A glucagon peptide-1 and glucagon receptor dual agonist polypeptide and uses thereof

Abstract

The application discloses a glucagon peptide-1 and glucagon receptor dual-agonistic polypeptide and application thereof, and the polypeptide structure is shown in the following general formula (I). The dual-agonistic polypeptide has dual-agonistic activity on human GLP-1 and glucagon receptors. The GLP-1 / glucagon receptor dual-agonistic polypeptide can effectively reduce blood sugar and weight, and has obvious lipid-regulating effect. The GLP-1 / glucagon receptor dual-agonistic polypeptide is stable in vivo, and is suitable to be used as an active component of drugs for diabetes, obesity, hyperlipidemia and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to a glucagon glucagon-like peptide-1 and glucagon receptor dual agonist polypeptide and its application, and belongs to the technical field of polypeptides. BACKGROUND

[0002] Obesity and its related metabolic syndrome have become a global public health problem, and the incidence and course development of many metabolic syndromes such as type 2 diabetes (T2DM), non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), and abnormal blood lipid metabolism are closely related to obesity. GLP-1 is a glucose-dependent hypoglycemic polypeptide hormone secreted by small intestinal L cells, and the most important function is to promote insulin secretion. GLP-1 can inhibit appetite and delay gastric emptying to achieve weight loss. Although GLP-1 has excellent hypoglycemic effect and certain weight loss effect, if better weight loss effect is needed, the dosage generally needs to be increased, and large doses of GLP-1 drugs can easily cause gastrointestinal side effects, poor tolerance and narrow therapeutic window. Therefore, there is still a need for more safe and tolerable therapeutic agents that can effectively reduce body weight and control blood glucose.

[0003] Glucagon is a hormone produced in the alpha cells of the pancreas, which acts on the liver to decompose glycogen in the liver and increase blood glucose in the body under stress such as cold and hunger. In addition to its blood glucose-raising effect, glucagon also has the effects of promoting lipolysis, fat oxidation, and fever in the body (Diabetologia, 2017, 60, 1851-1861). Long-term administration can present a weight loss effect by increasing energy metabolism, but the beneficial effects of glucagon on energy metabolism cannot be applied due to its inherent blood glucose-raising effect. Peripheral administration of GLP-1 and glucagon in animals and humans can simultaneously achieve the effects of reducing blood glucose and reducing body weight. SUMMARY

[0004] The present application provides a glucagon glucagon-like peptide-1 and glucagon receptor dual agonist polypeptide and its application, which retains the therapeutic effect of GLP-1 analogs on diabetes while having the beneficial effects of glucagon on metabolism, thereby producing a synergistic effect on sugar, lipid, and energy metabolism, and has more potential than single receptor agonists in the preparation of drugs for treating metabolic syndromes such as diabetes, obesity, and dyslipidemia.

[0005] Technical scheme: In order to achieve the above-mentioned purpose, the present application adopts the following technical scheme:

[0006] A glucagon-like peptide-1 and glucagon (GLP-1 / glucagon) receptor dual agonist polypeptide or a pharmaceutically acceptable salt thereof, the polypeptide having a structure according to the following general formula (I):

[0007]

[0008] wherein X1 is dextrorotatory serine or α-aminoisobutyric acid, X2 is KY or is absent, X3 is YL or is absent, X4 is SRRAQDFVQW or EEAVRLFIEW, and R1 is selected from one of the following structures:

[0009]

[0010] Preferably, the glucagon-like peptide-1 and glucagon receptor dual agonist polypeptide is selected from the following:

[0011] (1) Polypeptide 1

[0012]

[0013] (2) Polypeptide 2

[0014]

[0015] (3) Polypeptide 3

[0016]

[0017] (4) Polypeptide 4

[0018]

[0019] (5) Polypeptide 5

[0020]

[0021] (6) Polypeptide 6

[0022]

[0023] (7) Polypeptide 7

[0024]

[0025] (8) Polypeptide 8

[0026]

[0027] (9) Polypeptide 9

[0028]

[0029] (10) Polypeptide 10

[0030]

[0031] Preferably, the salt is a salt of the glucagon peptide-1 and glucagon receptor dual agonist polypeptide with one of the following compounds: acetic acid, salicylic acid, lauric acid, cinnamic acid, lactic acid, or succinic acid.

[0032] The method for synthesizing the glucagon peptide-1 and glucagon receptor dual agonist polypeptide comprises the following steps:

[0033] First, the resin is swelled, the Fmoc protecting group is removed, then the Fmoc-Ser-Rink amide-MBHA resin is synthesized, and then the peptide chain is elongated, the Lys side chain is modified, and finally the polypeptide on the resin is cleaved and purified.

[0034] The present application also provides a pharmaceutical composition comprising a therapeutically effective amount of at least one of the glucagon peptide-1 and glucagon receptor dual agonist polypeptides or pharmaceutically acceptable salts thereof, and a pharmaceutically acceptable carrier and / or excipient. The pharmaceutical composition can be any one of the pharmaceutically described tablets, capsules, inhalants, sprays, injections, films, patches, emulsions, or compound preparations.

[0035] The present application finally provides the use of the glucagon peptide-1 and glucagon receptor dual agonist polypeptides or pharmaceutically acceptable salts thereof, the pharmaceutical composition in the preparation of a medicament for treating metabolic diseases.

[0036] In particular aspects, the metabolic disease or disorder is diabetes, obesity, dyslipidemia. In particular aspects, the diabetes is T1DM, T2DM, or gestational diabetes. In particular aspects, the medicament is used to treat more than one metabolic disease or disorder, e.g., diabetes and obesity; obesity and dyslipidemia; diabetes and dyslipidemia; diabetes, dyslipidemia, and obesity.

[0037] Technical effects: Compared with the prior art, the present application provides a kind of polypeptide compound with GLP-1 / glucagon receptor dual agonism, the polypeptide retains the therapeutic effect of GLP-1 analogue on diabetes while having the beneficial effect of glucagon on metabolism, thereby having a synergistic effect on sugar, lipid and energy metabolism, effectively reducing blood sugar while significantly reducing weight loss and very excellent lipid-lowering effect.In addition, the GLP-1 / glucagon receptor dual agonism polypeptide compound provided by the present application is chemically stable and has pharmacokinetic characteristics supporting once-a-day or once-a-week administration.The dual-agonist polypeptide compound provided by the present application has a significantly better therapeutic effect on T2DM, obesity, dyslipidemia and other metabolic diseases than existing marketed drugs.Therefore, the dual-agonist polypeptide compound provided by the present application is suitable as an active ingredient for treating metabolic diseases such as diabetes, obesity and dyslipidemia. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 The acute hypoglycemic effect of single administration of each test substance on ICR mice is shown;

[0039] Figure 2 The percentage change in body weight of each test substance in DIO mice after 21 days of long-term administration is shown; DETAILED DESCRIPTION

[0040] The present application will be further illustrated below in conjunction with specific examples.

[0041] Unless otherwise defined herein, scientific and technical terms used in this application shall have the meanings commonly understood by one of ordinary skill in the art. Generally, the terms and methods described herein used in connection with chemistry, biology, pharmacology are well known and commonly used in the art.

[0042] In addition, the amino acids in the present application are abbreviated according to the naming rules of IUPAC-IUB as follows:

[0043] Alanine (Ala, A); Arginine (Arg, R); Asparagine (Asn, N); Aspartic acid (Asp, D); Cysteine (Cys, C); Glutamic acid (Glu, E); Glutamine (Gln, Q); Glycine (Gly, G); Histidine (His, H); Isoleucine (Ile, I); Leucine (Leu, L); Lysine (Lys, K); Methionine (Met, M); Phenylalanine (Phe, F); Proline (Pro, P); Serine (Ser, S); Threonine (Thr, T); Tryptophan (Trp, W); Tyrosine (Tyr, Y); Valine (Val, V).

[0044] In addition, all of the amino acid residues in the polypeptide compounds of the present application are preferably in the L configuration, unless explicitly indicated otherwise.

[0045] In addition, the "-NH2" moiety on the C-terminus of the sequence indicates an amide group (-CONH2) on the C-terminus.

[0046] In addition, the non-natural amino acid d-serine (S) and α-amino isobutyric acid (Aib) are used in the sequence of the present application in addition to the natural amino acids. d Ser, d S) and α-amino isobutyric acid (Aib)

[0047] The present application is illustrated by the following examples, which are not intended to limit the scope of the present application in any way.

[0048] Example 1

[0049] Synthesis of polypeptide 1

[0050]

[0051] (1) Swelling of the resin

[0052] Rink Amide MBHA resin with a loading of 0.36 mmol / g was weighed at 0.278 g (0.1 mmol equivalent), placed in a 25 mL reactor, and washed with 7 mL of DCM and methanol alternately once, 7 mL of DCM twice, then swelled with 7 mL of DCM for 1 h, and finally washed with 7 mL of DMF three times.

[0053] (2) Removal of the Fmoc protecting group of the resin

[0054] The swelled resin was transferred to a PSI-200 polypeptide synthesizer, 7 mL of 20% piperidine / DMF (v / v) was added, and the reaction was allowed to proceed at room temperature for 5 min. The deprotection solution was filtered off, the resin was washed once with 7 mL of DMF, 7 mL of 20% piperidine / DMF (v / v) was added again, and the reaction was allowed to proceed for 15 min. Finally, the resin was washed with 7 mL of DMF four times for 2 min each time, to obtain the Fmoc-protected Rink resin.

[0055] (3) Synthesis of Fmoc-Ser-Rink amide-MBHA Resin

[0056] Fmoc-Ser(tBu)-OH (0.4 mmol) was dissolved in 2 mL DMF, 3 mL DIC / HOBt (0.4 mmol / 0.44 mmol) condensing agent was added into the reactor, and the reaction was shaken at room temperature for 2 h. After the reaction solution was filtered off, the resin was washed with 7 mL DMF for 4 times. The reaction coupling was detected by Kaiser reagent. If it was not complete, the coupling was repeated twice.

[0057] (4) Extension of the peptide chain

[0058] According to the sequence of the peptide chain, the above-mentioned deprotection and coupling steps were repeated to sequentially connect the corresponding amino acids until the synthesis of the peptide chain was completed. Among them, the Lys at the side chain modification site was protected by Fmoc-Lys(Dde)-OH, and the His at the N terminus was protected by Boc-His(Boc)-OH.

[0059] (5) Modification of the Lys side chain

[0060] After the synthesis of the peptide chain was completed, 7 mL of 2% hydrazine hydrate / DMF (v / v) was added to selectively remove the Dde protecting group of Lys. After the Dde protecting group was removed, 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. Then, the Fmoc protecting group was removed using the same method as described above, and 0.4 mmol of palmitic acid, 0.4 mmol of DIC, and 0.44 mmol of HOBt were added for condensation reaction for 2 h. After the reaction was completed, the resin was washed with 7 mL of DMF for 4 times.

[0061] (6) Cleavage of the polypeptide

[0062] The resin with the polypeptide obtained above was transferred to a round-bottom flask, and 5 mL of the cleavage reagent Reagent R (TFA / phenylmethanethiol / phenol / EDT, 90:5:3:2, V / V) was used to cleave the resin. The reaction was carried out at a constant temperature of 30°C in an oil bath for 2 h. The cleavage solution was poured into 40 mL of ice ethyl ether, and the crude product was washed with 15 mL of ice ethyl ether for 3 times. Finally, the nitrogen was blown dry to obtain the crude peptide.

[0063] (7) Purification and characterization of the polypeptide

[0064] The crude target polypeptide was dissolved in water and filtered through a 0.25 μm filter membrane before purification on a Shimadzu preparative reverse-phase HPLC system. The chromatographic conditions were as follows: C18 reverse-phase preparative column (250 mm x 20 mm, 12 μm); mobile phase A: 0.1% TFA / water (v / v), mobile phase B: methanol (v / v); flow rate: 8 mL / min; detection wavelength: 214 nm. Elution was performed using a linear gradient (20% B to 70% B over 30 min), and the target peak was collected. After removal of the methanol, the pure product was obtained by lyophilization. The purity was greater than 98%, and the molecular weight of the target polypeptide was confirmed by LC-MS. The theoretical relative molecular mass was 4632.0. ESI-MS m / z: calculated value [M+3H] 3+ 1545.0, [M+4H] 4+ 1159.0; observed value [M+3H] 3+ 1544.5, [M+4H] 4+ 1158.6.

[0065] Example 2

[0066] Synthesis of polypeptide 2

[0067]

[0068] (1) Swelling of the resin

[0069] Rink Amide MBHA resin with a loading of 0.382 mmol / g was weighed at 0.262 g (0.1 mmol equivalent), and was placed in a 25 mL reactor. The resin was washed once with 7 mL of DCM and methanol alternately, twice with 7 mL of DCM, then swelled with 7 mL of DCM for 1 h, and finally washed with 7 mL of DMF for 3 times.

[0070] (2) Removal of the Fmoc protecting group of the resin

[0071] The swelled resin was transferred to a PSI-200 polypeptide synthesizer, and 7 mL of 20% piperidine / DMF (v / v) was added for reaction at room temperature for 5 min. The deprotection solution was filtered off, and the resin was washed once with 7 mL of DMF, then 7 mL of 20% piperidine / DMF (v / v) was added for reaction with the resin for 15 min. Finally, the resin was washed 4 times with 7 mL of DMF for 1.5 min each time, to obtain the Rink resin with the Fmoc protecting group removed.

[0072] (3) Synthesis of Fmoc-Ser-Rink amide-MBHA Resin

[0073] Fmoc-Ser(Boc)-OH (0.4 mmol) was dissolved in 3 mL of 10% DMF / DMSO (v / v), 2 mL of DIC / HOBt (0.4 mmol / 0.44 mmol) was added as a condensing agent, and after pre-activation for 30 min, the activated amino acid was added to the reactor, and the reaction was shaken at room temperature for 2 h. After the reaction solution was filtered off, the resin was washed with 7 mL of DMF four times, and the Kaiser reagent was used to determine whether the coupling was complete. If not, the coupling was repeated twice.

[0074] (4) Extension of the peptide chain

[0075] According to the sequence of the peptide chain, the above-mentioned deprotection and coupling steps were repeated to sequentially connect the corresponding amino acids until the synthesis of the peptide chain was completed. Among them, the Lys at position 12 can use Fmoc-Lys(Alloc)-OH, Fmoc-Lys(Dde)-OH, Fmoc-Lys(Mtt)-OH or Fmoc-Lys(ivDde)-OH, etc. In this example, Fmoc-Lys(Dde)-OH protection strategy was used, and Boc-His(Boc)-OH was used for the His at the N-terminus.

[0076] (5) Modification of the side chain of Lys

[0077] After the synthesis of the peptide chain was completed, 7 mL of 2% hydrazine hydrate / DMF (v / v) was added to selectively remove the Dde protecting group of Lys at position 12. 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 were added, and the condensation reaction was shaken for 2 h. After the Fmoc protecting group was removed, 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 shaken for 2 h. After the Fmoc protecting group was removed, 0.4 mmol of Fmoc-Glu-OtBu, 0.4 mmol of DIC and 0.44 mmol of HOBt were added, and the condensation reaction was shaken for 2 h. After the Fmoc protecting group was removed, 0.4 mmol of octadecanedioic acid mono-tert-butyl ester, 0.4 mmol of DIC and 0.44 mmol of HOBt were added, and the condensation reaction was shaken for 2 h. After the reaction was completed, the resin was washed with 7 mL of DMF four times.

[0078] (6) Cleavage of the polypeptide

[0079] The resin with the polypeptide was transferred to a round bottom flask, and the resin was cleaved using cleavage reagent Reagent R (TFA / benzyl mercaptide / phenol / EDT, 90:5:3:2, V / V) 5 mL, and reacted at 30°C in an oil bath for 2 h. The cleavage solution was poured into 40 mL of ice ethyl ether, and the crude product was washed with 15 mL of ice ethyl ether for 3 times after being frozen and centrifuged. Finally, the product was dried by nitrogen blowing. The crude peptide was obtained.

[0080] (7) Purification and characterization of polypeptides

[0081] The crude target polypeptide was dissolved in water, filtered by a 0.25 μm microporous filter, and then purified by a Shimadzu preparative reverse-phase HPLC system. The chromatographic conditions were as follows: a C18 reverse-phase preparative column (250 mm x 20 mm, 12 μm); mobile phase A: 0.1% TFA / water (V / V), mobile phase B: methanol (V / V); flow rate: 8 mL / min; detection wavelength: 214 nm. The linear gradient (20% B to 80% B / 30 min) was used for elution, and the target peak was collected. After removing methanol, the pure product 0.10 g was obtained by freeze-drying. The purity was greater than 98%, and the molecular weight of the target polypeptide was confirmed by LC-MS. The theoretical relative molecular mass was 4980.5. ESI-MS m / z: calculated value [M+3H] 3+ 1661.2, [M+4H] 4+ 1246.1; observed value [M+3H] 3+ 1660.5, [M+4H] 4+ 1245.6.

[0082] Example 3

[0083] Synthesis of polypeptide 3

[0084]

[0085] The synthesis method was the same as in Example 1. The pure product 0.16 g was obtained by collecting the target peak and freeze-drying. The purity was greater than 98%, and the molecular weight of the target polypeptide was confirmed by MS. The theoretical relative molecular mass was 4616.2. ESI-MS m / z: calculated value [M+3H] 3+ 1539.7, [M+4H] 4+ 1155.1; observed value [M+3H] 3+ 1539.1, [M+4H] 4+ 1154.6.

[0086] Example 4

[0087] Synthesis of polypeptide 4

[0088]

[0089] The synthesis method was the same as in Example 2. The target peak was collected and lyophilized to obtain 0.17 g of pure product with a purity greater than 98%. The molecular weight of the target peptide was confirmed by MS. The theoretical relative molecular mass is 4964.5. ESI-MS m / z: calculated value [M+3H] 3+ 1655.9, [M+4H] 4+ 1242.1; Observation [M+3H] 3+ 1655.5, [M+4H] 4+ 1241.6.

[0090] Example 5

[0091] Synthesis of polypeptide compounds

[0092]

[0093] The synthesis method was the same as in Example 1. The target peak was collected and lyophilized to obtain 0.12 g of pure product with a purity greater than 98%. The molecular weight of the target peptide was confirmed by MS. The theoretical relative molecular mass is 4631.2. ESI-MS m / z: calculated value [M+3H] 3+ 1544.7, [M+4H] 4+ 1158.8; Observation [M+3H] 3+ 1544.1, [M+4H] 4+ 1158.3.

[0094] Example 6

[0095] Synthesis of polypeptide 6 polypeptide compounds

[0096]

[0097] The synthesis method was the same as in Example 2. The target peak was collected and lyophilized to obtain 0.13 g of pure product with a purity greater than 98%. The molecular weight of the target polypeptide was confirmed by MS. The theoretical relative molecular mass is 4979.6. ESI-MS m / z: calculated value [M+3H] 3+ 1660.9, [M+4H] 4+ 1245.9; Observation [M+3H] 3+ 1660.2, [M+4H] 4+ 1245.4.

[0098] Example 7

[0099] Synthesis of polypeptide compounds

[0100]

[0101] The synthetic method is the same as Example 1. The pure product of 0.15 g with purity greater than 98% was collected by freeze-drying after collecting the target peak. The molecular weight of the target polypeptide was confirmed by MS. The theoretical relative molecular mass is 4614.2. ESI-MS m / z: calculated value [M+3H] 3+ 1539.1, [M+4H] 4+ 1154.6; observed value [M+3H] 3+ 1538.5, [M+4H] 4+ 1154.1.

[0102] Example 8

[0103] Synthesis of polypeptide 8 polypeptide compound

[0104] The synthetic method is the same as Example 2. The pure product of 0.14 g with purity greater than 98% was collected by freeze-drying after collecting the target peak. The molecular weight of the target polypeptide was confirmed by MS. The theoretical relative molecular mass is 4962.5. ESI-MS m / z: calculated value [M+3H] 3+ 1655.2, [M+4H] 4+ 1241.6; observed value [M+3H] 3+ 1654.5, [M+4H] 4+ 1241.1.

[0105] Example 9

[0106] Synthesis of polypeptide 9 polypeptide compound

[0107]

[0108] The synthetic method is the same as Example 1. The pure product of 0.15 g with purity greater than 98% was collected by freeze-drying after collecting the target peak. The molecular weight of the target polypeptide was confirmed by MS. The theoretical relative molecular mass is 4629.2. ESI-MS m / z: calculated value [M+3H] 3+ 1544.1, [M+4H] 4+ 1158.3; observed value [M+3H] 3+ 1543.5, [M+4H] 4+ 1157.9.

[0109] Example 10

[0110] Synthesis of polypeptide 10 polypeptide compound

[0111]

[0112] Synthesis method is the same as example 2, collect target peak freeze-dried pure product 0.12g, purity greater than 98%, by MS to confirm the molecular weight of the target polypeptide. The theoretical relative molecular mass is 4977.6. ESI-MS m / z: calculated value [M+3H] 3+ 1660.2, [M+4H] 4+ 1245.4; observed value [M+3H] 3+ 1659.8, [M+4H] 4+ 1244.9.

[0113] Example 11

[0114] Determination of agonist activity of polypeptide compounds on human GLP-1 receptor and glucagon receptor

[0115] The agonist effect of polypeptide compounds on the receptors, GLP-1 receptor and glucagon receptor agonist activity, is determined by measuring the cAMP response of HEK-293 cell lines stably expressing human GLP-1 receptor or glucagon receptor. Cells stably expressing GLP-1 receptor or glucagon receptor are divided into T175 flasks and grown overnight to near confluency in culture medium (DMEM / 10% FBS), then the culture medium is removed and the cells are washed with calcium and magnesium free PBS, then protease treated with Accutase enzyme. The detached cells are washed and resuspended in assay buffer (20 mM HEPES, 0.1% BSA, 2 mM IBMX, 1x HBSS) and the cell density is determined and 25 μL aliquots are dispensed into wells of a 96-well plate. For the measurement, 25 μL of test polypeptide compound in assay buffer is added to the wells, then incubated at room temperature for 30 minutes. The cAMP content of the cells is determined based on homogeneous time-resolved fluorescence (HTRF) using a kit from Cisbio. After addition of HTRF reagents diluted in lysis buffer (kit components), the plate is incubated for 30 minutes, then the fluorescence ratio at 665 / 620 nm is measured. The in vitro potency of agonists is quantified by detecting the concentration that causes 50% activation of the maximum response (EC 50 ).

[0116] The test data (nM) in the examples of the present patent application are shown in Table 1 below, although the test data is stated with a certain number of significant figures, it should not be considered to mean that the data has been determined to be accurate to the number of significant figures.

[0117] Table 1: Agonist activity of polypeptide compounds on human GLP-1 receptor and glucagon receptor

[0118]

[0119]

[0120] As shown in Table 1, all the polypeptide compounds showed dual agonistic activity to GLP-1 receptor and glucagon receptor, which indicated that these polypeptide compounds met the characteristics of the dual agonistic polypeptide described in the present patent. Meanwhile, polypeptide 1 compound showed better agonistic activity to GLP-1 receptor and glucagon receptor than GLP-1 and glucagon.

[0121] Example 12

[0122] Solubility and stability test of polypeptide compounds

[0123] Before testing the solubility and stability of polypeptide compounds, the purity of the polypeptide compounds was first determined by HPLC. Then, based on the determined % purity, 10 mg of polypeptide compound was dissolved in 1 mL solution in different buffer systems, and stirred gently for 2 hours. After centrifugation at 4500 rpm for 20 minutes, the supernatant was analyzed by HPLC to determine the peak area. Then, compared with the corresponding sample standard solution, the relative concentration of the test sample solution was calculated. For the stability test, an aliquot of the supernatant obtained by solubility was stored at 40°C for 7 days, and then the sample was centrifuged at 4500 rpm for 20 minutes, and the supernatant was analyzed by HPLC to determine the peak area. By comparing the peak area before the stability experiment (t0) and the peak area after 7 days of storage (t7), the "% remaining peptide" was obtained. The calculation formula is as follows: % remaining peptide = [(peak area t7) x 100] / peak area t0, and the stability is expressed as "% remaining peptide", and the calculation results are shown in Table 2 below.

[0124] Table 2: Solubility and stability of polypeptide compounds

[0125]

[0126]

[0127] As shown in Table 2, the solubility of the polypeptide compounds of the present application was greatly improved compared with natural GLP-1 and glucagon under the pH conditions of the body-acceptable injection solution, which possessed the characteristics of facilitating the preparation.

[0128] Example 13

[0129] Pharmacokinetic properties of polypeptide compounds in rats

[0130] SD rats were given 50 nmol / kg of liraglutide, semaglutide, polypeptide 1 and polypeptide 2 subcutaneous (s.c.) injection, and blood samples were collected at 0.25, 0.5, 1, 2, 4, 8, 16, 24 and 48 hours after administration. After protein precipitation with acetonitrile, plasma samples were analyzed by LC-MS. Pharmacokinetic parameters and half-life were calculated using WinonLin 5.2.1 (non-compartment model) (Table 3).

[0131] Table 3: Pharmacokinetic profiles of polypeptide compounds in rats

[0132] Samples T 1 / 2 (h)]]> C max (ng / mL) Liraglutide 2.3 489 Semaglutide 9.2 519 Polypeptide 1 4.1 472 Polypeptide 2 13.5 522

[0133] As shown in the results of Table 3, the in vivo half-life of the polypeptide compounds of the present application is significantly prolonged, which is superior to liraglutide or semaglutide, and has a pharmacokinetic characteristic supporting once-daily administration or once-weekly administration.

[0134] Example 14

[0135] Acute hypoglycemic activity of polypeptide compounds in mice

[0136] Male ICR mice were randomly divided into groups of 6. Only water was given, and the mice were fasted overnight. The blank group was given intraperitoneal injection of normal saline (10 mg / kg), and the administration group was divided into 2 groups, and the mice were given single intraperitoneal injection of 30 nmol / kg of semaglutide and polypeptide 1, respectively, in a non-fasting state. After 30 minutes, 3 g / kg of glucose solution was given to each group of mice. The blood glucose level was measured at -30, 0, 15, 30, 60, 120 min using a blood glucose meter

[0137] As Figure 1 As shown in the results, the acute glucose-lowering experiment in ICR mice showed that the polypeptide 1 polypeptide compound significantly improved the glucose tolerance level of the mice, and had excellent hypoglycemic effect, which was superior to semaglutide.

[0138] Example 15

[0139] Effect of polypeptide compounds on blood lipids and body weight of diet-induced obese (DIO) mice

[0140] Male C57BL / 6J mice, weighing about 22 g, were fed with high-fat diet D12492 of Research Diets for 18 weeks to establish a DIO mouse model. Before the start of the administration, the DIO mice in each group were randomly divided by weight, and a total of 3 groups were divided, each group of 6, namely the saline group (blank control group), the positive control group (semaglutide), and the test sample group (polypeptide 1). The mice in each group were subcutaneously injected with saline (10 mg / kg) once every two days, semaglutide (30 nmol / kg), and polypeptide 1 (30 nmol / kg) once a day, and the administration cycle was 21 days. The body weight change of the mice was recorded every day. After the end of the experiment, the mice in each group were sacrificed, blood was taken to prepare serum, and liver was taken to prepare homogenate, and the contents of triglyceride (TG) and total cholesterol (TC) in the liver and serum were measured.

[0141] As Figure 2 The results show that the polypeptide compound polypeptide 1 of the present application can reduce the body weight of the DIO mice by 34.8% at a dose of 30 nmol / kg for 3 weeks of continuous administration in vivo. Semaglutide can only reduce the body weight of the mice by 15.6%. The above results show that polypeptide 1 has excellent weight loss effect.

[0142] Table 4: Contents of total cholesterol (TC) and triglyceride (TG) in serum of DIO mice after 3 weeks of treatment

[0143]

[0144]

[0145] *** P < 0.001 compared with the blank control group; ### P < 0.001 compared with the semaglutide group (One-Way ANOVA, Tukey post hoc test), and the results are expressed as the average value ± SD of 6 mice in each group.

[0146] Table 5: Contents of total cholesterol (TC) and triglyceride (TG) in liver of DIO mice after 3 weeks of treatment

[0147] Samples (dose) Total cholesterol (mg / g) Triglycerides (mg / g) Blank control (saline group) 15.21±1.52 99.18±8.15 Semaglutide (30 nmol / kg) 12.28±1.14 76.12±4.15 Polypeptide 1 (30 nmol / kg) 6.89 ± 0.53***, ### ]] 38.65 ± 4.69***, ### ]]

[0148] * P < 0.05 compared with the blank control group; *** P < 0.001 compared with the blank control group; ### P < 0.001 compared with the semaglutide group (One-Way ANOVA, Tukey post hoc test), and the results are expressed as the average value ± SD of 6 mice in each group.

[0149] As shown in the results of Tables 4 and 5, the polypeptide compound polypeptide 1 of the present application can significantly reduce the triglyceride (TG) and total cholesterol (TC) contents in the serum and liver of the mice when administered continuously for 3 weeks in vivo in DIO mice, and the effect of polypeptide 1 on reducing serum and liver blood lipids is significantly stronger than that of the positive control semaglutide. The above results show that polypeptide 1 has a significantly better weight loss and liver and serum blood lipid reduction effect than semaglutide, indicating that the polypeptide compound of the present application has an exceptionally excellent weight loss and lipid regulation effect.

[0150] Finally, it should be noted that the above specific embodiments are only used to illustrate the technical solutions of the present application and not to limit it. Although the present application has been described in detail with reference to the examples, it should be understood by those skilled in the art that the technical solutions of the present application can be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present application, and they should be covered in the scope of the claims of the present application.

Claims

1. A glucagon peptide-1 and glucagon receptor dual agonist polypeptide or a pharmaceutically acceptable salt thereof, said polypeptide is selected from the following polypeptide 1 or polypeptide 2: (1) polypeptide 1 (2) polypeptide 2 or a pharmaceutically acceptable salt thereof. The salt is a salt of the glucagon peptide-1 and glucagon receptor dual agonist polypeptide with one of the following compounds: acetic acid, salicylic acid, lauric acid, cinnamic acid, lactic acid or succinic acid. The method comprises the following steps:

2. The glucagon glucagon peptide-1 and glucagon receptor dual agonist polypeptide or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, First, swell the resin, remove the Fmoc protecting group, then synthesize Fmoc-Ser-Rink amide-MBHA resin, and then extend the peptide chain, modify the Lys side chain, and finally cleave the polypeptide on the resin and purify it to obtain the polypeptide.

3. The method of synthesizing a glucagon peptide- 1 and glucagon receptor dual agonist polypeptide of claim 1, characterized in that, 4. A pharmaceutical composition comprising a therapeutically effective amount of at least one glucagon peptide-1 and glucagon receptor dual agonist polypeptide or a pharmaceutically acceptable salt thereof according to claim 1 and a pharmaceutically acceptable carrier and / or adjuvant. The metabolic disease is diabetes, obesity and / or dyslipidemia. The diabetes is T1DM, T2DM or gestational diabetes.

5. Use of the glucagon glucagon peptide-1 and glucagon receptor dual agonist polypeptide of claim 1 or a pharmaceutically acceptable salt thereof, the pharmaceutical composition of claim 4 in the manufacture of a medicament for the treatment of a metabolic disease, characterized in that, The diabetes is T1DM, T2DM or gestational diabetes.

6. Use according to claim 5, characterized in that, ​

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