A class of glp-1 / y2 receptor dual agonists and uses thereof

Abstract

The application provides a kind of GLP-1 / Y2 receptor dual agonist and its application, the dual agonist has dual agonist activity to human GLP-1 and Y2 receptor.The GLP-1 / Y2 receptor dual agonist of the application has significant weight loss effect while more effectively reducing blood sugar.The GLP-1 / Y2 receptor dual agonist provided by the application has stable chemical properties, has the pharmacokinetic characteristics of once a day or once a week subcutaneous injection administration in human body, realizes long-acting administration, and is suitable as active ingredient of drug for treating metabolic diseases, such as diabetes, obesity, dyslipidemia and other diseases.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a GLP-1 / Y2 receptor dual agonist and application thereof. BACKGROUND

[0002] Obesity and its related metabolic syndrome have become a global public health problem. The incidence and course development of many metabolic syndromes such as type 2 diabetes mellitus (T2DM), non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), and abnormal lipid metabolism are closely related to obesity. GLP-1 is a glucose-dependent hypoglycemic polypeptide hormone secreted by L cells in the small intestine, and its main function is to promote the secretion of insulin. Glucagon-like peptide-1 (GLP-1) can inhibit appetite and delay gastric emptying to achieve the effect of reducing body weight. Although GLP-1 has excellent hypoglycemic effect and certain weight loss effect, if better weight loss effect is to be achieved, the dosage generally needs to be increased, and large-dose administration of GLP-1 drugs is prone to cause gastrointestinal side effects, poor tolerance, and a 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] Neuropeptide Y (NPY) is a 36-amino acid peptide neurotransmitter, which is a member of the pancreatic polypeptide class of neurotransmitters / neuromodulators that have been shown to exist in both the peripheral and central nervous systems. NPY is one of the most potent orexigenic agents known and NPY has been shown to play an important role in regulating food intake in humans and animals. NPY receptors share four subtypes, Y1, Y2, Y4 and Y5, among which the neuropeptide-2 (Y2) receptor is widely distributed in the central nervous system of rodents and humans. In the hypothalamus, Y2 mRNA is localized in the arcuate nucleus, the preoptic nucleus and the dorsal medial nucleus. In the human brain, the Y2 receptor is the major NPY receptor subtype. In the arcuate nucleus, more than 80% of NPY neurons co-express Y2 receptor mRNA. Selective agonism of Y2 receptors can inhibit food intake and have a weight loss effect.

[0004] Peptide YY 3-36 (PYY 3-36 ) is a 34-amino acid linear peptide with Y2 receptor agonistic activity. The combined injection of GLP-1 and PYY 3-36 in animals and humans has a good hypoglycemic and weight loss effect, indicating that simultaneous agonism of GLP-1 and Y2 receptors can further utilize the appetite suppression effect brought by agonism of Y2 receptors to produce better weight loss effect while maintaining the hypoglycemic effect of GLP-1. Novo Nordisk reported a class of GLP-1 and short-chain PYY 3-36Hybrid peptides of analogs. However, such compounds are not modified for long-acting, and their in vivo stability is limited, and they cannot achieve long-acting administration (Angew. Chem. Int. Ed. Engl., 2021, 6; 60(15): 8268-8275). Brandon et al. disclosed a class of hybrid peptides of exendin-4 and short-chain PYY 3-36 analog, which has good GLP-1 and Y2 receptor dual agonistic activity, and is similar to the compound reported by Novo Nordisk. Such compounds are also not modified for long-acting, and their stability is poor (J. Med. Chem., 202, 28; 64(2): 1127-1138). SUMMARY

[0005] The present application aims to provide a class of GLP-1 / Y2 receptor dual agonists and their applications. The agonists have dual agonistic activity on human GLP-1 receptor and Y2 receptor and high stability, have pharmacokinetic characteristics that can be administered subcutaneously once a day or once a week in humans, and achieve long-acting administration. Their application in the preparation of drugs for treating metabolic syndrome, such as diabetes, obesity, and dyslipidemia, has greater potential.

[0006] To achieve the above-mentioned application purposes, the technical solutions of the present application are as follows:

[0007] A class of GLP-1 / Y2 receptor dual agonists, the amino acid sequence of the GLP-1 / Y2 receptor dual agonist is as follows: His-Xaa1-Glu-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Thr-Glu-Tyr-Leu-Glu-Glu-Xaa2-Ala-Ala-Xaa3-Glu-Phe-Ile-Glu-Trp-Leu-Ile-Lys-Gly-Lys-Xaa4-Xaa5-Lys-Pro-Glu-Ala-Pro-Gly-Xaa6-Asp-Ala-Ser-Pro-Glu-Glu-Xaa7-Asn-Arg-Tyr-Tyr-Ala-Xaa8-Leu-Arg-His-Tyr-Leu-Asn-Xaa9-Xaa 10 -Thr-Arg-Gln-Xaa 11 -Tyr-NH2,

[0008] Among them:

[0009] Xaa1 is selected from Ala, Gly or Aib;

[0010] Xaa2 is selected from Glu, Lys or a side chain-modified Lys;

[0011] Xaa3 is selected from Lys or a side chain-modified Lys;

[0012] Xaa4 is selected from Cys or a side chain-modified Cys-R1 or Cys-R2 or Cys-R3;

[0013] Xaa5 is selected from lie or Pro;

[0014] Xaa6 is selected from Glu or Lys;

[0015] Xaa7 is selected from Leu or Trp;

[0016] Xaa8 is selected from Ser or Asp;

[0017] Xaa9 is selected from Leu or Trp;

[0018] Xaa 10 is selected from Leu or Val;

[0019] Xaa 11 is selected from Arg or

[0020] wherein the side chain-modified Lys is selected from

[0021]

[0022]

[0023] wherein n is a natural number, and 12≤n≤20.

[0024] Preferably, the n is 14, 16, 18 or 20.

[0025] Preferably, the chemical structure of the Cys-R1 is as follows:

[0026]

[0027] The chemical structure of the Cys-R2 is as follows:

[0028]

[0029] The chemical structure of the Cys-R3 is as follows:

[0030]

[0031] Preferably, the amino acid sequence structure of the GLP-1 / Y2 receptor dual agonist is:

[0032] (1) SEQ ID NO: 1

[0033]

[0034] (2) SEQ ID NO: 2

[0035]

[0036] (3) SEQ ID NO: 3

[0037]

[0038] (4) SEQ ID NO: 4

[0039]

[0040] (5) SEQ ID NO: 5

[0041]

[0042] (6) SEQ ID NO: 6

[0043]

[0044] (7) SEQ ID NO: 7

[0045]

[0046] (8) SEQ ID NO: 8

[0047]

[0048] (9) SEQ ID NO: 9

[0049]

[0050] (10) SEQ ID NO: 10

[0051]

[0052] (11) SEQ ID NO: 11

[0053]

[0054] (12) SEQ ID NO: 12

[0055]

[0056] (13) SEQ ID NO: 13

[0057]

[0058] (14) SEQ ID NO: 14

[0059]

[0060] The present application also provides a class of pharmaceutically acceptable salts of GLP-1 / Y2 receptor dual agonists.

[0061] Preferably, the salt is formed between a GLP-1 / Y2 receptor dual agonist and one of the following compounds: acetic acid, salicylic acid, lauric acid, cinnamic acid, citric acid, oxalic acid, lactic acid, succinic acid.

[0062] The present application also provides a medicament prepared from a GLP-1 / Y2 receptor dual agonist, wherein the medicament is any one of a tablet, a capsule, an inhaler, a spray, an injection, a film, a patch, a cream, a suppository, or a compound preparation, and the medicament is composed of a GLP-1 / Y2 receptor dual agonist and pharmaceutically acceptable excipients, carriers or diluents.

[0063] The present application also provides a pharmaceutical composition containing a GLP-1 / Y2 receptor dual agonist, wherein the pharmaceutical composition is composed of any one of the GLP-1 / Y2 receptor dual agonists or a pharmaceutically acceptable salt thereof as an effective material, and a pharmaceutically acceptable carrier or diluent.

[0064] The present application also provides the use of a GLP-1 / Y2 receptor dual agonist or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition or a medicament thereof in the preparation of a medicament for treating a metabolic disease or disorder. 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 for treating more than one metabolic disease or disorder, for example, diabetes and obesity; diabetes and dyslipidemia; diabetes, dyslipidemia and obesity.

[0065] Compared with the prior art, the present application has the following advantages:

[0066] The present application provides a variant based on the sequence design of GLP-1 analogs, which retains the therapeutic effect of GLP-1 analogs on diabetes while having PYY 3-36The GLP-1 / Y2 receptor dual agonist has a beneficial effect on appetite suppression, has a dual agonistic activity on human GLP-1 receptor and Y2 receptor, has a synergistic effect on sugar, lipid and energy metabolism, has a stronger effect of reducing blood sugar and reducing weight, and has a significant advantage in the treatment of metabolic syndrome such as diabetes and obesity. In addition, the GLP-1 / Y2 receptor dual agonist has a very high stability, has a pharmacokinetic characteristic supporting subcutaneous injection administration once a day or once a week on a human body, realizes long-acting administration, and has greater potential in the preparation of a drug for treating metabolic syndrome such as diabetes, obesity, dyslipidemia and the like. BRIEF DESCRIPTION OF DRAWINGS

[0067] Figure 1 The figure shows the food intake inhibition effect of each test substance on C57BL / 6J mice after single administration;

[0068] Figure 2 The figure shows the percentage change in body weight of each test substance in DIO mice after long-term administration for 21 days. DETAILED DESCRIPTION

[0069] The application will be further described in detail below in combination with the drawings and specific examples.

[0070] Unless otherwise defined herein, scientific and technical terms used in this application shall have the meanings that are commonly understood by one of ordinary skill in the art. Generally, the nomenclature used herein for chemical, biological, and pharmacological associations is that conventionally used and commonly understood in the art.

[0071] In addition, the amino acids involved in the application are abbreviated according to the naming rules of IUPAC-IUB as follows:

[0072] 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).

[0073] In addition, unless specifically indicated, all amino acid residues in the polypeptide compounds of the application are preferably in the L configuration.

[0074] In addition, the "-NH2" part on the C-terminal of the sequence indicates an amide group (-CONH2) on the C-terminal.

[0075] In addition, an unnatural amino acid, alpha-aminoisobutyric acid (Aib), is used in the sequence of the present application in addition to natural amino acids.

[0076] Example 1

[0077] Synthesis of the polypeptide compound of SEQ ID NO: 1

[0078]

[0079] (1) Swelling of the resin

[0080] Rink Amide MBHA resin with a loading capacity 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.

[0081] (2) Removal of the Fmoc protecting group of the resin

[0082] The swelled resin was transferred to the PSI-200 polypeptide synthesizer, 7 mL of 20% piperidine / DMF (v / v) was added, and reaction was performed at room temperature for 5 min, the deprotection solution was filtered off, 7 mL of DMF was used to wash the resin once, 7 mL of 20% piperidine / DMF (v / v) deprotection solvent was added, and reaction was performed with the resin for 15 min, and finally 7 mL of DMF was used to wash the resin four times, each for 2 min, to obtain the Fmoc-protected Rink resin.

[0083] (3) Synthesis of Fmoc-Tyr-Rink amide-MBHA Resin

[0084] Fmoc-Tyr(tBu)-OH (0.4 mmol) was weighed, dissolved with 2 mL of DMF, 3 mL of DIC / HOBt (0.4 mmol / 0.44 mmol) condensing agent was added, and the mixture was added to the reactor, and reaction was performed at room temperature for 2 h, the reaction solution was filtered off, and the resin was washed with 7 mL of DMF four times, and Kaiser reagent was used to detect whether the reaction coupling was complete, and if not, two couplings were performed.

[0085] (4) Extension of the PYY partial peptide chain

[0086] According to the sequence of the PYY partial peptide chain (IKPEA PGEDA SPEEL NRYYA SLRHY LNLVT RQRY-NH2), the above deprotection and coupling steps were repeated to sequentially connect the corresponding amino acids until the synthesis of the peptide chain was completed.

[0087] (5) Synthesis of the linker arm

[0088] After the synthesis of the PYY moiety is completed, the synthesis of the linker moiety is continued by adding 0.4 mmol of Fmoc-AEEA-OH, 0.4 mmol of DIC and 0.44 mmol of HOBt, and shaking the condensation reaction for 2 h. After the Fmoc protecting group is removed, 0.4 mmol of Fmoc-AEEA-OH, 0.4 mmol of DIC and 0.44 mmol of HOBt are added again, and the condensation reaction is shaken for 2 h. After the Fmoc protecting group is removed, 0.4 mmol of 3-maleimidopropionic acid, 0.4 mmol of DIC and 0.44 mmol of HOBt are added, and the condensation reaction is shaken for 2 h.

[0089] (6) Cleavage of the PYY moiety containing the linker

[0090] The resin with the polypeptide attached is transferred to a round bottom flask, and the resin is cleaved using cleavage reagent Reagent R (TFA / benzyl mercaptan / phenol / EDT, 90:5:3:2, V / V) 5 mL, and the reaction is incubated at 30°C in an oil bath for 2 h. The cleavage solution is poured into 40 mL of ice ethyl ether, and the crude product is washed with 15 mL of ice ethyl ether 3 times after being frozen and centrifuged. Finally, the product is dried with nitrogen, and the crude peptide containing the PYY moiety with the linker is obtained.

[0091] (7) Synthesis of the GLP-1 moiety

[0092] Rink Amide MBHA resin with a loading capacity of 0.36 mmol / g is weighed at 0.278 g (0.1 mmol equivalent), and is placed in a 25 mL reactor. The resin is washed once with 10 mL of DCM and methanol alternately, and is washed twice with 10 mL of DCM. Then, the resin is swelled with 10 mL of DCM for 1 h, and is washed with 10 mL of DMF 3 times. The swelled resin is transferred to a PSI-200 polypeptide synthesizer, and 10 mL of 20% piperidine / DMF (v / v) is added for deprotection at room temperature for 5 min. The deprotection solution is filtered off, and the resin is washed once with 10 mL of DMF. Then, 10 mL of 20% piperidine / DMF (v / v) is added as a deprotection solution, and the reaction is performed for 15 min. Finally, the resin is washed 4 times with 10 mL of DMF for 1.5 min each time, and the Fmoc-protected Rink resin is obtained. Fmoc-Cys(Trt)-OH (0.4 mmol) is weighed, and is dissolved in 2 mL of 10% DMF / DMSO (v / v). Then, 3 mL of DIC / HOBt (0.4 mmol / 0.44 mmol) is added as a condensation reagent, and is added to the reactor. The reaction is shaken at room temperature for 2 h. After the reaction solution is filtered off, the resin is washed 4 times with 10 mL of DMF. Whether the reaction is complete is detected using a Kaiser reagent. If not, the coupling is performed twice.

[0093] (8) Extension of GLP-1 moiety peptide chain

[0094] According to the sequence of the peptide chain, repeat the above-mentioned deprotection and coupling steps to connect the corresponding amino acids in turn until the synthesis of the peptide chain is completed. Among them, the Lys at the side chain modification site is protected by Fmoc-Lys(Dde)-OH, and the His at the N-terminus is protected by Boc-His(Boc)-OH.

[0095] (9) Modification of Lys side chain of GLP-1 moiety

[0096] After the synthesis of the peptide chain is completed, 7 mL of 2% hydrazine hydrate / DMF (v / v) is added to selectively remove the Dde protecting group of Lys. After the Dde protecting group is removed, 0.4 mmol of Fmoc-Glu-OtBu, 0.4 mmol of DIC and 0.44 mmol of HOBt are added, and the reaction is shaken 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 are added again, and the condensation reaction is shaken for 2 h. After removing the Fmoc protecting group, 0.4 mmol of palmitic acid, 0.4 mmol of DIC and 0.44 mmol of HOBt are added for condensation reaction for 2 h. After the reaction is completed, the resin is washed with 7 mL of DMF for 4 times.

[0097] (10) Cleavage of GLP-1 moiety

[0098] The resin with the connected polypeptide obtained above is transferred to a round-bottom flask, and 5 mL of cleavage reagent Reagent R (TFA / benzyl mercaptan / phenol / EDT, 90:5:3:2, V / V) is used to cleave the resin. The reaction is carried out at a constant temperature of 30°C in an oil bath for 2 h. The cleavage solution is poured into 40 mL of ice ethyl ether, and after freezing centrifugation, the crude product is washed with 15 mL of ice ethyl ether for 3 times. Finally, it is dried by nitrogen blowing to obtain the crude peptide of GLP-1 moiety.

[0099] (11) Synthesis of target polypeptide

[0100] The crude peptide of 0.05 mmol of PYY moiety with a linker arm was dissolved in 2 mL of NMP, 0.005 mmol of DIPEA was added as a catalyst, after 20 minutes of reaction, 5 mL of 50% methanol / water (0.1% TFA) was added to stop the reaction, the reaction solution was filtered with a 0.25 μm microporous membrane and then purified by 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 90% B / 30 min), the target peak was collected, and the pure product was obtained after removing methanol 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 8490.6. ESI-MS m / z: calculated value [M+7H] 7+ 1213.9, [M+8H] 8+ 1062.3; observed value [M+7H] 7+ 1213.5, [M+8H] 8+ 1062.0.

[0101] Example 2

[0102] Synthesis of the polypeptide compound of SEQ ID NO: 2

[0103]

[0104] The synthesis method was the same as in Example 1, and the pure product was obtained after 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 8780.9. ESI-MS m / z: calculated value [M+7H] 7+ 1255.4, [M+8H] 8+ 1098.6; observed value [M+7H] 7+ 1255.1, [M+8H] 8+ 1098.3.

[0105] Example 3

[0106] Synthesis of the polypeptide compound of SEQ ID NO: 3

[0107]

[0108] The synthesis method was the same as in Example 1, and the pure product was obtained after 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 9361.5. ESI-MS m / z: calculated value [M+9H]9+ 1041.2, [M+10H] 10+ 937.2; observed [M+9H] 9+ 1040.9, [M+10H] 10+ 936.9.

[0109] Example 4

[0110] Synthesis of polypeptide compound of SEQ ID NO: 4

[0111]

[0112] The synthesis method is the same as Example 1. The pure product of 0.009 g is collected by freeze-drying of the target peak, with a purity greater than 98%. The molecular weight of the target polypeptide is confirmed by MS. The theoretical relative molecular mass is 8491.9. ESI-MS m / z: calculated value [M+7H] 7+ 1214.1, [M+8H] 8+ 1062.5; observed [M+7H] 7+ 1213.8, [M+8H] 8+ 1062.2.

[0113] Example 5

[0114] Synthesis of polypeptide compound of SEQ ID NO: 5

[0115]

[0116] The synthesis method is the same as Example 1. The pure product of 0.011 g is collected by freeze-drying of the target peak, with a purity greater than 98%. The molecular weight of the target polypeptide is confirmed by MS. The theoretical relative molecular mass is 8781.8. ESI-MS m / z: calculated value [M+7H] 7+ 1255.6, [M+8H] 8+ 1098.7; observed [M+7H] 7+ 1255.2, [M+8H] 8+ 1098.4.

[0117] Example 6

[0118] Synthesis of polypeptide compound of SEQ ID NO: 6

[0119]

[0120] The synthesis method is the same as Example 1. The pure product of 0.012 g is collected by freeze-drying of the target peak, with a purity greater than 98%. The molecular weight of the target polypeptide is confirmed by MS. The theoretical relative molecular mass is 9362.5. ESI-MS m / z: calculated value [M+9H] 9+1041.3, [M+10H] 10+ 937.3; observed [M+9H] 9+ 1041.1, [M+10H] 10+ 937.0.

[0121] Example 7

[0122] Synthesis of polypeptide compound of SEQ ID NO: 7

[0123]

[0124] The synthesis method is the same as that in Example 1, except that in step (9), the modification of the Lys side chain is different. After the synthesis of the GLP-1 moiety of the peptide chain is completed, 7 mL of 2% hydrazine hydrate / DMF (v / v) is added to selectively remove the Dde protecting group of Lys. After the Dde protecting group is removed, 0.4 mmol of Fmoc-AEEA-OH, 0.4 mmol of DIC, and 0.44 mmol of HOBt are added, and the condensation reaction is shaken for 2 h. After the Fmoc protecting group is removed, 0.4 mmol of Fmoc-AEEA-OH, 0.4 mmol of DIC, and 0.44 mmol of HOBt are added again, and the condensation reaction is shaken for 2 h. After the Fmoc protecting group is removed, 0.4 mmol of Fmoc-Glu-OtBu, 0.4 mmol of DIC, and 0.44 mmol of HOBt are added, and the condensation reaction is shaken for 2 h. After the Fmoc protecting group is removed, 0.4 mmol of octadecanedioic acid mono-tert-butyl ester, 0.4 mmol of DIC, and 0.44 mmol of HOBt are added, and the condensation reaction is shaken for 2 h. After the reaction is completed, the resin is washed with 7 mL of DMF for 4 times. The other synthesis method is the same as that in Example 1. The pure product is collected by freeze-drying of the target peak, and the purity is greater than 98%. The molecular weight of the target polypeptide is confirmed by MS. The theoretical relative molecular mass is 9129.3. ESI-MS m / z: calculated value [M+9H] 9+ 1015.4, [M+10H] 10+ 913.9; observed [M+9H] 9+ 1015.2, [M+10H] 10+ 913.7.

[0125] Example 8

[0126] Synthesis of polypeptide compound of SEQ ID NO: 8

[0127]

[0128] The synthetic method is the same as Example 7. The pure product of 0.011 g with purity greater than 98% was obtained by collecting the target peak and lyophilizing, and the molecular weight of the target polypeptide was confirmed by MS. The theoretical relative molecular mass is 9709.9. ESI-MS m / z: calculated value [M+9H] 9+ 1079.9, [M+10H] 10+ 972.0; observed value [M+9H] 9+ 1079.6, [M+10H] 10+ 971.8.

[0129] Example 9

[0130] Synthesis of the polypeptide compound of SEQ ID NO: 9

[0131]

[0132] The synthetic method is the same as Example 7. The pure product of 0.011 g with purity greater than 98% was obtained by collecting the target peak and lyophilizing, and the molecular weight of the target polypeptide was confirmed by MS. The theoretical relative molecular mass is 9709.9. ESI-MS m / z: calculated value [M+9H] 9+ 1015.5, [M+10H] 10+ 914.0; observed value [M+9H] 9+ 1015.2, [M+10H] 10+ 913.8.

[0133] Example 10

[0134] Synthesis of the polypeptide compound of SEQ ID NO: 10

[0135]

[0136] The synthetic method is the same as Example 7. The pure product of 0.011 g with purity greater than 98% was obtained by collecting the target peak and lyophilizing, and the molecular weight of the target polypeptide was confirmed by MS. The theoretical relative molecular mass is 9709.9. ESI-MS m / z: calculated value [M+9H] 9+ 1080.0, [M+10H] 10+ 972.1; observed value [M+9H] 9+ 1079.8, [M+10H] 10+ 971.9.

[0137] Example 11

[0138] Synthesis of the polypeptide compound of SEQ ID NO: 11

[0139]

[0140] The synthetic method is the same as Example 1. The pure product of 0.013 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 9532.6. ESI-MS m / z: calculated value [M+8H] 8+ 1120.0, [M+9H] 9+ 995.7; observed value [M+8H] 8+ 1119.7, [M+9H] 9+ 995.5.

[0141] Example 12

[0142] Synthesis of the polypeptide compound of SEQ ID NO: 12

[0143]

[0144] The synthetic method is the same as Example 1. The pure product of 0.013 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 9532.6. ESI-MS m / z: calculated value [M+8H] 8+ 1192.6, [M+9H] 9+ 1060.2; observed value [M+8H] 8+ 1192.2, [M+9H] 9+ 1059.5.

[0145] Example 13

[0146] Synthesis of the polypeptide compound of SEQ ID NO: 13

[0147]

[0148] The synthetic method is the same as Example 1. The pure product of 0.011 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 8867.9. ESI-MS m / z: calculated value [M+8H] 8+ 1109.5, [M+9H] 9+ 986.3; observed value [M+8H] 8+ 1109.4, [M+9H] 9+ 985.8.

[0149] Example 14

[0150] Synthesis of the polypeptide compound of SEQ ID NO: 14

[0151]

[0152] The synthetic procedure was the same as Example 1. The target peak was collected and lyophilized to give 0.011 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 was 9448.6. ESI-MS m / z: calcd [M+10H] 9448.6; found 9448.6 10+ 945.9, [M+11H] 11+ 860.0; obs [M+10H] 10+ 945.5, [M+11H] 11+ 859.8.

[0153] Example 15

[0154] Determination of agonist activity of polypeptide compounds on human GLP-1 receptor and Y2 receptor

[0155] The agonist effect of polypeptide compounds on the receptor was determined by a functional assay, GLP-1 receptor agonist activity was measured by determining the cAMP response of a HEK-293 cell line stably expressing human GLP-1 receptor. Cells stably expressing GLP-1 receptor were split into T175 flasks and grown overnight to near confluency in culture medium (DMEM / 10% FBS), then the medium was removed and the cells were washed with PBS without calcium and magnesium, then protease treated with Accutase enzyme. The detached cells were washed and resuspended in assay buffer (20 mM HEPES, 0.1% BSA, 2 mM IBMX, 1 x HBSS) and the cell density was determined and 25 μL aliquots were dispensed into the wells of a 96-well plate. For the measurement, 25 μL of test polypeptide compound in assay buffer was added to the wells, then incubated for 30 minutes at room temperature. The cAMP content of the cells was determined based on homogeneous time-resolved fluorescence (HTRF) using a kit from Cisbio. After addition of the HTRF reagents diluted in lysis buffer (kit components), the plate was incubated for 30 minutes, then the fluorescence ratio at 665 / 620 nm was measured. The in vitro potency of agonists was quantified by detecting the concentration (EC50) that caused 50% activation of the maximum response. 50

[0156] ​The agonistic effect of the compounds on Y2 receptor was determined using HEK-293 cells stably expressing human Y2 receptor and cAMP-sensitive calcium ion channel. First, the cells were cultured in culture medium (DMEM, 10% FBS, geneticin, geneticin, penicillin / streptomycin), and then 20 μL of cell suspension per well was added to a 384-well plate (20000 cells / well). Then the cells were pretreated with a calcium-sensitive dye at 37°C, 5% CO2for 50 minutes, followed by pretreatment at 25°C for 10 minutes. The test compound was serially diluted 10 times with 4 times the amount, and 750 nL of the test compound was transferred to the 384-well plate. Then the 384-well plate was taken out of the incubator and placed in the FLIPR Tetra System, and the fluorescence signal was measured (excitation 494 nm / emission 516 nm), and the in vitro potency of the agonist was quantified by detecting the concentration (EC50) causing 50% activation of the maximum response. 50 ) to measure the fluorescence signal.

[0157] 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 to mean that the data has been determined to be accurate to the number of significant figures.

[0158] Table 1 Agonistic activity of polypeptide compounds on human GLP-1 receptor and Y2 receptor

[0159]

[0160]

[0161] As shown in Table 1, all polypeptide compounds showed dual agonistic activity on GLP-1 receptor and Y2 receptor, indicating that these polypeptide compounds all have the characteristics of dual agonists. At the same time, some polypeptide compounds showed agonistic activity close to or better than GLP-1 and PYY 3-36 on GLP-1 receptor and Y2 receptor.

[0162] Example 16

[0163] Pharmacokinetic properties of polypeptide compounds in rats

[0164] Rats were administered 50 nmol / kg by subcutaneous (s.c.) injection, and blood samples were collected at 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, 16 h and 24 h after administration. After protein precipitation with acetonitrile, the plasma samples were analyzed by LC-MS. The pharmacokinetic parameters and half-life were calculated using WinonLin 5.2.1 (non-compartment model).

[0165] Table 2 Pharmacokinetic profiles of polypeptide compounds in rats

[0166] Sample T 1 / 2 (h)]]> C max (ng / mL) Liraglutide 3.3 462 SEQ ID NO: 11 4.4 459

[0167] As shown in Table 2, the in vivo half-life of the polypeptide compound of the present application is significantly prolonged, with pharmacokinetic characteristics supporting once-daily or once-weekly dosing.

[0168] Example 17

[0169] Effect of polypeptide compound on food intake of C57BL / 6J mice

[0170] Male C57BL / 6J mice were randomly divided into 3 groups, 6 mice in each group. The mice were fasted for 12 h before the experiment, the blank group was subcutaneously injected with normal saline (10 mg / kg), the administration group was divided into 2 groups, and the mice were subcutaneously injected with 25 nmol / kg of liraglutide and SEQ ID NO: 11, respectively, under non-fasting conditions. Then the mice were immediately given pre-weighed mouse feed, and the feed weight was weighed again at 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 8 h, 10 h, 12 h and 24 h, and the food intake of the mice at different time points was calculated.

[0171] As Figure 1 As shown in the results, the food intake experiment in C57BL / 6J mice showed that at 24 h, liraglutide could only reduce the food intake of mice by 30.2%, while the polypeptide compound of SEQ ID NO: 11 could reduce the food intake of mice by 72.6%, which was significantly better than liraglutide, indicating that the polypeptide compound of the present application had excellent food intake inhibition effect.

[0172] Example 18

[0173] Effect of polypeptide compound on blood glucose and body weight of diet-induced obese (DIO) mice

[0174] Male C57BL / 6J mice, weighing about 22 g, a total of 18 mice, were fed with D12492 high-fat feed from Research Diets for 18 weeks to establish a DIO mouse model. Before the start of administration, the DIO mice in each group were randomly divided by weight, and a total of 3 groups were formed, 6 mice in each group, namely the normal saline group (blank control group), the positive control group (liraglutide), and the test sample group (SEQ ID NO: 11). Each group of mice was subcutaneously injected with normal saline (10 mg / kg), liraglutide (25 nmol / kg), and SEQ ID NO: 11 (25 nmol / kg) twice a day, and the administration period was 21 days. The body weight of the mice was recorded every day. At the end of the experiment, the mice in each group were sacrificed, blood was taken to prepare serum, and the serum triglyceride (TG) and total cholesterol (TC) contents were measured.

[0175] As Figure 2 The results show that the polypeptide compound of the present application SEQ ID NO: 11 can reduce the weight of mice by 32.6% in DIO mice for 3 weeks of continuous administration, while liraglutide can only reduce the weight of mice by 16.5%, indicating that the weight loss effect of SEQ ID NO: 11 is significantly stronger than the positive control drug liraglutide.

[0176] Table 3 Serum triglyceride (TG) and total cholesterol (TC) content of DIO mice after 3 weeks of treatment

[0177] Sample (dose) Total cholesterol (mmol / L) Triglyceride (mmol / L) Blank control (saline group) 7.14±0.33 1.61±0.12 Liraglutide (25 nmol / kg) 5.97 ± 0.31 *** ]] 1.18 ± 0.08 *** ]] SEQ ID NO: 11 (10 nmol / kg) 4.15 ± 0.15 ***,### ]] 0.96 ± 0.10 ***,## ]]

[0178] *** : P < 0.001 compared with the blank control group; ## : P < 0.01 compared with the liraglutide group; ### : P < 0.001 compared with the liraglutide group (One-Way ANOVA, Tukey post hoc test), the results are expressed as the average value ± SD of 6 mice in each group.

[0179] As shown in Table 3, the polypeptide compound of the present application SEQ ID NO: 11 can significantly reduce the serum triglyceride (TG) and total cholesterol (TC) content of mice in DIO mice for 3 weeks of continuous administration, and the lipid-lowering effect of the polypeptide compound of the present application is significantly stronger than the positive control drug liraglutide.

Claims

1. A GLP-1 / Y2 receptor dual agonist, characterized in that, The amino acid sequence structure of the GLP-1 / Y2 receptor dual agonist is as follows: 。 2. A GLP-1 / Y2 receptor dual agonist, characterized in that, The amino acid sequence structure of the GLP-1 / Y2 receptor dual agonist is as follows: 。 3. A pharmaceutically acceptable salt of a GLP-1 / Y2 receptor dual agonist, characterized in that: The GLP-1 / Y2 receptor dual agonist is the GLP-1 / Y2 receptor dual agonist as described in any one of claims 1-2.

4. A pharmaceutically acceptable salt of a GLP-1 / Y2 receptor dual agonist according to claim 3, characterized in that the salt is a salt formed by the GLP-1 / Y2 receptor dual agonist and one of the following compounds: hydrochloric acid, acetic acid, salicylic acid, lauric acid, cinnamic acid, citric acid, oxalic acid, lactic acid, and succinic acid.

5. A pharmaceutical preparation made from a GLP-1 / Y2 receptor dual agonist according to any one of claims 1-2, characterized in that, The pharmaceutical preparation is a tablet, capsule, inhaler, spray, injection, film, patch, emulsion, suppository, or combination preparation, and the pharmaceutical preparation consists of the GLP-1 / Y2 receptor dual agonist and pharmaceutically acceptable excipients.

6. A pharmaceutical composition containing a dual GLP-1 / γ2 receptor agonist, characterized in that, The pharmaceutical composition uses the GLP-1 / Y2 receptor dual agonist as the active ingredient in any one of claims 1-2, or a pharmaceutically acceptable salt of the GLP-1 / Y2 receptor dual agonist as described in claim 3 or 4, plus a pharmaceutically acceptable carrier or diluent.

7. Use of the GLP-1 / Y2 receptor dual agonist of any one of claims 1-2, or a pharmaceutically acceptable salt of the GLP-1 / Y2 receptor dual agonist of any one of claims 3-4, or the agent of claim 5, or the pharmaceutical composition of claim 6, in the preparation of a medicament for treating metabolic diseases, said metabolic diseases being diabetes, obesity, or dyslipidemia.

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