Ser -Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2
合成方法同实施例4，收集目标峰冻干得纯品0.17g。The synthetic method was the same as that in Example 4, and the target peak was collected and lyophilized to obtain 0.17g of pure product.
实施例11Example 11
SEQ ID NO:11多肽化合物的合成Synthesis of SEQ ID NO: 11 Polypeptide Compound
His-D-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Lys(AEEA-AEEA-γ-Glu-CO-(CH 2) 16-COOH)-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2 His-D-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Lys(AEEA-AEEA-γ-Glu-CO-(CH 2 ) 16 -COOH)-Asp -Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2
合成方法同实施例4，收集目标峰冻干得纯品0.16g。The synthetic method was the same as that in Example 4, and the target peak was collected and lyophilized to obtain 0.16 g of pure product.
实施例12Example 12
SEQ ID NO:12多肽化合物的合成Synthesis of SEQ ID NO:12 polypeptide compound
His-Aib-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Lys(AEEA-AEEA-γ-Glu-CO-(CH 2) 16-COOH)-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2 His-Aib-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Lys(AEEA-AEEA-γ-Glu-CO-(CH 2 ) 16 -COOH)-Asp-Ser -Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2
合成方法同实施例4，收集目标峰冻干得纯品0.14g。The synthetic method was the same as that of Example 4, and the target peak was collected and lyophilized to obtain 0.14g of pure product.
实施例13Example 13
SEQ ID NO:13多肽化合物的合成Synthesis of SEQ ID NO:13 polypeptide compound
His-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Lys(γ-Glu-CO-(CH 2) 14-CH 3)-NH 2 His-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp- Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Lys(γ-Glu-CO-(CH 2 ) 14 -CH 3 )-NH 2
合成方法同实施例1，收集目标峰冻干得纯品0.13g。The synthetic method was the same as that of Example 1, and the target peak was collected and lyophilized to obtain 0.13 g of pure product.
实施例14Example 14
SEQ ID NO:14多肽化合物的合成Synthesis of SEQ ID NO:14 polypeptide compound
His-D-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Lys(γ-Glu-CO-(CH 2) 14-CH 3)-NH 2 His-D-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu- Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Lys(γ-Glu-CO-(CH 2 ) 14 -CH 3 )-NH 2
合成方法同实施例1，收集目标峰冻干得纯品0.15g。The synthetic method was the same as that of Example 1, and the target peak was collected and lyophilized to obtain 0.15 g of pure product.
实施例15Example 15
SEQ ID NO:15多肽化合物的合成Synthesis of SEQ ID NO:15 polypeptide compound
His-Aib-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro- Pro-Pro-Ser-Lys(γ-Glu-CO-(CH 2) 14-CH 3)-NH 2 His-Aib-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp- Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Lys(γ-Glu-CO-(CH 2 ) 14 -CH 3 )-NH 2
合成方法同实施例1，收集目标峰冻干得纯品0.16g。The synthetic method was the same as that of Example 1, and the target peak was collected and lyophilized to obtain 0.16 g of pure product.
实施例16Example 16
SEQ ID NO:16多肽化合物的合成Synthesis of SEQ ID NO:16 polypeptide compound
His-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Lys(AEEA-AEEA-γ-Glu-CO-(CH 2) 16-COOH)-NH 2 His-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp- Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Lys(AEEA-AEEA-γ-Glu-CO-(CH 2 ) 16 -COOH)-NH 2
合成方法同实施例4，收集目标峰冻干得纯品0.15g。The synthesis method was the same as that in Example 4, and the target peak was collected and lyophilized to obtain 0.15 g of pure product.
实施例17Example 17
SEQ ID NO:17多肽化合物的合成Synthesis of SEQ ID NO:17 polypeptide compound
His-D-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Lys(AEEA-AEEA-γ-Glu-CO-(CH 2) 16-COOH)-NH 2 His-D-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu- Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Lys(AEEA-AEEA-γ-Glu-CO-(CH 2 ) 16 -COOH) -NH2
合成方法同实施例4，收集目标峰冻干得纯品0.16g。The synthetic method was the same as that in Example 4, and the target peak was collected and lyophilized to obtain 0.16 g of pure product.
实施例18Example 18
SEQ ID NO:18多肽化合物的合成Synthesis of SEQ ID NO:18 polypeptide compound
His-Aib-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Lys(AEEA-AEEA-γ-Glu-CO-(CH 2) 16-COOH)-NH 2 His-Aib-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp- Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Lys(AEEA-AEEA-γ-Glu-CO-(CH 2 ) 16 -COOH)-NH 2
合成方法同实施例4，收集目标峰冻干得纯品0.15g。The synthesis method was the same as that in Example 4, and the target peak was collected and lyophilized to obtain 0.15 g of pure product.
实施例19Example 19
多肽化合物对GLP-1受体、GCG受体和GIP受体的激动活性测定Determination of Agonistic Activity of Polypeptide Compounds on GLP-1 Receptor, GCG Receptor and GIP Receptor
通过功能测定法来确定多肽化合物对受体的激动作用，所述测定法测量稳定表达人GLP-1受体、GCG受体或GIP受体的HEK-293细胞系的cAMP响应。将稳定表达上述三种受体的细胞分入T175培养瓶并在培养基(DMEM/10％FBS)中过夜生长至接近汇合状态，然后除去培养基，并用无钙和镁的PBS洗涤细胞，然后用Accutase酶进行蛋白酶处理。洗涤脱离的细胞并将其重悬于测定缓冲液(20mM HEPES,0.1％BSA,2mM IBMX,1×HBSS)中，并确定细胞密度，并将25μL的等分试样分装至96孔板的孔中。为了测量，将25μL的测试多肽化合物在测定缓冲液中的溶液添加到孔中，然后室温温育30分钟。用Cisbio的试剂盒，基于均相时间分辨荧光(HTRF)来确定细胞的cAMP含量。添加稀释于裂解缓冲液(试剂盒组分)中的HTRF试剂后，将平板温育1小时，然后测量665/620nm处的荧光比。通过检测引起最大响应的50％激活的浓度(EC 50)来对激动剂的体外效力进行量化。 The agonistic effect of polypeptide compounds on the receptor is determined by functional assays that measure the cAMP response of HEK-293 cell lines stably expressing the human GLP-1 receptor, GCG receptor or GIP receptor. Cells stably expressing the above three receptors were split into T175 flasks and grown overnight in medium (DMEM/10% FBS) to near confluence, then the medium was removed and cells were washed with PBS without calcium and magnesium, then Protease treatment with Accutase enzyme. Detached cells were washed and resuspended in assay buffer (20 mM HEPES, 0.1% BSA, 2 mM IBMX, 1 x HBSS) and cell density was determined and aliquots of 25 μL were aliquoted into 96-well plates. in the hole. For measurements, 25 [mu]L of a solution of the test polypeptide compound in assay buffer was added to the wells, followed by incubation at room temperature for 30 minutes. The cAMP content of cells was determined based on homogeneous time-resolved fluorescence (HTRF) using a kit from Cisbio. After adding HTRF reagent diluted in lysis buffer (kit component), the plate was incubated for 1 hour and then the fluorescence ratio at 665/620 nm was measured. The in vitro potency of agonists is quantified by measuring the concentration that elicits 50% activation of the maximal response ( EC50 ).
将本专利申请实施例中的检测数据(nM)显示于下表1中，虽然用一定数量的有效数字来陈述检测数据，但不应该认为表示数据已确定精确为有效数字的数。The detection data (nM) in the examples of the present patent application are shown in Table 1 below. Although the detection data is stated with a certain number of significant figures, it should not be considered that the data has been determined to be exactly the number of significant figures.
表1：多肽化合物对人GLP-1受体、GCG受体及GIP受体的EC 50值(以nM表示) Table 1: EC 50 values of polypeptide compounds for human GLP-1 receptor, GCG receptor and GIP receptor (expressed in nM)
样品sample EC 50(GLP-1受体) EC 50 (GLP-1 receptor) EC 50(GCG受体) EC 50 (GCG receptor) EC 50(GIP受体) EC 50 (GIP receptor)
GLP-1GLP-1 0.0780.078 2498.62498.6 1412.41412.4
GCGGCG 3.63.6 0.0650.065 1521.81521.8
GIPGIP 1625.81625.8 458.2458.2 0.0350.035
SEQ ID NO：1SEQ ID NO: 1 0.0520.052 0.120.12 2598.62598.6
SEQ ID NO：2SEQ ID NO: 2 0.0650.065 0.190.19 3815.13815.1
SEQ ID NO：3SEQ ID NO: 3 0.0310.031 0.0580.058 3158.63158.6
SEQ ID NO：4SEQ ID NO: 4 0.0710.071 0.350.35 2598.62598.6
SEQ ID NO：5SEQ ID NO: 5 0.0750.075 0.100.10 2815.12815.1
SEQ ID NO：6SEQ ID NO: 6 0.0650.065 0.0980.098 3158.63158.6
SEQ ID NO：7SEQ ID NO: 7 0.0210.021 0.0430.043 4215.64215.6
SEQ ID NO：8SEQ ID NO: 8 0.0250.025 0.0480.048 6869.56869.5
SEQ ID NO：9SEQ ID NO: 9 0.0180.018 0.0380.038 5358.15358.1
SEQ ID NO：10SEQ ID NO: 10 0.0350.035 0.0510.051 3359.53359.5
SEQ ID NO：11SEQ ID NO: 11 0.0410.041 0.0600.060 4015.64015.6
SEQ ID NO：12SEQ ID NO: 12 0.0330.033 0.0480.048 5869.55869.5
SEQ ID NO：13SEQ ID NO: 13 0.0490.049 0.0590.059 2869.92869.9
SEQ ID NO：14SEQ ID NO: 14 0.0350.035 0.0620.062 3215.63215.6
SEQ ID NO：15SEQ ID NO: 15 0.0610.061 0.0520.052 3015.33015.3
SEQ ID NO：16SEQ ID NO: 16 0.0770.077 0.0600.060 3225.63225.6
SEQ ID NO：17SEQ ID NO: 17 0.0750.075 0.0560.056 3568.63568.6
SEQ ID NO：18SEQ ID NO: 18 0.0720.072 0.0610.061 3348.53348.5
如表1所示，所有多肽化合物对GLP-1受体的激动活性都高于天然GLP-1，并且绝大多数多肽化合物对GCG受体的激动活性也高于天然GCG，同时所有多肽化合物都表现出了更弱的GIP受体的激动活性。As shown in Table 1, the agonistic activity of all polypeptide compounds on GLP-1 receptor is higher than that of natural GLP-1, and the agonistic activity of most polypeptide compounds on GCG receptor is also higher than that of natural GCG. Shows weaker agonistic activity of GIP receptors.
实施例20Example 20
多肽化合物的溶解度和稳定性测试Solubility and Stability Testing of Peptide Compounds
在测试多肽化合物的溶解度和稳定性之前，首先使用HPLC确定其纯度。然后，基于确定的％纯度，在不同的缓冲体系中，溶解10mg多肽化合物在1mL溶液中，温和搅拌2小时。使用4500rpm离心20分钟后，取上清液进HPLC分析，确定峰面积。然后与相应样品标准溶液比对，计算得到受试样品溶液的相对浓度。对于稳定性测试，将溶解度获得的上清液的等分试样在40℃储存7天，然后样品在4500rpm离心20分钟，上清液进HPLC分析，确定峰面积。通 过比较稳定性实验开始前的峰面积(t 0)和存储7天后的峰面积(t 7)，得到“％剩余肽”。按以下公式计算：％剩余肽＝[(峰面积t 7)×100]/峰面积t 0，稳定性表示为“％剩余肽”，计算结果如下表2所示。 Before testing the solubility and stability of the polypeptide compounds, their purity was first determined using HPLC. Then, based on the determined % purity, 10 mg of the polypeptide compound was dissolved in 1 mL of the solution in various buffer systems with gentle stirring for 2 hours. After centrifugation at 4500 rpm for 20 minutes, the supernatant was taken for HPLC analysis to determine the peak area. Then compare it with the corresponding sample standard solution, and calculate the relative concentration of the tested sample solution. For stability testing, aliquots of the supernatant obtained for solubility were stored at 40°C for 7 days, then the samples were centrifuged at 4500 rpm for 20 minutes and the supernatant was analyzed by HPLC to determine peak areas. The "% peptide remaining" was obtained by comparing the peak area before the start of the stability experiment (t 0 ) with the peak area after 7 days of storage (t 7 ). Calculated according to the following formula: % remaining peptide=[(peak area t 7 )×100]/peak area t 0 , the stability is expressed as “% remaining peptide”, and the calculation results are shown in Table 2 below.
表2：多肽化合物的溶解度和稳定性Table 2: Solubility and Stability of Peptide Compounds
Figure PCTCN2021131369-appb-000003
Figure PCTCN2021131369-appb-000003
SEQ ID NO：6SEQ ID NO: 6 9494 100100 ＞8>8 ＞8>8
SEQ ID NO：7SEQ ID NO: 7 9393 100100 ＞8>8 ＞8>8
SEQ ID NO：8SEQ ID NO: 8 9595 9898 ＞8>8 ＞8>8
SEQ ID NO：9SEQ ID NO: 9 9090 9999 ＞8>8 ＞8>8
SEQ ID NO：10SEQ ID NO: 10 8989 9999 ＞8>8 ＞8>8
SEQ ID NO：11SEQ ID NO: 11 8888 100100 ＞8>8 ＞8>8
SEQ ID NO：12SEQ ID NO: 12 9393 9898 ＞8>8 ＞8>8
SEQ ID NO：13SEQ ID NO: 13 9595 9797 ＞8>8 ＞8>8
SEQ ID NO：14SEQ ID NO: 14 9494 100100 ＞8>8 ＞8>8
SEQ ID NO：15SEQ ID NO: 15 9292 100100 ＞8>8 ＞8>8
SEQ ID NO：16SEQ ID NO: 16 9494 9999 ＞8>8 ＞8>8
SEQ ID NO：17SEQ ID NO: 17 9090 9898 ＞8>8 ＞8>8
SEQ ID NO：18SEQ ID NO: 18 8888 100100 ＞8>8 ＞8>8
如表2结果显示，本发明的多肽化合物与天然GLP-1和GCG相比，在机体可接受的注射液pH条件下的溶解性大幅改善，具备了有利于制剂的特性。本发明的多肽化合物在pH4.5也具有高溶解性，该特性可能允许用于与胰岛素或胰岛素衍生物的组合治疗的共制剂。此外，在pH 4.5和中性pH条件下本发明的多肽化合物也具有很高的稳定性。As shown in Table 2, compared with natural GLP-1 and GCG, the solubility of the polypeptide compound of the present invention is greatly improved under the pH condition of the injection solution acceptable to the body, and has the characteristics that are beneficial to the formulation. The polypeptide compounds of the present invention also have high solubility at pH 4.5, a property that may allow co-formulation for combination therapy with insulin or insulin derivatives. In addition, the polypeptide compounds of the present invention also have high stability under pH 4.5 and neutral pH conditions.
实施例21Example 21
多肽化合物对DPP-IV和NEP酶的稳定性Stability of Polypeptide Compounds to DPP-IV and NEP Enzymes
受试样品于37℃与纯化的人DPP-IV或NEP酶共孵0，2，4，8小时，使用HPLC法测定各时间点溶液中的残留样品峰面积，计算样品半衰期，结果如表3所示。The test sample was incubated with purified human DPP-IV or NEP enzyme at 37°C for 0, 2, 4, and 8 hours. The peak area of the residual sample in the solution at each time point was measured by HPLC, and the half-life of the sample was calculated. The results are shown in the table. 3 shown.
表3：多肽化合物在DPP-IV酶或NEP酶体系中的半衰期(以h表示)Table 3: Half-life of polypeptide compounds in DPP-IV enzyme or NEP enzyme system (expressed in h)
样品sample 半衰期(DPP-IV中)Half-life (in DPP-IV) 半衰期(NEP中)Half-life (in NEP)
GLP-1GLP-1 1.41.4 2.02.0
GCGGCG 1.21.2 1.71.7
SEQ ID NO：2SEQ ID NO: 2 ＞8>8 ＞8>8
SEQ ID NO：3SEQ ID NO: 3 ＞8>8 ＞8>8
SEQ ID NO：5SEQ ID NO: 5 ＞8>8 ＞8>8
SEQ ID NO：6SEQ ID NO: 6 ＞8>8 ＞8>8
SEQ ID NO：7SEQ ID NO: 7 ＞8>8 ＞8>8
SEQ ID NO：8SEQ ID NO: 8 ＞8>8 ＞8>8
SEQ ID NO：9SEQ ID NO: 9 ＞8>8 ＞8>8
SEQ ID NO：11SEQ ID NO: 11 ＞8>8 ＞8>8
SEQ ID NO：12SEQ ID NO: 12 ＞8>8 ＞8>8
SEQ ID NO：13SEQ ID NO: 13 ＞8>8 ＞8>8
SEQ ID NO：14SEQ ID NO: 14 ＞8>8 ＞8>8
SEQ ID NO：15SEQ ID NO: 15 ＞8>8 ＞8>8
SEQ ID NO：17SEQ ID NO: 17 ＞8>8 ＞8>8
SEQ ID NO：18SEQ ID NO: 18 ＞8>8 ＞8>8
如表3结果显示，本发明的多肽化合物在含DPP-IV酶溶液和NEP酶溶液体系中的半衰期均超过8个小时，说明可以有效耐受DPP-IV和NEP酶的降解。As shown in Table 3, the half-life of the polypeptide compound of the present invention in both DPP-IV enzyme solution and NEP enzyme solution system exceeds 8 hours, indicating that it can effectively withstand the degradation of DPP-IV and NEP enzymes.
实施例22Example 22
多肽化合物在大鼠体内的药代动力学性质Pharmacokinetic properties of polypeptide compounds in rats
大鼠给予50nmol/kg的皮下(s.c.)注射给药，在给药后0.25，0.5，1，2，4，8，16，24，36和48小时收集血样。使用乙腈沉淀蛋白质后，用LC-MS分析血浆样品。用WinonLin5.2.1(非房室模型)计算药代参数和半衰期(表4)。Rats were administered a subcutaneous (s.c.) injection of 50 nmol/kg and blood samples were collected at 0.25, 0.5, 1, 2, 4, 8, 16, 24, 36 and 48 hours post-dose. Plasma samples were analyzed by LC-MS after protein precipitation using acetonitrile. Pharmacokinetic parameters and half-life were calculated using WinonLin 5.2.1 (non-compartmental model) (Table 4).
表4：多肽化合物在大鼠体内的药代动力学概貌Table 4: Pharmacokinetic profile of polypeptide compounds in rats
样品sample T 1/2(h) T 1/2 (h) C max(ng/mL) Cmax (ng/mL)
LiraglutideLiraglutide 2.32.3 489489
SemaglutideSemaglutide 9.29.2 519519
SEQ ID NO:8SEQ ID NO: 8 3.93.9 539539
SEQ ID NO:9SEQ ID NO: 9 4.64.6 551551
SEQ ID NO:11SEQ ID NO: 11 11.511.5 541541
SEQ ID NO:12SEQ ID NO: 12 12.612.6 561561
如表4结果显示，本发明的多肽化合物的体内半衰期显著延长，具有支持每天一次给药或每周一次给药的药代动力学特征。As shown in Table 4, the in vivo half-life of the polypeptide compound of the present invention is significantly prolonged, and has the pharmacokinetic characteristics supporting once-daily or once-weekly administration.
实施例23Example 23
多肽化合物对糖尿病模型小鼠(db/db小鼠)血糖的影响Effects of polypeptide compounds on blood glucose in diabetic model mice (db/db mice)
雄性db/db小鼠，随机分组，每组6只。空白组皮下注射给予生理盐水(10mg/kg)，给药组分为6组，小鼠实验期间自由进食和饮水，小鼠非空腹状态下分别皮下单次注射25nmol/kg的liraglutide，semaglutide，SEQ ID NO:8，SEQ ID NO:9，SEQ ID NO:11，SEQID NO:12。在给药前0h，以及给药后4，6，24和48h用血糖仪测量各组小鼠血糖水平。Male db/db mice were randomly divided into groups of 6 mice. The blank group was subcutaneously injected with normal saline (10 mg/kg), and the administration group was divided into 6 groups. During the experiment, the mice were free to eat and drink, and the mice were subcutaneously injected with 25 nmol/kg of liraglutide, semaglutide, SEQ in a non-fasting state. ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12. Blood glucose levels of mice in each group were measured with a blood glucose meter at 0 h before administration and at 4, 6, 24 and 48 h after administration.
如图1结果所示，在db/db小鼠体内的降血糖实验结果表明，本发明的多肽化合物显示出了优于阳性对照药liraglutide和semaglutide的长效降血糖活性。As shown in the results in Figure 1, the results of the hypoglycemic experiments in db/db mice show that the polypeptide compounds of the present invention show a long-acting hypoglycemic activity superior to that of the positive control drugs liraglutide and semaglutide.
实施例24Example 24
多肽化合物对饮食诱导肥胖(DIO)小鼠血糖和体重的影响Effects of peptide compounds on blood glucose and body weight in diet-induced obesity (DIO) mice
雄性C57BL/6J小鼠，体重22g左右，模型组共42只，用Research Di ets公司的D12492高脂饲料饲养18周造DIO小鼠模型。空白对照组6只采用标准鼠饲料饲养(对照标准饮食组)。在给药开始前，各组DIO小鼠按照体重随机分组，共分为7组，每组6只，分别为生理盐水组(对照高脂饮食组)、阳性对照组(liraglutide和semaglutide)和受试样品组(SEQID NO:8、9、11、12)。对照标准饮食组和对照高脂饮食组每天两次皮下注射生理盐水(10mg/kg)，liraglutide，SEQ ID NO:8，SEQ ID NO:9组每天两次皮下注射(25nmol/kg)，semaglutide，SEQ ID NO:11，SEQ ID NO:12组每天一次皮下注射(25nmol/kg)，给药周期21天。每天记录小鼠体重变化，实验开始前和结束时使用核磁共振(NMR)来测量体脂量，在实验结束后，各组小鼠禁食12h后，口服给予葡萄糖(1.5g/kg)，并使用血糖仪测定给糖后15，30，60和120min各组小鼠的血糖数值(表5)。Male C57BL/6J mice, weighing about 22 g, with a total of 42 models in the model group, were fed with D12492 high-fat diet from Research Diets for 18 weeks to establish the DIO mouse model. The blank control group was fed with standard rat chow (control standard diet group). Before the start of administration, the DIO mice in each group were randomly divided into 7 groups according to their body weight, with 6 mice in each group, namely the normal saline group (control high-fat diet group), the positive control group (liraglutide and semaglutide) and the control group. Test sample group (SEQ ID NO: 8, 9, 11, 12). The control standard diet group and the control high-fat diet group were subcutaneously injected twice a day with normal saline (10mg/kg), liraglutide, SEQ ID NO: 8, and SEQ ID NO: 9 groups were subcutaneously injected twice a day (25nmol/kg), semaglutide, The SEQ ID NO: 11 and SEQ ID NO: 12 groups were subcutaneously injected (25 nmol/kg) once a day, and the dosing cycle was 21 days. The body weight changes of the mice were recorded every day, and nuclear magnetic resonance (NMR) was used to measure body fat before and at the end of the experiment. After the end of the experiment, the mice in each group were fasted for 12 h and then orally administered glucose (1.5 g/kg) and The blood glucose values of each group of mice were measured at 15, 30, 60 and 120 min after glucose administration using a blood glucose meter (Table 5).
表5：DIO小鼠在3周给药周期内的体重和体脂变化Table 5: Changes in body weight and body fat in DIO mice over a 3-week dosing cycle
样品(剂量)sample (dose) 整体体重变化(％)Overall body weight change (%) 体脂变化(％)Body Fat Change (%)
对照标准饮食control standard diet +1.3％(±0.6％)+1.3% (±0.6%) +6.1％(±3.2％)+6.1% (±3.2%)
对照高脂饮食control high-fat diet +0.4％(±0.4％)+0.4% (±0.4%) +2.1％(±1.0％)+2.1% (±1.0%)
Liraglutide(25nmol/kg每天两次)Liraglutide (25nmol/kg twice daily) -14.2％(±2.6％) *** -14.2% (±2.6%) *** -29.2％(±3.1％) *** -29.2% (±3.1%) ***
SEQ ID NO:8(25nmol/kg每天两次)SEQ ID NO: 8 (25nmol/kg twice daily) -28.3％(±3.2％) ***,### -28.3%(±3.2%) ***,### -54.3％(±4.4％) ***,### -54.3% (±4.4%) ***,###
SEQ ID NO:9(25nmol/kg每天两次)SEQ ID NO: 9 (25nmol/kg twice daily) -27.8％(±2.5％) ***,### -27.8%(±2.5%) ***,### -52.9％(±5.2％) ***,### -52.9%(±5.2%) ***,###
Semaglutide(25nmol/kg每天一次)Semaglutide (25nmol/kg once a day) -15.7％(±3.1％) *** -15.7% (±3.1%) *** -30.4％(±3.6％) *** -30.4% (±3.6%) ***
SEQ ID NO:11(25nmol/kg每天一次)SEQ ID NO: 11 (25nmol/kg once a day) -27.5％(±2.3％) ***,### -27.5%(±2.3%) ***,### -52.3％(±3.3％) ***,### -52.3%(±3.3%) ***,###
SEQ ID NO:12(25nmol/kg每天一次)SEQ ID NO: 12 (25nmol/kg once a day) -26.2％(±1.8％) ***,### -26.2% (±1.8%) ***,### -50.6％(±4.8％) ***,### -50.6%(±4.8%) ***,###
***：与对照高脂饮食组相比P<0.001； ###：与liraglutide和semaglutide组比P<0.001 *** : P<0.001 compared with the control high-fat diet group; ### : P<0.001 compared with the liraglutide and semaglutide groups
如表5结果显示，本发明的多肽化合物的在DIO小鼠体内连续给药3周，可以显著降低小鼠的体重和体脂含量，并且本发明的多肽化合物的作用显著强于阳性对照药 liraglutide和semaglutide。As shown in Table 5, continuous administration of the polypeptide compound of the present invention in DIO mice for 3 weeks can significantly reduce the body weight and body fat content of the mice, and the effect of the polypeptide compound of the present invention is significantly stronger than that of the positive control drug liraglutide and semaglutide.
如图2结果所示，降血糖实验结果表明，本发明的多肽化合物显示出了与阳性对照药liraglutide和semaglutide相当的降血糖活性。As shown in Fig. 2, the results of the hypoglycemic experiment show that the polypeptide compound of the present invention exhibits hypoglycemic activity equivalent to that of the positive control drugs liraglutide and semaglutide.
实施例25Example 25
多肽化合物对db/db小鼠糖化血红蛋白(HbA1c)和空腹血糖的影响Effects of polypeptide compounds on glycosylated hemoglobin (HbA1c) and fasting blood glucose in db/db mice
雄性db/db小鼠，随机分组，每组6只。适应性饲养一周后，尾部取血测量治疗开始前初始HbA1c数值和空腹血糖数值。空白组每天两次皮下注射给予生理盐水(10mg/kg)，给药组分为6组，分别皮下注射25nmol/kg的liraglutide(每天两次)，semaglutide(每天一次)，SEQ ID NO：8(每天两次)，SEQ ID NO：9(每天两次)，SEQ ID NO：11(每天一次)，SEQ ID NO：12(每天一次)。治疗周期为5周，治疗结束后小鼠禁食过夜后测量空腹血糖数值，同时取血测量HbA1c(％)数值(表6、7)。Male db/db mice were randomly divided into groups of 6 mice. After one week of adaptive feeding, tail blood was taken to measure the initial HbA1c value and fasting blood glucose value before the start of treatment. The blank group was subcutaneously injected with normal saline (10 mg/kg) twice a day, and the administration group was divided into 6 groups. twice daily), SEQ ID NO: 9 (twice daily), SEQ ID NO: 11 (once daily), SEQ ID NO: 12 (once daily). The treatment period was 5 weeks. After the treatment, the mice were fasted overnight to measure the fasting blood glucose value, and at the same time, blood was taken to measure the HbA1c (%) value (Tables 6 and 7).
表6：db/db小鼠在5周给药周期内的HbA1c(％)变化Table 6: Changes in HbA1c (%) in db/db mice during a 5-week dosing cycle
样品(剂量)sample (dose) HbA1c％(治疗前)HbA1c% (before treatment) HbA1c％(治疗后)HbA1c% (after treatment)
生理盐水normal saline 5.3±0.45.3±0.4 6.9±0.76.9±0.7
Liraglutide(25nmol/kg每天两次)Liraglutide (25nmol/kg twice daily) 5.2±0.35.2±0.3 5.3±0.25.3±0.2
SEQ ID NO：8(25nmol/kg每天两次)SEQ ID NO: 8 (25nmol/kg twice daily) 5.4±0.55.4±0.5 5.2±0.45.2±0.4
SEQ ID NO：9(25nmol/kg每天两次)SEQ ID NO: 9 (25nmol/kg twice daily) 5.1±0.25.1±0.2 5.1±0.35.1±0.3
Semaglutide(25nmol/kg每天一次)Semaglutide (25nmol/kg once a day) 5.6±0.55.6±0.5 5.8±0.35.8±0.3
SEQ ID NO：11(25nmol/kg每天一次)SEQ ID NO: 11 (25nmol/kg once daily) 5.4±0.65.4±0.6 5.3±0.45.3±0.4
SEQ ID NO：12(25nmol/kg每天一次)SEQ ID NO: 12 (25nmol/kg once daily) 5.7±0.45.7±0.4 5.6±0.3％5.6±0.3%
如表6结果显示，本发明的多肽化合物的在db/db小鼠体内连续给药5周，可以抑制HbA1c数值的增高，说明具有很好的血糖控制作用。As shown in Table 6, continuous administration of the polypeptide compound of the present invention in db/db mice for 5 weeks can inhibit the increase of HbA1c value, indicating that it has a good blood sugar control effect.
表7：db/db小鼠在5周给药周期内的空腹血糖变化Table 7: Changes in fasting blood glucose in db/db mice during a 5-week dosing cycle
样品(剂量)sample (dose) 空腹血糖(％)Fasting blood glucose (%)
生理盐水normal saline +7.3％(±0.9％)+7.3% (±0.9%)
Liraglutide(25nmol/kg每天两次)Liraglutide (25nmol/kg twice daily) -2.6％(±0.3％) *** -2.6%(±0.3%) ***
SEQ ID NO：8(25nmol/kg每天两次)SEQ ID NO: 8 (25nmol/kg twice daily) -6.6％(±0.6％) ***，## -6.6% (±0.6%) ***,##
SEQ ID NO：9(25nmol/kg每天两次)SEQ ID NO: 9 (25nmol/kg twice daily) -5.9％(±0.4％) ***，## -5.9% (±0.4%) ***,##
Semaglutide(25nmol/kg每天一次)Semaglutide (25nmol/kg once a day) -3.1％(±0.2％) *** -3.1%(±0.2%) ***
SEQ ID NO：11(25nmol/kg每天一次)SEQ ID NO: 11 (25nmol/kg once daily) -6.4％(±0.3％) ***，## -6.4% (±0.3%) ***,##
SEQ ID NO：12(25nmol/kg每天一次)SEQ ID NO: 12 (25nmol/kg once daily) -5.8％(±0.5％) ***，## -5.8% (±0.5%) ***,##
***：与生理盐水组相比P＜0.001； ##：与liraglutide和semaglutide组比P＜0.01 *** : P<0.001 compared with the normal saline group; ## : P<0.01 compared with the liraglutide and semaglutide groups
如表7结果显示，本发明的多肽化合物的在db/db小鼠体内连续给药5周，可以显著降低db/db小鼠空腹血糖数值，说明具有很高的血糖控制作用，并且本发明的多肽化合物的作用显著强于阳性对照药liraglutide和semaglutide。As shown in Table 7, continuous administration of the polypeptide compound of the present invention in db/db mice for 5 weeks can significantly reduce the fasting blood glucose value of db/db mice, indicating that it has a high blood sugar control effect, and the The effect of polypeptide compounds was significantly stronger than that of positive control drugs liraglutide and semaglutide.
实施例26Example 26
多肽化合物的免疫原性Immunogenicity of Polypeptide Compounds
采用来自50例中国人捐赠者的外周血单个核细胞(PBMC)进行了诱导T细胞增殖的免疫原性实验。PBMC在AIMV培养基中培养，并添加到24孔板(2mL)中以达到最终浓度～3×10 6cells/mL，然后通过在AIMV培养基中添加liraglutide，semaglutide，SEQ ID NO:8，SEQ ID NO:9来刺激PBMC。24孔板在37℃的二氧化碳培养箱(5％)中培养8天。第5天、第6天、第7天和第8天，将培养板各孔的细胞转移到96孔板上。用[3H]-胸腺嘧啶核苷对培养物进行处理，再培养18小时，并测定每个孔的每分钟计数(cpm)。刺激指数(SI)是通过将每个供体的试验孔的增殖反应(cpm)除以培养基处理(cpm)的增殖反应来计算的，大于2.0的SI被视为阳性。通过将整个时间过程(5-8天)内有阳性反应的捐赠者数量占接受测试的捐赠者总数的百分比来计算捐赠者的响应百分比。 Immunogenicity experiments for the induction of T cell proliferation were performed using peripheral blood mononuclear cells (PBMCs) from 50 Chinese donors. PBMCs were cultured in AIMV medium and added to 24-well plates (2 mL) to reach a final concentration of ~3 x 10 6 cells/mL, followed by addition of liraglutide, semaglutide, SEQ ID NO: 8, SEQ ID NO: 8, in AIMV medium ID NO: 9 to stimulate PBMCs. The 24-well plate was incubated in a carbon dioxide incubator (5%) at 37°C for 8 days. On days 5, 6, 7, and 8, cells from each well of the plate were transferred to 96-well plates. Cultures were treated with [3H]-thymidine, incubated for an additional 18 hours, and counts per minute (cpm) per well were determined. Stimulation index (SI) was calculated by dividing the proliferative response (cpm) for each donor's test wells by the proliferative response to the medium treatment (cpm), with an SI greater than 2.0 considered positive. Donor response percentages were calculated by dividing the number of positive donors over the entire time course (5-8 days) as a percentage of the total number of donors tested.
如图3结果所示，本发明的多肽化合物的捐赠者反应比例低于liraglutide和semaglutide，说明本发明的多肽化合物具有低免疫原性。As shown in Fig. 3, the donor response ratio of the polypeptide compound of the present invention is lower than that of liraglutide and semaglutide, indicating that the polypeptide compound of the present invention has low immunogenicity.
最后所应说明的是，以上具体实施方式仅用以说明本发明的技术方案而非限制，尽管参照实例对本发明进行了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的精神和范围，其均应涵盖在本发明的权利要求范围当中。Finally, it should be noted that the above specific embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to examples, those of ordinary skill in the art should understand that the technical solutions of the present invention can be Modifications or equivalent substitutions without departing from the spirit and scope of the technical solutions of the present invention should be included in the scope of the claims of the present invention.
Figure PCTCN2021131369-appb-000004
Figure PCTCN2021131369-appb-000004
Figure PCTCN2021131369-appb-000005
Figure PCTCN2021131369-appb-000005
Figure PCTCN2021131369-appb-000006
Figure PCTCN2021131369-appb-000006
Figure PCTCN2021131369-appb-000007
Figure PCTCN2021131369-appb-000007
Figure PCTCN2021131369-appb-000008
Figure PCTCN2021131369-appb-000008
Figure PCTCN2021131369-appb-000009
Figure PCTCN2021131369-appb-000009

Claims (10)

  1. 一类GLP-1/GCG受体双重激动多肽化合物，其特征在于，所述GLP-1/GCG受体双重激动多肽化合物的氨基酸序列通式为：A class of GLP-1/GCG receptor dual agonist polypeptide compounds, characterized in that the general amino acid sequence formula of the GLP-1/GCG receptor dual agonist polypeptide compound is:
    His-Xaa 1-Xaa 2-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Xaa 3-Xaa 4-Tyr-Xaa 5-Xaa 6-Xaa 7-Xaa 8-Xaa 9-Ala-Xaa 10-Xaa 11-Phe-Ile-Glu-Trp-Leu-Xaa 12-Xaa 13-Gly-Xaa 14-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Xaa 15 His-Xaa 1 -Xaa 2 -Gly-Thr-Tyr-Thr-Asn-Asp-Val-Xaa 3 -Xaa 4 -Tyr-Xaa 5 -Xaa 6 -Xaa 7 -Xaa 8 -Xaa 9 -Ala-Xaa 10 - Xaa 11 -Phe-Ile-Glu-Trp-Leu-Xaa 12 -Xaa 13 -Gly-Xaa 14 -Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Xaa 15
    其中：in:
    Xaa 1取自Ser，D-Ser或Aib； Xaa 1 is taken from Ser, D-Ser or Aib;
    Xaa 2取自Glu或Gln； Xaa 2 is taken from Glu or Gln;
    Xaa 3取自Thr或Ser； Xaa 3 is taken from Thr or Ser;
    Xaa 4取自Glu、Lys或侧链被修饰的Lys； Xaa 4 is taken from Glu, Lys or Lys whose side chain is modified;
    Xaa 5取自Leu、Lys或侧链被修饰的Lys； Xaa 5 is taken from Leu, Lys or Lys whose side chain is modified;
    Xaa 6取自Glu或Asp； Xaa 6 is taken from Glu or Asp;
    Xaa 7取自Glu或Ser； Xaa 7 is taken from Glu or Ser;
    Xaa 8取自Glu或Arg； Xaa 8 is taken from Glu or Arg;
    Xaa 9取自Ala或Arg； Xaa 9 is taken from Ala or Arg;
    Xaa 10取自Lys或Gln； Xaa 10 from Lys or Gln;
    Xaa 11取自Glu或Asp； Xaa 11 is taken from Glu or Asp;
    Xaa 12取自Ile或Lys； Xaa 12 taken from Ile or Lys;
    Xaa 13取自Lys或Asn； Xaa 13 from Lys or Asn;
    Xaa 14取自Lys或Gly； Xaa 14 from Lys or Gly;
    Xaa 15取自-NH 2或侧链被修饰的Lys； Xaa 15 is taken from -NH 2 or Lys whose side chain is modified;
    其中，侧链被修饰的Lys是Lys(γ-Glu-CO-(CH 2) n-CH 3)或Lys(AEEA-AEEA-γ-Glu-CO-(CH 2) n-COOH)，Lys(γ-Glu-CO-(CH 2) n-CH 3)的结构式如下式所示： Wherein, the Lys whose side chain is modified is Lys(γ-Glu-CO-(CH 2 ) n -CH 3 ) or Lys(AEEA-AEEA-γ-Glu-CO-(CH 2 ) n -COOH), Lys( The structural formula of γ-Glu-CO-(CH 2 ) n -CH 3 ) is shown below:
    Figure PCTCN2021131369-appb-100001
    Figure PCTCN2021131369-appb-100001
    Lys(AEEA-AEEA-γ-Glu-CO-(CH 2) n-COOH)的结构式如下式所示： The structural formula of Lys(AEEA-AEEA-γ-Glu-CO-(CH 2 ) n -COOH) is shown below:
    Figure PCTCN2021131369-appb-100002
    Figure PCTCN2021131369-appb-100002
    其中，n为自然数，且12≤n≤20。Among them, n is a natural number, and 12≤n≤20.
  2. 根据权利要求1所述的一类GLP-1/GCG受体双重激动多肽化合物，其特征在于，所述n是14、16、18或20。The class of GLP-1/GCG receptor dual agonist polypeptide compounds according to claim 1, wherein the n is 14, 16, 18 or 20.
  3. 根据权利要求1所述的一类GLP-1/GCG受体双重激动多肽化合物，其特征在于，所述氨基酸序列是下列序列之一：A class of GLP-1/GCG receptor dual agonist polypeptide compounds according to claim 1, wherein the amino acid sequence is one of the following sequences:
    (1)(1)
    His-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys(γ-Glu-CO-(CH 2) 14-CH 3)-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2 His-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys(γ-Glu-CO-(CH 2 ) 14 -CH 3 )-Tyr-Leu-Asp-Ser-Arg- Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2
    (2)(2)
    His-D-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys(γ-Glu-CO-(CH 2) 14-CH 3)-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2 His-D-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys(γ-Glu-CO-(CH 2 ) 14 -CH 3 )-Tyr-Leu-Asp-Ser- Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2
    (3)(3)
    His-Aib-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys(γ-Glu-CO-(CH 2) 14-CH 3)-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2 His-Aib-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys(γ-Glu-CO-(CH 2 ) 14 -CH 3 )-Tyr-Leu-Asp-Ser-Arg- Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2
    (4)(4)
    His-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys(AEEA-AEEA-γ-Glu-CO-(CH 2) 16-COOH)-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2 His-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys(AEEA-AEEA-γ-Glu-CO-(CH 2 ) 16 -COOH)-Tyr-Leu-Asp-Ser -Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2
    (5)(5)
    His-D-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys(AEEA-AEEA-γ-Glu-CO-(CH 2) 16-COOH)-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2 His-D-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys(AEEA-AEEA-γ-Glu-CO-(CH 2 ) 16 -COOH)-Tyr-Leu-Asp -Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2
    (6)(6)
    His-Aib-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys(AEEA-AEEA-γ-Glu-CO-(CH 2) 16-COOH)-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2 His-Aib-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys(AEEA-AEEA-γ-Glu-CO-(CH 2 ) 16 -COOH)-Tyr-Leu-Asp-Ser -Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2
    (7)(7)
    His-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Lys(γ-Glu-CO-(CH 2) 14-CH 3)-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2 His-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Lys(γ-Glu-CO-(CH 2 ) 14 -CH 3 )-Asp-Ser-Arg- Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2
    (8)(8)
    His-D-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Lys(γ-Glu-CO-(CH 2) 14-CH 3)-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2 His-D-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Lys(γ-Glu-CO-(CH 2 ) 14 -CH 3 )-Asp-Ser- Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2
    (9)(9)
    His-Aib-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Lys(γ-Glu-CO-(CH 2) 14-CH 3)-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2 His-Aib-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Lys(γ-Glu-CO-(CH 2 ) 14 -CH 3 )-Asp-Ser-Arg- Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2
    (10)(10)
    His-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Lys(AEEA-AEEA-γ-Glu-CO-(CH 2) 16-COOH)-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2 His-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Lys(AEEA-AEEA-γ-Glu-CO-(CH 2 ) 16 -COOH)-Asp-Ser -Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2
    (11)(11)
    His-D-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Lys(AEEA-AEEA-γ-Glu-CO-(CH 2) 16-COOH)-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn- Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2 His-D-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Lys(AEEA-AEEA-γ-Glu-CO-(CH 2 ) 16 -COOH)-Asp -Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2
    (12)(12)
    His-Aib-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Lys(AEEA-AEEA-γ-Glu-CO-(CH 2) 16-COOH)-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2 His-Aib-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Lys(AEEA-AEEA-γ-Glu-CO-(CH 2 ) 16 -COOH)-Asp-Ser -Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2
    (13)(13)
    His-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Lys(γ-Glu-CO-(CH 2) 14-CH 3)-NH 2 His-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp- Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Lys(γ-Glu-CO-(CH 2 ) 14 -CH 3 )-NH 2
    (14)(14)
    His-D-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Lys(γ-Glu-CO-(CH 2) 14-CH 3)-NH 2 His-D-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu- Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Lys(γ-Glu-CO-(CH 2 ) 14 -CH 3 )-NH 2
    (15)(15)
    His-Aib-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Lys(γ-Glu-CO-(CH 2) 14-CH 3)-NH 2 His-Aib-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp- Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Lys(γ-Glu-CO-(CH 2 ) 14 -CH 3 )-NH 2
    (16)(16)
    His-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Lys(AEEA-AEEA-γ-Glu-CO-(CH 2) 16-COOH)-NH 2 His-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp- Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Lys(AEEA-AEEA-γ-Glu-CO-(CH 2 ) 16 -COOH)-NH 2
    (17)(17)
    His-D-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Lys(AEEA-AEEA-γ-Glu-CO-(CH 2) 16-COOH)-NH 2 His-D-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu- Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Lys(AEEA-AEEA-γ-Glu-CO-(CH 2 ) 16 -COOH) -NH2
    (18)(18)
    His-Aib-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Lys(AEEA-AEEA-γ-Glu-CO-(CH 2) 16-COOH)-NH 2。 His-Aib-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp- Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Lys(AEEA-AEEA-γ-Glu-CO-(CH 2 ) 16 -COOH)-NH 2 .
  4. 权利要求1中所述GLP-1/GCG受体双重激动多肽化合物的药学上可接受的盐。A pharmaceutically acceptable salt of the GLP-1/GCG receptor dual agonist polypeptide compound of claim 1.
  5. 根据权利要求4所述的GLP-1/GCG受体双重激动多肽化合物的药学上可接受的盐，其特在于，所述盐为GLP-1/GCG受体双重激动多肽化合物与下述化合物中的一种所形成的盐：氢溴酸、盐酸、甲磺酸、磷酸、乙磺酸、甲酸、对甲苯磺酸、乙酸、乙酰乙酸、丙酮酸、果胶酯酸、丁酸、己酸、苯磺酸、庚酸、十一烷酸、苯甲酸、水杨酸、月桂酸、2-(4-羟基苯甲酰基)苯甲酸、肉桂酸、樟脑酸、环戊烷丙酸、3-羟基-2-萘甲酸、樟脑磺酸、二葡糖酸、烟酸、扑酸、丙酸、过硫酸、、苦味酸、3-苯基丙酸、特戊酸、衣康酸、2-羟基乙磺酸、氨基磺酸、十二烷基硫酸、三氟甲磺酸、萘二磺酸、2-萘磺酸、柠檬酸、扁桃酸、抗坏血酸、酒硬脂酸、石酸、草酸、乳酸、琥珀酸、丙二酸、半硫酸、苹果酸、马来酸、藻酸、富马酸、D-葡糖酸、甘油磷酸、葡庚酸、天冬氨酸、硫氰酸、磺基水杨酸。The pharmaceutically acceptable salt of the GLP-1/GCG receptor dual agonist polypeptide compound according to claim 4, wherein the salt is one of the GLP-1/GCG receptor dual agonist polypeptide compound and the following compounds A salt formed of: hydrobromic acid, hydrochloric acid, methanesulfonic acid, phosphoric acid, ethanesulfonic acid, formic acid, p-toluenesulfonic acid, acetic acid, acetoacetic acid, pyruvic acid, pectin ester acid, butyric acid, caproic acid, Benzenesulfonic acid, heptanoic acid, undecanoic acid, benzoic acid, salicylic acid, lauric acid, 2-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, camphoric acid, cyclopentanepropionic acid, 3-hydroxy -2-Naphthoic acid, camphorsulfonic acid, digluconic acid, niacin, pamoic acid, propionic acid, persulfuric acid, picric acid, 3-phenylpropionic acid, pivalic acid, itaconic acid, 2-hydroxyethyl Sulfonic acid, sulfamic acid, dodecyl sulfuric acid, trifluoromethanesulfonic acid, naphthalenedisulfonic acid, 2-naphthalenesulfonic acid, citric acid, mandelic acid, ascorbic acid, wine stearic acid, tartaric acid, oxalic acid, lactic acid, Succinic acid, malonic acid, hemisulfuric acid, malic acid, maleic acid, alginic acid, fumaric acid, D-gluconic acid, glycerophosphoric acid, glucoheptanoic acid, aspartic acid, thiocyanic acid, sulfosalicylic acid acid.
  6. 含有GLP-1/GCG受体双重激动多肽化合物的药物组合物，其特征在于，包括：权利要求1中所述GLP-1/GCG受体双重激动多肽化合物或权利要求4中所述药学上可接受的盐，以及药学上可接受的载体或稀释剂。A pharmaceutical composition containing a GLP-1/GCG receptor dual agonist polypeptide compound, characterized in that it comprises: the GLP-1/GCG receptor dual agonist polypeptide compound described in claim 1 or the pharmaceutically acceptable compound described in claim 4 accepted salts, as well as pharmaceutically acceptable carriers or diluents.
  7. 含有权利要求1中所述GLP-1/GCG受体双重激动多肽化合物的药剂，其特征在于，包括：GLP-1/GCG受体双重激动多肽化合物及药学上可接受的药用辅料、载体或稀释剂。A medicament containing the GLP-1/GCG receptor dual agonist polypeptide compound described in claim 1, characterized in that it comprises: a GLP-1/GCG receptor dual agonist polypeptide compound and a pharmaceutically acceptable pharmaceutical excipient, carrier or thinner.
  8. 根据要求7所述的药剂，其特征在于，所述药剂是药剂学上所说的胶囊、片剂、喷雾剂、吸入剂、注射剂、贴剂、乳剂、膜剂、散剂或者复方制剂，药剂由GLP-1/GCG受体双重激动多肽化合物和药学上可接受的药用辅料、载体或稀释剂组成。The medicament according to claim 7, characterized in that, the medicament is a capsule, tablet, spray, inhalation, injection, patch, emulsion, film, powder or compound preparation mentioned in pharmacy, and the medicament is composed of It is composed of a GLP-1/GCG receptor dual agonist polypeptide compound and a pharmaceutically acceptable pharmaceutical excipient, carrier or diluent.
  9. GLP-1/GCG受体双重激动多肽化合物、其药学上可接受的盐、及其药物组合物或其药剂在制备用于治疗代谢性疾病或病症的药物中的应用。Use of GLP-1/GCG receptor dual agonist polypeptide compounds, pharmaceutically acceptable salts thereof, and pharmaceutical compositions or agents thereof in the preparation of medicaments for the treatment of metabolic diseases or disorders.
  10. 根据权利要求9所述的应用，其特征在于，所述代谢性疾病或病症为糖尿病、NAFLD、NASH、高血脂或肥胖。The use according to claim 9, wherein the metabolic disease or disorder is diabetes, NAFLD, NASH, hyperlipidemia or obesity.
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