A glucagon analogue containing β - amino acid or a pharmaceutically acceptable salt thereof and its application

Abstract

The present invention provides a glucagon analogue containing β - amino acids or a pharmaceutically acceptable salt thereof and its application. By replacing α - amino acids with corresponding β - amino acids at different amino acid sites, the glucagon analogue of the present invention has a glucagon receptor (GCGR) agonistic potency equivalent to or higher than that of natural glucagon, and at the same time has significantly improved plasma stability and liver microsome stability, and thus has better drug - like properties.

Description

Technical Field

[0001] The present invention relates to the field of biopharmaceuticals. Specifically, the present invention relates to a glucagon analogue and its application. Background Art

[0002] Proglucagon is a precursor polypeptide composed of 158 amino acids. This polypeptide is processed in tissues into a variety of hormones and peptides with different functions, including glucagon (Glu), glucagon-like peptide-1 (GLP-1), glucagon-like peptide-2 (GLP-2), and oxyntomodulin (OXM). These hormones and peptides play important roles in physiological functions such as blood glucose homeostasis, insulin secretion, gastric emptying, intestinal growth, and food intake.

[0003] Among the above hormones and peptides, glucagon binds to the glucagon receptor (GCGR) on hepatocytes, causing the liver to release the glucose stored in the form of glycogen, thereby helping to maintain blood glucose levels. When these stored glucose are depleted, glucagon stimulates the liver to synthesize additional glucose through glycogenogenesis. These glucose are released into the bloodstream, thus preventing the occurrence of hypoglycemia. Structurally, glucagon corresponds to the amino acid sequence segment from positions 53 to 81 of proglucagon, consisting of 29 amino acids, with the sequence His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr (SEQ ID NO. 1).

[0004] In view of the important physiological role of glucagon, various analogues of glucagon have been proposed in the art. Most of these analogues are obtained by substituting or modifying amino acid residues of natural glucagon, and can mimic the action of natural glucagon to activate the glucagon receptor. Thus, as glucagon receptor agonists, they play biological regulatory functions such as increasing the metabolic rate, promoting high oxygen consumption, and increasing energy consumption. In addition, glucagon analogues can also delay gastric emptying and reduce appetite, which is beneficial for further helping to control body weight.

[0005] However, whether it is natural glucagon or existing artificially constructed glucagon analogs, most of them have problems such as enzymatic instability in the body and poor metabolic kinetics (for example, the plasma half-life may be less than 10 minutes), which hinder their therapeutic applications for conditions such as obesity and impaired glucose tolerance. Summary of the Invention

[0006] To solve the above technical problems, the object of the present invention is to provide a glucagon analog, which has GCGR agonist activity and, compared with natural glucagon and existing analogs, has enhanced stability, specificity and potency. It is expected that these improvements will optimize the therapeutic efficacy of metabolic disorders.

[0007] The technical solution of the present invention is as follows.

[0008] On the one hand, the present invention provides a glucagon analog or a pharmaceutically acceptable salt thereof, and the glucagon analog or the pharmaceutically acceptable salt thereof comprises an amino acid sequence. Relative to the amino acid sequence shown in SEQ ID NO. 1, the amino acid sequence comprised by the glucagon analog or the pharmaceutically acceptable salt thereof has α-amino acids at one or more positions selected from the 2nd, 3rd, 5th, 10th, 13th, 16th, 17th, 21st and 24th positions replaced with β-amino acids.

[0009] As is well known in the art, "β-amino acid" is similar to the naturally occurring α-amino acid, but its amino group is bonded to the β-carbon (C3) rather than the α-carbon (C2), and the structure is shown as follows:

[0010]

[0011] Preferably, in the glucagon analog or the pharmaceutically acceptable salt thereof provided by the present invention, the β-amino acid may have the structure shown in Formula I:

[0012]

[0013] In Formula I, the substituents R1, R2, R3, R4 and R5 may independently be hydrogen (H) or a substituted group, and a cyclic structure may optionally be formed between the substituent and the adjacent carbon.

[0014] Preferably, R1, R2, R3, R4 and R5 are independently selected from hydrogen (H) or a substituted group, and the substituted groups may be the same or different from each other; and / or, any two of R1, R2, R3, R4 and R5, together with the α-carbon (C2) and / or β-carbon (C3) to which they are bonded, may form a cyclic structure.

[0015] Furthermore, the β-amino acid may have the structure shown in Formula II:

[0016]

[0017] In Formula II, the substituents R1, R2 and R5 can each independently be selected from hydrogen (H) or a substituted group, and the substituted groups can be the same or different from each other and / or, any two of R1, R2 and R5, together with the β-carbon (C3) to which they are bonded, can form a cyclic structure.

[0018] Preferably, in Formula II, R5 is hydrogen (H), and the cyclic structure can contain R1 or R2. Preferably, R5 is hydrogen (H). Preferably, in Formula II, R1 or R2 is hydrogen (H).

[0019] According to a specific embodiment of the present invention, the side chain group of the β-amino acid can be the side chain group (R) in its corresponding α-amino acid, except for the positioning of the amino group in the backbone and the insertion of the methylene unit. The β-amino acid can be present in the amino acid sequence as follows:

[0020]

[0021] In the context of the present invention, "a plurality of" sites can be 2 or more, such as 3, 4, 5, 6, 7, 8, 9 or more.

[0022] In the context of the present invention, the numbering of the amino acid sites is based on the amino acid site numbering of SEQ ID NO. 1.

[0023] In the context of the present invention, SEQ ID NO. 1 shows the amino acid sequence of native glucagon:

[0024] His- Ser - Gln -Gly- Thr -Phe-Thr-Ser-Asp- Tyr -Ser-Lys- Tyr -Leu-Asp- Ser - Arg -Arg-Ala-Gln- Asp -Phe-Val- Gln -Trp-Leu-Met-Asn-Thr (SEQ ID NO. 1)

[0025] Compared with the natural glucagon amino acid sequence shown in SEQ ID NO. 1, in the amino acid sequence contained in the glucagon analog or its pharmaceutically acceptable salt provided by the present invention, at one or more positions corresponding to positions 2, 3, 5, 10, 13, 16, 17, 21, and 24 of SEQ ID NO. 1 (for example, at 2-9 specified positions), the corresponding β-amino acids are β-Ser (beta-Ser; β-S; βS), β-Gln (beta-Gln; β-Q; βQ), β-Thr (beta-Thr; β-T; βT), β-Tyr (beta-Tyr; β-Y; βY), β-Arg (beta-Arg; β-R; βR), or β-Asp (beta-Asp; β-D; βD). The "corresponding β-amino acid" means the side chain group of the β-amino acid, i.e., the side chain group in the α-amino acid. For example, in the amino acid sequence contained in the glucagon analog or its pharmaceutically acceptable salt provided by the present invention, the side chain group in β-Ser is the side chain group of natural α-Ser, with the only difference being that in β-Ser, the side chain group is bonded to the β (C3) carbon.

[0026] Glucagon analogs:

[0027] Preferably, the present invention provides a glucagon analog, which may be a polypeptide containing β-amino acids.

[0028] 1. β - Amino acids:

[0029] As described above, relative to the amino acid sequence shown in SEQ ID NO. 1, the polypeptide may have corresponding β-amino acids at one or more positions selected from positions 2, 3, 5, 10, 13, 16, 17, 21, and 24.

[0030] Preferably, the glucagon analog provided by the present invention has corresponding β-amino acids at 1 or 2 positions selected from positions 2, 3, 5, 10, 13, 16, 17, 21, and 24 relative to the amino acid sequence shown in SEQ ID NO. 1 (for example, βY at position 10, βS at position 16, βQ at position 24); or has a specific combination of β-amino acids (for example, βS at positions 2 and 16, or βQ at positions 3 and 24).

[0031] According to the specific embodiments of the present invention, the glucagon analog provided by the present invention is a polypeptide containing β-amino acids, and relative to the amino acid sequence shown in SEQ ID NO. 1, the polypeptide has corresponding β-amino acids at positions selected from the following:

[0032] (1) Position 10: βY;

[0033] (2) Position 16: βS;

[0034] (3) Position 24: βQ;

[0035] (4) Position 21, βD;

[0036] (5) Position 5: βT;

[0037] (6) Position 13: βY;

[0038] (7) Position 17: βR;

[0039] (8) Position 2: βS;

[0040] (9) Positions 2 and 16: βS;

[0041] (10) Positions 3 and 24: βQ;

[0042] (11) Position 3: βQ;

[0043] (12) Position 16: βS, and Position 24: βQ; or

[0044] (13) Position 3: βQ, and Position 16: βS.

[0045] 2. Other amino acid modifications:

[0046] Furthermore, the glucagon analog provided by the present invention is a polypeptide containing β - amino acids. Relative to the amino acid sequence shown in SEQ ID NO. 1, the polypeptide further has amino acid substitutions at one or more sites selected from positions 7, 10, 17, 18, 20, 21, 24, 27, 28, and 29.

[0047] Preferably, the glucagon analog provided by the present invention is a polypeptide containing β - amino acids. Relative to the amino acid sequence shown in SEQ ID NO. 1, the polypeptide further has amino acid substitutions at 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 sites selected from positions 7, 10, 17, 18, 20, 21, 24, 27, 28, and 29; preferably, it has amino acid substitutions at 4, 5, 6, 7, 8, 9, or 10 sites selected from positions 7, 10, 17, 18, 20, 21, 24, 27, 28, and 29.

[0048] More preferably, the amino acid substitution may be: T7I; Y10K; R17A; R18A; Q20A; Q20E; D21E; Q24K; Q24A; M27E; M27L; N28S; N28D; or T29E.

[0049] According to the specific embodiments of the present invention, the glucagon analog provided by the present invention is a polypeptide containing β - amino acids. Relative to the amino acid sequence shown in SEQ ID NO. 1, the polypeptide has amino acid substitutions at positions selected from the following:

[0050] (1) R17A; Q20E; D21E; Q24K; M27E; N28S;

[0051] (2) T7I; Y10K; R17A; Q20E; D21E; Q24K; M27E; N28S;

[0052] (3) T7I; Y10K; R18A; N28D;

[0053] (4) Q20A; M27L; N28D; T29E; or

[0054] (5) Q20A; Q24A; M27L; N28D; T29E.

[0055] 3. Unnatural amino acids:

[0056] Furthermore, the glucagon analog provided by the present invention is a polypeptide containing β - amino acids. Relative to the amino acid sequence shown in SEQ ID NO. 1, the polypeptide further has unnatural amino acids at one or more positions selected from the 2nd, 3rd, and 16th positions, that is, the corresponding natural amino acids are replaced by unnatural amino acids.

[0057] Preferably, the glucagon analog provided by the present invention is a polypeptide containing β - amino acids. Relative to the amino acid sequence shown in SEQ ID NO. 1, the polypeptide has unnatural amino acids at 1 or 2 positions selected from the 2nd, 3rd, and 16th positions.

[0058] More preferably, the unnatural amino acid may be: acetyl - 2,4 - diamino butyric acid (Dab(Ac)); or α - amino isobutyric acid (Aib).

[0059] According to the specific embodiments of the present invention, the glucagon analog provided by the present invention is a polypeptide containing β - amino acids. Relative to the amino acid sequence shown in SEQ ID NO. 1, the polypeptide has unnatural amino acids at positions selected from the following:

[0060] (1) 16th position: Aib;

[0061] (2) 2nd position: Aib;

[0062] (3) 3rd position: Dab(Ac); or

[0063] (4) 2nd and 16th positions: Aib.

[0064] More specifically, the present invention provides a glucagon analogue or a pharmaceutically acceptable salt thereof, the glucagon analogue or the pharmaceutically acceptable salt thereof comprising an amino acid sequence which, relative to the amino acid sequence shown in SEQ ID NO. 1, has corresponding β - amino acids, specific amino acid substitutions and / or unnatural amino acids at certain positions. The disclosed glucagon analogues incorporate β - amino acids, targeted substitutions and unnatural residues to confer resistance to proteolytic degradation, an extended half - life and enhanced receptor specificity.

[0065] Examples of the amino acid sequences comprised by the glucagon analogue or the pharmaceutically acceptable salt thereof provided by the present invention are shown in Table 1:

[0066] Table 1. Amino acid sequences of polypeptides

[0067]

[0068] According to a specific embodiment of the present invention, the glucagon analogue provided by the present invention is a polypeptide, such as any one of the polypeptides numbered P001 to P019 shown in Table 1, the amino acid sequence of which is shown in any one of SEQ ID NOs. 5 - 23.

[0069] Pharmaceutically acceptable salts of glucagon analogs:

[0070] Preferably, the present invention further provides a pharmaceutically acceptable salt of a glucagon analogue, the glucagon analogue being as described above.

[0071] The pharmaceutically acceptable salts provided by the present invention may be inorganic acid salts or organic acid salts. Suitable inorganic acid salts are, for example, hydrochloride, sulfate, phosphate, hydrobromide, hydroiodide, etc. Suitable organic acid salts are, for example, formate, acetate, propionate, benzoate, maleate, mesylate, fumarate, trichloroacetate, trifluoroacetate, citrate, etc. In addition, the pharmaceutically acceptable salts provided by the present invention may also be inorganic base salts or organic base salts. Suitable inorganic base salts are, for example, sodium salt, potassium salt, lithium salt, calcium salt, magnesium salt, ammonium salt, aluminum salt, zinc salt, copper salt, manganese salt, etc. Suitable organic base salts are salts formed with organic bases, for example, salts formed with methylamine, dimethylamine, trimethylamine, piperidine, morpholine or tris-(2-hydroxyethyl)amine, etc.

[0072] On the other hand, the present invention provides a pharmaceutical composition, which comprises the glucagon analogue or its pharmaceutically acceptable salt provided by the present invention, and optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, adjuvant or excipient.

[0073] Depending on the application requirements, the pharmaceutical composition may be in the form of tablets, capsules, syrups, tinctures, inhalants, sprays, injections, films, patches, powders, granules, emulsions, suppositories or compound preparations.

[0074] Optionally, the pharmaceutical composition may further comprise other therapeutic agents that can be used in combination with the glucagon analogue or its pharmaceutically acceptable salt provided by the present invention. The other therapeutic agents may be natural glucagon or synthetic glucagon analogues. Among them, the synthetic glucagon analogue may be an agonist of G protein-coupled receptor (GPCR), such as GCGR, GLP1R, GIPR agonist.

[0075] In yet another aspect, the present invention provides the use of the glucagon analogue or its pharmaceutically acceptable salt or the pharmaceutical composition in the preparation of a glucagon receptor agonist.

[0076] Compared with the prior art, the present invention provides a novel glucagon analogue, which contains β-amino acids.

[0077] Relative to the amino acid sequence of natural glucagon, in the amino acid sequence contained in the glucagon analogue or its pharmaceutically acceptable salt provided by the present invention, the α-amino acid at a specific amino acid site in natural glucagon is replaced with the corresponding β-amino acid at this site.

[0078] Experimental results have shown that modified glucagon analogs with similar or better glucagon receptor agonist activity can be obtained by substituting α - amino acids with corresponding β - amino acids at different amino acid sites. For example, by using β - amino acids, the resulting glucagon analogs have comparable or higher agonist potency for the glucagon receptor, while also exhibiting significantly improved plasma stability and liver microsomal stability. Therefore, the glucagon analogs provided by the present invention have better drug - like properties. In addition, compared with the glucagon - like peptide 1 receptor (GLP1R) and the gastric inhibitory polypeptide receptor (GIPR), the glucagon analogs provided by the present invention have higher specificity for the glucagon receptor, thus minimizing off - target effects. Detailed Description of the Invention

[0079] The present invention will be described below with reference to specific examples. Those skilled in the art can understand that these examples are only used to illustrate the present invention and do not limit the scope of the present invention in any way.

[0080] Unless otherwise specified, the experimental methods in the following examples are all conventional methods. Unless otherwise specified, the raw materials, reagent materials, etc. used in the following examples are all commercially available products. Among them:

[0081] Glucagon and exemplary glucagon analogs:

[0082] 1. Glucagon: HSQGTFTSDYSKYLDSRRAQDFVQWLMNT (SEQ ID NO. 1);

[0083] 2. Dacic glucagon: HSQGTFTSDYSKYLDX 16 ARAEEFVKWLEST, X 16 = Aib (SEQ ID NO.2);

[0084] 3. IUB288: HX2QGTFISDKSKYLDX 16 RAAQDFVQWLMDT, X2 = Aib, X 16 = Aib (SEQ IDNO. 3);

[0085] 4. IUB76: HSX3GTFTSDYSKYLDX 16 RRAADFVAWLLDE, X3 = Dab(Ac), X 16 = Aib (SEQ IDNO. 4).

[0086] Example 1 Synthesis of polypeptides containing β - amino acids

[0087] The polypeptides containing β - amino acids provided in Table 1 were generated on an automatic solid - phase peptide synthesizer using Fmoc coupling chemical reactions. During the synthesis, the CTC resin concentration was 0.51 mmol / g and the target synthesis amount was 0.2 mmol.

[0088] Resin activation and initial coupling:

[0089] Dissolve 1 g of triphenylvinyl alcohol resin in 10 ml of dichloromethane and shake at 400 rpm for 30 minutes at 25 °C to activate the triphenylvinyl alcohol resin, obtaining a resin solution. Connect the first amino acid to the trichloroethylene resin using 4.0 equivalents of Fmoc - protected amino acid. If a resin with a lower degree of substitution is required, the amount of amino acid can be reduced.

[0090] Amino acid preparation and coupling:

[0091] Dissolve 1 g of amino acid in 10 ml of dichloromethane. If the amino acid does not dissolve completely, a small amount of DMF can be added to obtain an amino acid solution. Then, add the amino acid solution to the resin solution, and then add 4.0 equivalents of diacetyl acetic acid. Shake at 400 rpm for 1 hour at 25 °C. Add HPLC - grade methanol (0.8 mL per gram of resin) to cap the remaining active trialkyl groups, shake at 25 °C for 15 minutes, filter the mixture, and wash it three times with DCM (about 10 mL per gram of resin) and twice with DMF (about 10 mL per gram of resin). After washing, the synthesized resin is directly used for the next step.

[0092] Fmoc de - protection:

[0093] Remove the Fmoc group with 20% v / v piperidine in DMF, and then shake at 400 rpm for 10 minutes. After washing and filtering, the resin is directly used for the following coupling reaction.

[0094] Amino acid sequential coupling:

[0095] Perform one coupling for each natural amino acid (4 equivalents, with appropriate protecting groups on the side chain) using Oxyma (4 equivalents, Cas: 3849 - 21 - 6) and DIC (8 equivalents, Cas: 693 - 13 - 0) in 2.0 ml of DMF (150 mg of resin) at 50 °C and shake at 400 rpm for 45 minutes. Perform one coupling for each unnatural amino acid (4 equivalents, with appropriate protecting groups on the side chain) using Oxyma (4 equivalents, Cas: 3849 - 21 - 6) and DIC (8 equivalents, Cas: 693 - 13 - 0) in 2.0 ml of DMF (150 mg of resin) at 50 °C and shake at 400 rpm for 1 hour.

[0096] Post - coupling processing:

[0097] Wash four times with DMF (3 mL each time). After washing and filtering, the resin is used for the next round of Fmoc deprotection and coupling. Repeat the above operations until all amino acids are coupled. After all couplings are completed, remove the Fmoc of the last amino acid with the same operation as before. After washing and filtering, the resin is directly used for the cleavage and full deprotection of the amino acid side chains.

[0098] Global deprotection and cleavage:

[0099] Deprotect and cleave the final polypeptide under reducing conditions by shaking in a solution of TFA:H2O:thioanisole:DODT (Cas: 14970 - 87 - 7) (37:1:1:1) at 40 °C for 2 hours. Remove the resin by vacuum filtration, and precipitate the crude peptide in the filtrate with cold diethyl ether (50 mL) and incubate at -20 °C for 30 minutes.

[0100] Finally, centrifuge the mixture at 4400 g for 5 minutes.

[0101] Purification and characterization:

[0102] Wash the polypeptide particles twice with cold diethyl ether (30 mL), and then directly purify them by HPLC. The crude peptide is purified by reverse - phase high - performance liquid chromatography, and the purification conditions are shown in Table 2 below. The fractions collected by HPLC are analyzed by matrix - assisted laser desorption / ionization time - of - flight mass spectrometry (MALDI - TOF MS) in positive - ion or negative - ion reflectron mode on a Bruker UltraFlexXtreme IIMALDI - TOF mass spectrometer. The fractions are directly lyophilized to obtain the final solid dry matter.

[0103] Table 2. Purification of polypeptides

[0104]

[0105] Example 2 Potency characterization of β - amino acid - containing polypeptides against class B G protein - coupled receptors GCGR, GLP1R, and GIPR

[0106] Detect the in vitro agonist potency of the β - amino acid - containing polypeptides provided by the present invention against glucagon receptor (GCGR), glucagon - like peptide 1 receptor (GLP1R), and gastric inhibitory polypeptide receptor (GIPR) by detecting the cAMP content using a homogeneous time - resolved fluorescence (HTRF) assay.

[0107] 1. Reagents and cells

[0108] Control polypeptide:

[0109] GLP1 (7-37): Purchased from MedChemExpress (MCE);

[0110] GIP (1-42): Purchased from Tocris Bioscience;

[0111] Glucagon hydrochloride: Purchased from Selleck Chemicals.

[0112] Buffer:

[0113] Hanks balanced salt solution supplemented with 20 mmol / l HEPES, 0.1% (wt / vol.) BSA, and 0.5 mmol / l 3-isobutyl-1-methylxanthine (IBMX) (pH 7.4);

[0114] Recombinant expression cells:

[0115] Cells stably overexpressing GLP1R, GIPR, and GCGR were constructed using the Flp-In™ system (Thermo Fisher Scientific, Loughborough, UK) and named Flp In-293-GLP1R cells, Flp In-293-GIPR cells, and Flp In-CHO-GCGR cells; they were cryopreserved in liquid nitrogen and thawed before use.

[0116] 2. Experimental procedure

[0117] Polypeptide dilution:

[0118] Using the above buffer, the polypeptides containing β-amino acids of the present invention and the control polypeptides were respectively diluted and serially diluted to obtain a series of dilution solutions with 10 concentration gradients. The series of dilution solutions were added to the wells of the plate.

[0119] Cell preparation:

[0120] The cells stably overexpressing GCGR, namely Flp In-CHO-GCGR cells, were resuspended in the above buffer and added to the wells of the plate to be mixed with the polypeptide.

[0121] cAMP quantification:

[0122] Incubate at 37 °C for 30 min. Then, use the cAMP dynamic 2 kit (Cisbio, Codolet, France) to measure the cellular cAMP level according to the manufacturer's instructions. After 1 h, read the plate in an EnVision plate reader (PerkinElmer, Waltham, MA, USA).

[0123] Receptor cross-detection:

[0124] Referring to the above experimental procedure, use Flp In-293-GLP1R cells and Flp In-293-GIPR cells to detect the in vitro agonistic potency of the β-amino acid-containing polypeptide and the control polypeptide on two other receptors. The polypeptide detection concentration is 1 nM.

[0125] 3. Data analysis

[0126] Use GraphPad Prism (San Diego, CA, USA) software to calculate the EC50 value by non-linear regression. The results are shown in Tables 3 and 4 below.

[0127] Table 3. Agonistic potency of polypeptides on the glucagon receptor

[0128]

[0129] As shown in Table 3, the β-amino acid-containing polypeptides provided by the present invention show strong agonistic activity on the glucagon receptor (GCCR) under the in vitro test conditions, inducing a significant increase in glucose production. The observed efficacy demonstrates their equivalent function or superior performance compared to natural glucagon, confirming their potential application in metabolic disorders and the like that require glucagon receptor regulation.

[0130] Table 4. Agonistic potency of polypeptides on the glucagon-like peptide 1 receptor and the gastric inhibitory polypeptide receptor

[0131]

[0132] As shown in Table 4, the β-amino acid-containing polypeptides provided by the present invention have higher selectivity for the glucagon receptor GCGR compared to the other two G protein-coupled receptors GLP1R and GIPR, showing a binding affinity of ≥10-fold in the above agonist comparison assay. This receptor-specific activity profile will minimize off-target interactions while maintaining efficacy in blood glucose homeostasis regulation.

[0133] Example 3 Characterization of plasma stability and hepatic microsomal metabolic stability of β-amino acid-containing polypeptides

[0134] Detect the plasma stability and hepatic microsomal metabolic stability of the polypeptide containing β - amino acid provided by the present invention in humans and mice.

[0135] 1. Detection of plasma stability

[0136] Experimental procedure:

[0137] Prepare 1 mM working solutions of the polypeptide containing β - amino acid of the present invention and the control polypeptide in DMSO. Take 4 μL of each working solution and add it to 796 μL of pre - incubated plasma (human or mouse) to obtain a final concentration of 5 μM. Aliquot the resulting plasma into new reaction tubes at 50 μL each, place the reaction tubes in a 37 °C water bath, and shake at 60 rpm for incubation for 15 min (t15), 30 min (t30), 60 min (t60), and 120 min (t120) respectively. Terminate the reaction by adding 50 μL of HCl (1 N) and 300 μL of room - temperature quenching solution (acetonitrile containing 3% formaldehyde and internal standards (500 nM labetalol and 2 μM ketoprofen)) to the plasma at the corresponding time points. Prepare 0 min (T0) samples by adding the resulting plasma to new reaction tubes containing 50 μL of HCl (1 N) and 300 μL of room - temperature quenching solution.

[0138] After the reaction, vortex the reaction tubes for 5 min; then centrifuge at 3,220 g for 30 min at 4 °C to precipitate the protein. Take 100 μL of the supernatant and transfer it to a new 96 - well plate with 100 μL of water in each well for LC - MS / MS analysis.

[0139] Data analysis:

[0140] Determine the peak area ratio of each analyte from the extracted ion chromatogram; calculate the percentage of the remaining polypeptide at each time point using the following formula:

[0141] Remaining percentage t min (%) = (Peak area t min / Peak area 0 min ) × 100

[0142] In this formula, the remaining percentage t min is the ratio of the peak area of the polypeptide of the present invention or the control polypeptide at t min to the initial T0 peak area (peak area 0 min ). Infer the half - life ( t 1 / 2) from the resulting degradation kinetics.

[0143] 2. Detection of hepatic microsomal metabolic stability

[0144] Experimental procedure:

[0145] First, prepare the stock solutions as shown in Table 5.

[0146] Table 5. Preparation of the stock solution

[0147] Then, the following experiments were carried out separately.

[0148] a) System with cofactors (NADPH and UDPGA):

[0149] 10 μL of 20 mg / mL liver microsomes, 40 μL of 10 mM NADPH, and 40 μL of 20 mM UDPGA were added to the stock solution, and the final concentrations of microsomal protein, NADPH, and UDPGA were 0.5 mg / mL, 1 mM, and 2 mM, respectively.

[0150] b) System without cofactors (NADPH and UDPGA):

[0151] 10 μL of 20 mg / mL liver microsomes and 80 μL of ultrapure water were added to the stock solution, and the final concentration of microsomal protein was 0.5 mg / mL.

[0152] The stock solution of liver microsomes with or without cofactors was aliquoted into glass tubes at 398 μL each, and then the glass tubes were placed in a water bath and shaken for about 10 min. 2 μL of the solution of the polypeptide containing β - amino acid and the control polypeptide of the present invention (final concentration 1 μM) was added. The resulting reaction solution was aliquoted into each well of a new microtube at 50 µL, reacted at 37 °C, and then the reaction was terminated at 0, 15, 30, 45, and 60 min by adding 1 volume of HCl (1 M) and 4 volumes of acetonitrile containing internal standards (3% formic acid, 200 nM labetalol, and 2 μM ibuprofen). Then, the resulting samples were centrifuged at 3,220 g for 40 min, 100 μL of the supernatant was mixed with 100 μL of ultrapure H2O, and LC - MS / MS analysis was performed.

[0153] Data analysis:

[0154] The peak area was determined from the extracted ion chromatogram; a linear regression curve of the natural logarithm of the remaining percentage of the test drug vs. the incubation time was plotted, and the negative slope was determined from this curve, which was the clearance rate constant ( k ). The in vitro half - life ( t 1 / 2) and the intrinsic clearance rate ( CL int) were calculated as follows:

[0155] t 1 / 2 = 0.693 / k

[0156] CL int = k× (Incubation volume (μL) / Microsomal protein amount (mg))

[0157] The results of three repeated measurements are shown in Table 6 below.

[0158] Table 6. Plasma stability and microsomal stability of the polypeptide

[0159]

[0160] As shown in Table 6, compared with glucagon, the polypeptides containing β - amino acids provided by the present invention show significantly longer in vitro half - lives under the same metabolic conditions, thus proving that they have stronger metabolic stability.

[0161] The above description of the specific embodiments of the present invention does not limit the present invention. Those skilled in the art can make various changes or deformations according to the present invention, as long as they do not depart from the spirit of the present invention, they shall fall within the scope of the appended claims of the present invention.

Claims

1. A glucagon analogue or a pharmaceutically acceptable salt thereof, characterized in that, The glucagon analog is a polypeptide comprising β-amino acids, and the amino acid sequence of the polypeptide is shown in SEQ ID NO. 7 or SEQ ID NO.

22.

2. The glucagon analogue or a pharmaceutically acceptable salt thereof according to claim 1, wherein The pharmaceutically acceptable salt is an inorganic acid salt or an organic acid salt, or an inorganic base salt or an organic base salt.

3. The glucagon analogue or a pharmaceutically acceptable salt thereof according to claim 2, wherein The inorganic acid salt is hydrochloride, sulfate, phosphate, hydrobromide or hydroiodide; or, The organic acid salt is formate, acetate, propionate, benzoate, maleate, mesylate, fumarate, trichloroacetate, trifluoroacetate or citrate.

4. The glucagon analogue or a pharmaceutically acceptable salt thereof according to claim 2, wherein, The inorganic base salt is sodium salt, potassium salt, lithium salt, calcium salt, magnesium salt, ammonium salt, aluminum salt, zinc salt, copper salt or manganese salt; or, The organic base salt is a salt formed with methylamine, dimethylamine, trimethylamine, piperidine, morpholine or tris-(2-hydroxyethyl)amine.

5. A pharmaceutical composition, the pharmaceutical composition comprising the glucagon analog or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, and optionally a pharmaceutically acceptable carrier, excipient or adjuvant.

6. The pharmaceutical composition according to claim 5, characterized in that, The pharmaceutical composition is in the form of tablets, capsules, syrups, tinctures, inhalants, sprays, injections, films, patches, powders, granules, emulsions, suppositories or compound preparations.

7. The pharmaceutical composition according to claim 5 or 6, characterized in that, The pharmaceutical composition further comprises other therapeutic agents.

8. The pharmaceutical composition according to claim 7, characterized in that, The other therapeutic agent is an agonist of the G protein-coupled receptor GPCR.

9. The pharmaceutical composition according to claim 7, wherein The other therapeutic agent is an agonist of GCGR, GLP1R and / or GIPR.

Images