Polypeptide and application thereof

Abstract

Belongs to the technical field of biology. The polypeptide is established on the basis of mechanism research on ETBR agonists, and belongs to ETBR high-selectivity agonists. According to the polypeptide, the half-life period of the medicine is prolonged through structure-function relationship research and structural modification, the in-vivo exposure amount is increased, the administration frequency is reduced, the side effect in animal experiments is lower than that of IRL-1620, and the safety window of the medicine is larger. The medicine can be used for treating diseases related to ETBR receptors, such as rehabilitation treatment of patients with cerebral arterial thrombosis in an acute stage and a recovery stage, and better recovery of the patients with cerebral arterial thrombosis is promoted.

Description

Peptides and their applications

[0001] This application claims priority to the Chinese patent application filed with the China Patent Office on April 26, 2023, with application number 202310461975.3 and invention name “Polypeptides and Their Applications”, the entire contents of which are incorporated by reference into this application.

[0002] This application claims priority to the Chinese patent application filed with the China Patent Office on December 27, 2023, with application number 202311825930.6 and invention name “Polypeptides and Their Applications”, the entire contents of which are incorporated by reference into this application. Technical Field

[0003] The present invention relates to the field of biotechnology, and in particular to polypeptides and applications thereof. Background Art

[0004] Currently, the main treatments and medications for stroke include vascular intervention, thrombolysis, antiplatelet therapy, anticoagulation, fibrinolytic therapy, and neuroprotective agents. Vascular intervention and thrombolysis are generally performed very early after the onset of a stroke and must be performed in a hospital with sufficient capacity. Antiplatelet and anticoagulant drugs are generally used in conjunction with thrombolytics at a later stage, but their effectiveness is limited and carries a certain risk of bleeding. Neuroprotective agents have great potential and are generally used for acute treatment. However, there are no marketed therapeutics specifically designed to promote recovery in the subacute and recovery phases of stroke patients. Previous studies have shown that endothelin B receptor agonists have great potential in promoting long-term recovery and post-stroke rehabilitation in stroke patients.

[0005] Endothelin (ET) is a 21-amino acid polypeptide divided into three subtypes: ET-1, ET-2, and ET-3. These subtypes share structural homology with the snake venom sarafotoxins b and c (S6b and S6c). Endothelin is the most potent and long-lasting vasoactive peptide known, exerting potent vasoconstriction and promoting smooth muscle cell migration and proliferation. It acts on a wide range of systems in the body and is closely linked to diseases such as hypertension, congestive heart failure, diabetes, cancer, and fibrosis.

[0006] Endothelins primarily exert their biological effects by binding to endothelin receptors (ETRs) on target cell membranes. They play a crucial role in the development and progression of numerous diseases, including cardiovascular and cerebrovascular diseases, kidney disease, diabetes, autoimmune diseases, and tumors. Human ETRs are divided into two subtypes, ETA and ETB, both of which are G-protein-coupled receptors (GPCRs), but with distinct functions. ETA is considered a primary vasoconstrictor and growth-promoting receptor, while ETB inhibits cell growth and vasoconstriction in the vascular system and is referred to as a "scavenger receptor" due to its involvement in the clearance of ET-1. Endothelins and their receptors are diversely distributed across tissues, mediating complex biological effects through distinct subtypes and signaling systems. They are closely linked to a wide range of diseases, with ETA and ETB receptors playing a crucial role.

[0007] Studies have shown that ETBR agonists have great potential in promoting long-term recovery and post-rehabilitation in stroke patients. Among the ETBR agonist compounds discovered so far, sovateltide (IRL-1620, Suc-DEEAVYFAHLDIIW) is the only ETBR agonist that has advanced to clinical research. However, due to its short half-life of less than 2 minutes and low exposure, IRL-1620 requires repeated administration in preclinical and clinical studies, which greatly reduces its sustained efficacy and patient compliance.

[0008] Summary of the Invention

[0009] In view of this, the present invention provides polypeptides and uses thereof.

[0010] The present invention provides polypeptides and their applications. The polypeptides discovered in this patent are based on mechanistic research on ETBR agonists and are highly selective agonists of ETBR. Through structure-activity relationship research and structural modification, the polypeptides developed in this patent extend the half-life and in vivo exposure of the drug, reduce the number of dosing times, have fewer side effects than IRL-1620 in animal experiments, and have a wider safety window. The compounds of the present invention can be used to treat diseases related to ETBR receptors, such as rehabilitation issues in patients in the recovery period of ischemic stroke, and promote better recovery of stroke patients.

[0011] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0012] The present invention provides a polypeptide, characterized in that it comprises:

[0013] (I), having the structure shown in Formula I; or

[0014] X1-X2-X3-(A5)-X4-X5-(A1)-X6(A2)-X7-X8-X9-(A3)-X10-X11-(A4)-X12-X13-X14-X15-X16

[0015] Formula I

[0016] (II) a sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the structure shown in (I); or

[0017] (III) a sequence with a structural similarity of more than 90% to that shown in (I); or

[0018] (IV), a pharmaceutically acceptable salt, solvate, chelate or non-covalent complex having a structure as shown in Formula I; and / or

[0019] (V), a prodrug based on the structure of Formula I; and / or

[0020] (VI), any mixture comprising (IV) and / or (V);

[0021] Preferably, X1 is selected from Suc, Fum, MeO-Fum, Ac, SA, MA, FA, A1-D or A1-NH-C2H4-NH-Suc;

[0022] Preferably, X2 is selected from D, E or K (A1);

[0023] The X1 and X2 as a whole can be:

[0024] Preferably, X3 is selected from E, D or K (A1);

[0025] Preferably, X4 is selected from E, E(C16) or D;

[0026] Preferably, X5 is selected from A, K, R, H, E or D;

[0027] Preferably, X6 is selected from V, I, K, Dab, Orn, L, F, W or C;

[0028] Preferably, X7 is selected from Y, F or W;

[0029] Preferably, X8 is selected from F, Y or W;

[0030] Preferably, X9 is selected from A, K, R, H, D, E, Dab or Orn;

[0031] Preferably, X10 is selected from H, K or R;

[0032] Preferably, X11 is selected from K, L, L(N-Me), I, V, Cit, H, R, L-2-amino-4-guanidinobutyric acid or Orn;

[0033] Preferably, X12 is selected from D or E;

[0034] Preferably, X13 is selected from I, I(N-Me), 2-Abu, L, P or V;

[0035] Preferably, X14 is selected from I, 2-Abu, L or V;

[0036] Preferably, X15 is selected from W, F, Y or

[0037] Preferably, X16 is selected from OH or NH2;

[0038] Preferably, said A5 is selected from C or is absent;

[0039] Preferably, said A1, A2, A3 or A4 independently comprises (Y1-Y2-Y3-Y4);

[0040] Preferably, the Y1 is selected from -NH-(CH2-CH2-O)m1-(CH2)m2-CO-, L / D-Glu or absent, wherein L / D-Glu is connected to the side chain amino group of the polypeptide main chain amino acid through an α- or γ-carboxyl group and is connected to the carboxyl group of Y2 through an amino group;

[0041] Preferably, the Y2 is selected from -NH-(CH2-CH2-O)m3-(CH2)m4-CO-, or does not exist;

[0042] Preferably, the Y3 is selected from -NH-(CH2-CH2-O)m5-(CH2)m6-CO-, L / D-Glu or absent, wherein L / D-Glu is connected to the amino group of Y2 through an α- or γ-carboxyl group and is connected to the carboxyl group of Y4 through an amino group;

[0043] Preferably, the Y4 is selected from D-Biotin, the structure shown in Formula 2, HOOC-(CH2) n -CO, CH3-(CH2) n -CO or not present;

[0044] wherein m1, m2, m3, m4, m5 or m6 is an integer from 1 to 10;

[0045] wherein n is selected from an integer from 10 to 25;

[0046] Preferably, A1, A2, A3 or A4 can only exist alone or not at all; and

[0047] The polypeptide of formula I is not Suc-DEEAVYFAHLDIIW.

[0048] In some specific embodiments of the present invention,

[0049] Preferably, X1 is selected from Suc, Fum, MeO-Fum, Ac, SA, MA, FA(ODDA-γE-AEEA-AEEA-)D or (ODDA-γE-AEEA-AEEA-)NH-C2H4-NH-Suc;

[0050] Preferably, X2 is D or K(A1);

[0051] Preferably, X1 and X2 as a whole can be:

[0052] Preferably, X3 is E or K (A1);

[0053] Preferably, X4 is selected from E or E(C16);

[0054] Preferably, X5 is selected from A, K or E;

[0055] Preferably, X6 is selected from V, K, Dab, L, F or C;

[0056] Preferably, X7 is Y;

[0057] Preferably, X8 is F;

[0058] Preferably, X9 is selected from A, K or E;

[0059] Preferably, X10 is H;

[0060] Preferably, X11 is selected from K, L, L(N-Me), R, Cit, L-2-amino-4-guanidinobutyric acid or Orn;

[0061] Preferably, X12 is D;

[0062] Preferably, X13 is selected from I, I(N-Me), 2-Abu, P or V;

[0063] Preferably, X14 is 1;

[0064] Preferably, the X15 is selected from W or

[0065] Preferably, the A1 is selected from -γE-AEEA-AEEA-ODDA, ODDA-γE-AEEA-AEEA-, Biotin, -AEEA-AEEA-γE-ODDA or is absent;

[0066] Preferably, said A2 is selected from -γE-AEEA-AEEA-ODDA or is absent;

[0067] Preferably, said A3 is selected from -γE-AEEA-AEEA-ODDA or is absent;

[0068] Preferably, the A4 is selected from -AEEA-AEEA-γE-ODDA, -γE-AEEA-AEEA-C18, -γE-AEEA-AEEA-C16, -γE-AEEA-AEEA-C12, -γE-AEEA -AEEA-B, -γE-AEEA-AEEA-ODDA, CPAK-MPA-, -AEEA-γE-C18, -AEEA-γE-C16, -AEEA-γE-C12, -AEEA-E-C12 or absent.

[0069] In some specific embodiments of the present invention, the structure represented by Formula I is cyclized by any of the following:

[0070] (I), introducing C to form a disulfide bond; and / or

[0071] (II) The carboxyl group of the D or E side chain forms an amide bond with the amino group of the K or Dab side chain.

[0072] In some specific embodiments of the present invention, the cyclization comprises any of the following:

[0073] (I) inserting C between X3 and X4, and replacing X6 with C, to form a disulfide bond cyclization through the two Cs; and / or

[0074] (II), E of X3 and K or Dab of X6 form an amide bond cyclization; and / or

[0075] (III) E of X5 and K of X11 form an amide bond to cyclize.

[0076] In some specific embodiments of the present invention, the polypeptide sequence is as follows:

[0077] The present invention also provides a method for preparing the polypeptide, comprising the following steps:

[0078] Step 1: obtaining a peptide resin of the polypeptide by solid phase peptide synthesis;

[0079] Step 2: cleavage and purification to obtain the polypeptide.

[0080] Specifically, the present invention provides a method for preparing polypeptide BX-34, comprising the following steps:

[0081] Step 1: Weigh 0.50 g (0.25 mmol) of Fmoc-Trp(Boc)-Wang Resin into a reactor and swell with 10 mL of DCM for 10 min. Filter and wash twice with DMF. Add 25% 4-methylpiperidine / DMF (volume ratio) and react for 30 min to remove the Fmoc group. Filter and wash the resin four times with DMF and twice with DCM. Ninhydrin detection reveals a blue resin solution. Weigh Fmoc-Ile-OH (1 mmol, 4 eq.) and HBTU (1 mmol, 4 eq.) and dissolve them in DMF. Add DIEA (2 mmol, 8 eq.), mix thoroughly, and add to the resin. React with magnetic stirring at room temperature (25°C ± 5°C) for 1 h. Filter and wash three times with DMF, MeOH, and DCM. Ninhydrin detection reveals a colorless and transparent resin and a light yellow solution, indicating complete reaction. Add 25% 4-methylpiperidine / DMF (volume ratio) to remove the Fmoc group. The coupling of Ile, Asp(OtBu), Lys(Dde), His(Trt), Ala, Phe, Tyr(tBu), Val, Ala, Glu(OtBu), Glu(OtBu), Asp(OtBu), and OtBu-Fum was completed in a sequential cycle. Subsequently, 2% hydrazine hydrate / DMF was added to the peptide resin for 10 min × 3 times to remove the Dde from the Lys side chain, followed by the condensation of AEEA, AEEA, Glu-α-OtBu, and OtBu-ODDA.

[0082] Step 2: Drain the resin and add 15 mL of pre-chilled lysis buffer (TFA:TIS:H2O = 95:2.5:2.5). Stir and react at room temperature (25°C ± 5°C) for 3 h. Drain the reaction solution and wash the resin twice with a small amount of TFA. Drain the reaction solution and add approximately 200 mL of pre-chilled methyl tert-butyl ether to precipitate a white precipitate. Centrifuge (5000 rpm × 5 min) and discard the supernatant. Add methyl tert-butyl ether to the precipitate, vortex, centrifuge, and discard the supernatant. Dry the precipitate in a vacuum desiccator for 12 h to obtain the crude product.

[0083] Step 3: The crude peptide was dissolved in about 1% ammonia water, filtered, and the filtrate was purified by preparative liquid phase and gradient elution to obtain the pure target compound. The target compound components with a purity of >95% were combined, and the acetonitrile was removed by decompression at about 40°C using a water pump and a rotary evaporator. The resulting concentrate was frozen into a solid with liquid nitrogen, and then freeze-dried in a freeze dryer for 48 hours to obtain the target compound, and the molecular weight was identified by mass spectrometry.

[0084] In some specific embodiments of the present invention, the preparative column used for purification includes C18-10-100, 30×250 mm, and the flow rate used is 25 mL / min; the preparative liquid phase includes phase A: 0.1% TFA / water, phase B: 0.1% TFA / 90% acetonitrile / water.

[0085] In some specific embodiments of the present invention, the method for preparing the polypeptide BX-34 can also prepare BX-5, F-BX-5, BX-7, BX-8, BX-43, BX-45, BX-51, BX-52, BX-53, BX-54, BX-58, BX-59, BX-60, BX-61, BX-66, BX-67, BX-6 8. BX-69, BX-70, BX-71, BX-72, BX-73, BX-74, BX-75, BX-78, BX-86, BX-101, BX- 105, BX-106, BX-107, BX-108, BX-109, BX-110, BX-111, BX-112, BX-229 and BX-238.

[0086] Specifically, the present invention also provides a method for preparing the polypeptide BX-76, comprising the following steps:

[0087] Step 1: Complete the synthesis of the main chain by referring to the synthesis method of BX-34;

[0088] Step 2: Under nitrogen protection, add 0.3 equivalents of tetrakistriphenylphosphine palladium and 12 equivalents of phenylsilane in DCM to the peptide resin and react at room temperature (25°C ± 5°C) for 2 hours to remove the allyl ester of the Glu side chain and the allyloxycarbonyl of the Lys side chain; filter with suction, wash with DCM solvent 5 times, wash with DMF 3 times, add 2 equivalents of PYBOP, 2 equivalents of HOBt and 4 equivalents of DIEA, dissolve in DMF, and react at room temperature (25°C ± 5°C) for 5 hours; filter with suction, wash with DMF 3 times, wash with DCM 3 times, and detect with ninhydrin. If the reaction is incomplete, repeat the condensation once;

[0089] Step 3: Then, refer to the BX-34 method to perform cleavage and purification to obtain the pure target compound, and identify the molecular weight by mass spectrometry.

[0090] In some specific embodiments of the present invention, the method for preparing the polypeptide BX-76 can also prepare BX-48, BX-49, BX-50, BX-62, BX-63, BX-64, BX-65, BX-77, BX-79, BX-100, BX-103, BX-104, BX-99, and BX-102.

[0091] Specifically, the present invention also provides a method for preparing the polypeptide BX-56, comprising the following steps:

[0092] Step 1: Complete the synthesis of the main chain by referring to the synthesis method of BX-34;

[0093] Step 2: Add 5 equivalents of iodine in DMF to the peptide resin and react at room temperature (25°C ± 5°C) for 6 hours to remove the Trt protecting group on the Cys side chain and simultaneously oxidize it. Filter, wash six times with DMF, three times with DCM, and dry.

[0094] Step 3: Refer to the synthesis method of BX-34, cleave and purify to obtain the pure target compound, and identify the molecular weight by mass spectrometry.

[0095] In some specific embodiments of the present invention, the method for preparing the polypeptide BX-56 can also be used to prepare BX-57.

[0096] Specifically, the present invention also provides a method for preparing the polypeptide BX-95, comprising the following steps:

[0097] Step 1: Refer to the synthesis method of BX-34 to obtain the main chain pure peptide, and refer to the synthesis method of BX-34 to obtain the CPAK pure peptide;

[0098] Step 2: Dissolve the backbone pure peptide and the CPAK pure peptide in equivalent amounts in 50% acetonitrile aqueous solution, adjust the pH to 8-9 with triethylamine, and react at room temperature (25°C ± 5°C) until complete;

[0099] Step 3: Purify according to the method of BX-34 to obtain the pure target compound, and identify the molecular weight by mass spectrometry.

[0100] Based on the above research, the present invention also provides a polypeptide prepared by the preparation method.

[0101] The present invention also provides the use of the polypeptide in preparing medicines for treating diseases related to ETBR receptors.

[0102] In some specific embodiments of the present invention, the ETBR receptor-related diseases include one or more of stroke, Alzheimer's disease, spinal cord injury, aortic valve stenosis or neonatal hypoxic-ischemic encephalopathy;

[0103] The stroke includes ischemic stroke and / or hemorrhagic stroke.

[0104] The present invention also provides a medicine comprising the polypeptide and a pharmaceutically acceptable adjuvant or auxiliary agent.

[0105] The present invention also provides a pharmaceutical composition comprising the drug and any other effective ingredients.

[0106] The present invention also provides a method for treating a disease associated with ETBR receptors, comprising administering to a subject any of the following:

[0107] (I), the polypeptide; and / or

[0108] (II), the drug; and / or

[0109] (III) the pharmaceutical composition.

[0110] In some specific embodiments of the present invention, the ETBR receptor-related diseases include one or more of stroke, Alzheimer's disease, spinal cord injury, aortic valve stenosis or neonatal hypoxic-ischemic encephalopathy;

[0111] The stroke includes ischemic stroke and / or hemorrhagic stroke.

[0112] The peptides discovered in this patent are based on mechanistic research on ETBR agonists and are highly selective ETBR agonists. Through structure-activity relationship studies and structural modifications, the peptides developed in this patent extend the drug's half-life and in vivo exposure, reduce the number of dosing times, and exhibit fewer side effects than IRL-1620 in animal studies, with a wider safety window. The compounds of this invention can be used to treat diseases associated with ETBR receptors, such as rehabilitation therapy for patients recovering from ischemic stroke, promoting better recovery in stroke patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0113] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for describing the embodiments or the prior art.

[0114] Figure 1 shows the EC50 determination of the activity of the polypeptides in Example 3 against the human ETB receptor; wherein A shows the EC50 of BX-15; B shows the EC50 of BX-6; C shows the EC50 of BX-10; D shows the EC50 of BX-21; E shows the EC50 of BX-22; and F shows the EC50 of BX-5;

[0115] Figure 2 shows the EC50 determination of the activity of the polypeptides in Example 3 against the human ETB receptor; wherein A shows the EC50 of BX-9; B shows the EC50 of BX-8; C shows the EC50 of BX-7; D shows the EC50 of F-BX-5; E shows the EC50 of BX-13-2; and F shows the EC50 of BX-31;

[0116] Figure 3 shows the EC50 determination of the activity of the polypeptides in Example 3 against the human ETB receptor; wherein A shows the EC50 of BX-32; B shows the EC50 of BX-38; C shows the EC50 of BX-43; D shows the EC50 of BX-45; E shows the EC50 of QP-1; and F shows the EC50 of QP-3.

[0117] Figure 4 shows the EC50 determination of the activity of the polypeptides in Example 3 against the human ETB receptor; wherein A shows the EC50 of BX-24; B shows the EC50 of BX-53; C shows the EC50 of BX-54; D shows the EC50 of BX-58; E shows the EC50 of FBX-8; and F shows the EC50 of BX-34;

[0118] Figure 5 shows the EC50 determination of the activity of the polypeptides in Example 3 against the human ETB receptor; wherein A shows the EC50 of BX-48; B shows the EC50 of BX-49; and C shows the EC50 of BX-50;

[0119] Figure 6 shows the EC50 determination of the activity of the polypeptides on the human ETB receptor in Example 3; wherein A shows the EC50 of BX-69 and its determination data; B shows the EC50 of BX-70 and its determination data; C shows the EC50 of BX-71 and its determination data;

[0120] Figure 7 shows the EC50 determination of the activity of the polypeptides in Example 3 against the human ETB receptor; wherein A shows the EC50 of BX-76 and its determination data; B shows the EC50 of BX-95 and its determination data; C shows the EC50 of BX-99 and its determination data;

[0121] Figure 8 shows the EC50 determination of the activity of the polypeptides in Example 6 on canine ETB receptor; wherein A shows the EC50 of Dog-BX-34 and its determination data; B shows the EC50 of Dog-BX-76 and its determination data; C shows the EC50 of Dog-BX-74 and its determination data;

[0122] Figure 9 shows the EC50 determination of the activity of the polypeptides in Example 6 on canine ETB receptor; wherein A shows the EC50 and its determination data of Dog-BX-99; B shows the EC50 and its determination data of Dog-BX-86; C shows the EC50 and its determination data of Dog-BX-69;

[0123] Figure 10 shows the EC50 determination of the canine ETB receptor activity of the polypeptides in Example 6; wherein A shows the EC50 of Dog-BX-70 and its determination data; B shows the EC50 of Dog-BX-71 and its determination data; C shows the EC50 of Dog-BX-95 and its determination data;

[0124] Figure 11 shows the EC50 determination of the activity of the polypeptides in Example 4 on rat ETB receptors; wherein A shows the EC50 of Rat-BX-34 and its determination data; B shows the EC50 of Rat-F-BX-5 and its determination data; C shows the EC50 of Rat-BX-69 and its determination data;

[0125] Figure 12 shows the EC50 determination of the activity of the polypeptides in Example 4 on rat ETB receptors; wherein A shows the EC50 of Rat-BX-99 and its determination data; B shows the EC50 of Rat-BX-71 and its determination data; C shows the EC50 of Rat-BX-95 and its determination data;

[0126] Figure 13 shows the EC50 determination of the activity of the polypeptides in Example 4 on rat ETB receptor; wherein A shows the EC50 of Rat-BX-74 and its determination data; B shows the EC50 of Rat-BX-76 and its determination data;

[0127] Figure 14 shows the EC50 determination of the activity of the polypeptides in Example 5 on the mouse ETB receptor; wherein A shows the EC50 and its determination data of Mouse-BX-34; B shows the EC50 and its determination data of Mouse-BX-69; C shows the EC50 and its determination data of Mouse-BX-70;

[0128] Figure 15 shows the EC50 determination of the activity of the polypeptides in Example 5 on the mouse ETB receptor; wherein A shows the EC50 and its determination data of Mouse-BX-71; B shows the EC50 and its determination data of Mouse-BX-95; C shows the EC50 and its determination data of Mouse-F-BX-5;

[0129] FIG16 shows the effect of the polypeptide BX34 in Example 7 on cerebral blood perfusion in dMACO model mice;

[0130] Figure 17 shows the EC50 determination of the activity of the polypeptides in Example 3 against the human ETB receptor; wherein A shows the EC50 of BX-223; B shows the EC50 of BX-224; and C shows the EC50 of BX-225-Na;

[0131] Figure 18 shows the EC50 determination of the activity of the polypeptides in Example 3 against the human ETB receptor; wherein A shows the EC50 of BX-253; B shows the EC50 of BX-254; and C shows the EC50 of BX-256;

[0132] Figure 19 shows the EC50 determination of the activity of the polypeptides in Example 3 against the human ETB receptor; wherein A shows the EC50 of BX-221; B shows the EC50 of BX-56; and C shows the EC50 of BX-238;

[0133] Figure 20 shows the EC50 determination of the activity of the polypeptides in Example 3 against the human ETB receptor; wherein A shows the EC50 of BX-229; B shows the EC50 of BX-255;

[0134] Figure 21 shows the EC50 determination of the canine ETB receptor activity of the polypeptides in Example 6; wherein A shows the EC50 and its determination data of Dog-BX-224; B shows the EC50 and its determination data of Dog-BX-225; C shows the EC50 and its determination data of Dog-BX-253;

[0135] Figure 22 shows the EC50 determination of the activity of the polypeptides in Example 6 against canine ETB receptors; wherein A shows the EC50 and its determination data of Dog-BX-254; B shows the EC50 and its determination data of Dog-BX-255; C shows the EC50 and its determination data of Dog-BX-256;

[0136] Figure 23 shows the EC50 determination of the canine ETB receptor activity of the polypeptides in Example 6; wherein A shows the EC50 and its determination data of Dog-BX-56; B shows the EC50 and its determination data of Dog-BX-229-Na; C shows the EC50 and its determination data of Dog-BX-238;

[0137] Figure 24 shows the EC50 determination of the activity of the polypeptides in Example 4 on rat ETB receptors; wherein A shows the EC50 of Rat-BX-223 and its determination data; B shows the EC50 of Rat-BX-224 and its determination data; C shows the EC50 of Rat-BX-225 and its determination data;

[0138] Figure 25 shows the EC50 determination of the activity of the polypeptides in Example 4 on rat ETB receptor; wherein A shows the EC50 and its determination data of Rat-BX-255; B shows the EC50 and its determination data of Rat-BX-254; C shows the EC50 and its determination data of Rat-BX-253;

[0139] Figure 26 shows the EC50 determination of the activity of the polypeptides in Example 4 on rat ETB receptors; wherein A shows the EC50 and its determination data of Rat-BX-56; B shows the EC50 and its determination data of Rat-BX-238; C shows the EC50 and its determination data of Rat-BX-229;

[0140] FIG27 shows the EC50 determination of the activity of the polypeptides in Example 4 on rat ETB receptor; specifically showing the EC50 of Rat-BX-256 and its determination data;

[0141] Figure 28 shows the EC50 determination of the activity of the polypeptides in Example 5 on the mouse ETB receptor; wherein A shows the EC50 and its determination data for Mouse-BX-223; B shows the EC50 and its determination data for Mouse-BX-224; C shows the EC50 and its determination data for Mouse-BX-225;

[0142] Figure 29 shows the EC50 determination of the activity of the polypeptides in Example 5 on the mouse ETB receptor; wherein A shows the EC50 and its determination data of Mouse-BX-253; B shows the EC50 and its determination data of Mouse-BX-254; C shows the EC50 and its determination data of Mouse-BX-255;

[0143] Figure 30 shows the EC50 determination of the activity of the polypeptides in Example 5 on the mouse ETB receptor; wherein A shows the EC50 and its determination data of Mouse-BX-256; B shows the EC50 and its determination data of Mouse-BX-238; C shows the EC50 and its determination data of Mouse-BX-56;

[0144] FIG31 shows the EC50 determination of the activity of the polypeptides in Example 5 on mouse ETB receptor; wherein A shows the EC50 of Mouse-BX-229 and its determination data;

[0145] Figure 32 shows the Garcia JH score on day 7 after modeling and administration in Example 13;

[0146] Figure 33 shows the Garcia JH score on day 14 after modeling and administration in Example 13;

[0147] FIG34 shows the changes in VEGF-A protein expression in Example 13;

[0148] FIG35 shows the changes in BDNF protein expression in Example 13. DETAILED DESCRIPTION

[0149] The present invention discloses polypeptides and their applications. Those skilled in the art can refer to the disclosure herein and appropriately modify process parameters to achieve the desired results. It should be noted that all similar substitutions and modifications apparent to those skilled in the art are considered encompassed by the present invention. The methods and applications of the present invention have been described through preferred embodiments. It is apparent that those skilled in the art can modify or appropriately alter and combine the methods and applications described herein to implement and apply the technology of the present invention without departing from the disclosure, spirit, and scope of the present invention.

[0150] The structures of some polypeptides of the present invention and the results of biological experiments are as follows:

[0151] Table 1 Peptide structure and activity on human ETB receptor

[0152] Table 6 Structures of some peptides and their activity on human ETA receptor

[0153] Table 7 Structures of some peptides and their activity on canine ETB receptor

[0154] Table 8 Structures of some peptides and their activity on rat ETB receptor

[0155] Table 9 Structures of some peptides and their activity on mouse ETB receptor

[0156] Table 10 Chinese explanation of abbreviations or full English names

[0157] General Note: The hyphen "-" in the middle of the compound number can be omitted. In some experiments, the hyphen is omitted, for example, BX229 is equivalent to BX-229. Unless otherwise specified, the peptide is in the trifluoroacetate form by default. Some compounds will be marked with a special salt form after the number, such as BX-229-Na, and BX229Na both represent the sodium salt form of BX-229.

[0158] Table 11 Main reagents and solvents

[0159] Unless otherwise specified, the polypeptides provided by the present invention and the raw materials and reagents used in their applications can be purchased from the market.

[0160] The present invention will be further described below in conjunction with the embodiments:

[0161] Preparation Example: Synthesis of BX-34 Series Compounds

[0162] 1. Synthesis of BX-34

[0163] 0.50 g (0.25 mmol) of Fmoc-Trp(Boc)-Wang Resin was placed in a reactor and swelled for 10 min with 10 mL of DCM. The mixture was filtered and washed twice with DMF. 25% 4-methylpiperidine / DMF (volume ratio) was added and reacted for 30 min to remove the Fmoc group. The resin was filtered and washed four times with DMF and twice with DCM. Ninhydrin detection indicated a blue resin solution. Fmoc-Ile-OH (1 mmol, 4 eq.) and HBTU (1 mmol, 4 eq.) were weighed and dissolved in DMF. DIEA (2 mmol, 8 eq.) was added and mixed thoroughly. The resin was added to the resin and reacted with magnetic stirring at room temperature (25°C ± 5°C) for 1 h. The mixture was filtered and washed three times with DMF, MeOH, and DCM. Ninhydrin detection indicated that the resin was colorless and transparent, and the solution was light yellow, indicating complete reaction. 25% 4-methylpiperidine / DMF (volume ratio) was added to remove the Fmoc group. The coupling cycle was repeated sequentially to complete the coupling of Ile, Asp(OtBu), Lys(Dde), His(Trt), Ala, Phe, Tyr(tBu), Val, Ala, Glu(OtBu), Glu(OtBu), Asp(OtBu), and OtBu-Fum. Subsequently, 2% hydrazine hydrate / DMF was added to the peptide resin for 10 min three times to remove the Dde from the Lys side chain. AEEA, AEEA, Glu-α-OtBu, and OtBu-ODDA were then condensed in sequence. The peptide resin was drained and 15 mL of pre-chilled lysis buffer (TFA:TIS:H2O = 95:2.5:2.5) was added. The reaction was stirred at room temperature (25°C ± 5°C) for 3 h. The reaction solution was then withdrawn and the resin was washed twice with a small amount of TFA and withdrawn. The reaction mixture was combined and about 200 mL of pre-cooled methyl tert-butyl ether was added to precipitate a white precipitate. The mixture was centrifuged (5000 rpm × 5 min), the supernatant was discarded, and the precipitate was added with methyl tert-butyl ether again, vortexed, centrifuged, and the supernatant was discarded. The precipitate was placed in a vacuum desiccator and dried for 12 hours to obtain a crude product. The crude peptide was dissolved in about 1% ammonia water, filtered, and the filtrate was purified by preparative liquid phase. Preparative column: C18-10-100, 30 × 250 mm. Flow rate: 25 mL / min. Phase A: 0.1% TFA / water, Phase B: 0.1% TFA / 90% acetonitrile / water. Gradient elution was performed to obtain the pure target compound. The target compound fractions with a purity > 95% were combined, and the acetonitrile was removed by decompression at about 40°C using a water pump and a rotary evaporator. The resulting concentrate was frozen into a solid with liquid nitrogen and then freeze-dried in a freeze dryer for 48 hours to obtain the target compound. The molecular weight was correct ([M+3] 3+ =850.8).

[0164] 2. Synthesis of BX-5

[0165] The target compound was obtained by referring to the synthesis method of BX-34. The molecular weight was correct ([M+1] + =1837.3).

[0166] 3. Synthesis of F-BX-5

[0167] The target compound was obtained by referring to the synthesis method of BX-34. The molecular weight was correct ([M+2] 2+ =918).

[0168] 4. Synthesis of BX-7

[0169] The target compound was obtained by referring to the synthesis method of BX-34. The molecular weight was correct ([M+1] + =1807.4).

[0170] 5. Synthesis of BX-8

[0171] The target compound was obtained by referring to the synthesis method of BX-34. The molecular weight was correct ([M+1] + =1792.1).

[0172] 6. Synthesis of BX-43

[0173] The target compound was obtained by referring to the synthesis method of BX-34. The molecular weight was correct ([M+1] + =1851.4).

[0174] 7. Synthesis of BX-45

[0175] The target compound was obtained by referring to the synthesis method of BX-34. The molecular weight was correct ([M+1] + =1893.2).

[0176] 8. Synthesis of BX-51

[0177] The target compound was obtained by referring to the synthesis method of BX-34. The molecular weight was correct ([M+2] 2+ =1268.5).

[0178] 9. Synthesis of BX-52

[0179] The target compound was obtained by referring to the synthesis method of BX-34. The molecular weight was correct ([M+2] 2+ =946.9).

[0180] 10. Synthesis of BX-53

[0181] The target compound was obtained by referring to the synthesis method of BX-34. The molecular weight was correct ([M+1] + =1881.5).

[0182] 11. Synthesis of BX-54

[0183] The target compound was obtained by referring to the synthesis method of BX-34. The molecular weight was correct ([M+2] 2+ =1306.2).

[0184] 12. Synthesis of BX-58

[0185] The target compound was obtained by referring to the synthesis method of BX-34. The molecular weight was correct ([M+1] + =1907.3).

[0186] 13. Synthesis of BX-59

[0187] The target compound was obtained by referring to the synthesis method of BX-34. The molecular weight was correct ([M+2] 2+ =939.9).

[0188] 14. Synthesis of BX-60

[0189] The target compound was obtained by referring to the synthesis method of BX-34. The molecular weight was correct ([M+2] 2+ =1305.6).

[0190] 15. Synthesis of BX-61

[0191] The target compound was obtained by referring to the synthesis method of BX-34. The molecular weight was correct ([M+2] 2+ =1270.4).

[0192] 16. Synthesis of BX-66

[0193] The target compound was obtained by referring to the synthesis method of BX-34. The molecular weight was correct ([M+2] 2+ =1297.2).

[0194] 17. Synthesis of BX-67

[0195] The target compound was obtained by referring to the synthesis method of BX-34. The molecular weight was correct ([M+2] 2+ =1283.3).

[0196] 18. Synthesis of BX-68

[0197] The target compound was obtained by referring to the synthesis method of BX-34. The molecular weight was correct ([M+2] 2+ =1297.6).

[0198] 19. Synthesis of BX-69

[0199] The target compound was obtained by referring to the synthesis method of BX-34. The molecular weight was correct ([M+3] 3+ =841.1).

[0200] 20. Synthesis of BX-70

[0201] The target compound was obtained by referring to the synthesis method of BX-34. The molecular weight was correct ([M+3] 3+ =831.7).

[0202] 21. Synthesis of BX-71

[0203] The target compound was obtained by referring to the synthesis method of BX-34. The molecular weight was correct ([M+3] 3+ =825.8).

[0204] 22. Synthesis of BX-72

[0205] The target compound was obtained by referring to the synthesis method of BX-34. The molecular weight was correct ([M+3] 3+ =1653.4).

[0206] 23. Synthesis of BX-73

[0207] The target compound was obtained by referring to the synthesis method of BX-34. The molecular weight was correct ([M+2] 2+ =1066).

[0208] 24. Synthesis of BX-74

[0209] The target compound was obtained by referring to the synthesis method of BX-34. The molecular weight was correct ([M+3] 3+ =851.6).

[0210] 25. Synthesis of BX-75

[0211] The target compound was obtained by referring to the synthesis method of BX-34. The molecular weight was correct ([M+3] 3+ =855.7).

[0212] 26. Synthesis of BX-78

[0213] The target compound was obtained by referring to the synthesis method of BX-34. The molecular weight was correct ([M+3] 3+ =847.1).

[0214] 27. Synthesis of BX-86

[0215] The target compound was obtained by referring to the synthesis method of BX-34. The molecular weight was correct ([M+3] 3+ =850.8).

[0216] 28. Synthesis of BX-101

[0217] The target compound was obtained by referring to the synthesis method of BX-34. The molecular weight was correct ([M+3] 3+ =846.3).

[0218] 29. Synthesis of BX-105

[0219] The target compound was obtained by referring to the synthesis method of BX-34. The molecular weight was correct ([M+1] + =1848.2).

[0220] 30. Synthesis of BX-106

[0221] The target compound was obtained by referring to the synthesis method of BX-34. The molecular weight was correct ([M+1] + =1837.3).

[0222] 31. Synthesis of BX-107

[0223] The target compound was obtained by referring to the synthesis method of BX-34. The molecular weight was correct ([M+3] 3+ =860.7).

[0224] 32. Synthesis of BX-108

[0225] The target compound was obtained by referring to the synthesis method of BX-34. The molecular weight was correct ([M+3] 3+ =856.7).

[0226] 33. Synthesis of BX-109

[0227] The target compound was obtained by referring to the synthesis method of BX-34. The molecular weight was correct ([M+3] 3+ =856.3).

[0228] 34. Synthesis of BX-110

[0229] The target compound was obtained by referring to the synthesis method of BX-34. The molecular weight was correct ([M+3] 3+ =850.7).

[0230] 35. Synthesis of BX-111

[0231] The target compound was obtained by referring to the synthesis method of BX-34. The molecular weight was correct ([M+1] + =1845.5).

[0232] 36. Synthesis of BX-112

[0233] The target compound was obtained by referring to the synthesis method of BX-34. The molecular weight was correct ([M+1] + =1845.3).

[0234] 37. Synthesis of BX-229

[0235] 0.50 g (0.25 mmol) of Fmoc-Trp(Boc)-Wang Resin was placed in a reactor and swelled for 10 min with 10 mL of DCM. The mixture was filtered and washed twice with DMF. 25% 4-methylpiperidine / DMF (volume ratio) was added and reacted for 30 min to remove the Fmoc group. The resin was filtered and washed four times with DMF and twice with DCM. Ninhydrin detection indicated a blue resin solution. Fmoc-Ile-OH (1 mmol, 4 eq.) and HBTU (1 mmol, 4 eq.) were weighed and dissolved in DMF. DIEA (2 mmol, 8 eq.) was added and mixed thoroughly. The resin was added to the resin and reacted with magnetic stirring at room temperature (25°C ± 5°C) for 1 h. The mixture was filtered and washed three times with DMF, MeOH, and DCM. Ninhydrin detection indicated that the resin was colorless and transparent, and the solution was light yellow, indicating complete reaction. 25% 4-methylpiperidine / DMF (volume ratio) was added to remove the Fmoc group. The coupling of Val, Asp(OtBu), Lys(Dde), His(Trt), Ala, Phe, Tyr(tBu), Val, Ala, Glu(OtBu), Glu(OtBu), Asp(OtBu), and OtBu-Fum was completed in a sequential cycle. Subsequently, 2% hydrazine hydrate / DMF was added to the peptide resin for 10 min three times to remove the Dde from the Lys side chain. AEEA, Glu-α-OtBu, and C12-OH were then condensed in sequence. The peptide resin was drained and 15 mL of pre-chilled lysis buffer (TFA:TIS:H2O = 95:2.5:2.5) was added. The reaction was stirred at room temperature (25°C ± 5°C) for 3 h. The reaction solution was then withdrawn and the resin washed twice with a small amount of TFA and withdrawn. The reaction mixture was combined and about 200 mL of pre-cooled methyl tert-butyl ether was added to precipitate a white precipitate. The mixture was centrifuged (5000 rpm × 5 min), the supernatant was discarded, and the precipitate was added with methyl tert-butyl ether again, vortexed, centrifuged, and the supernatant was discarded. The precipitate was placed in a vacuum desiccator and dried for 12 hours to obtain a crude product. The crude peptide was dissolved in about 1% ammonia water, filtered, and the filtrate was purified by preparative liquid phase. Preparative column: C18-10-100, 30 × 250 mm. Flow rate: 25 mL / min. Phase A: 0.1% TFA / water, Phase B: 0.1% TFA / 90% acetonitrile / water. Gradient elution was performed to obtain the pure target compound. The target compound fractions with a purity of >95% were combined, and the acetonitrile was removed by decompression at about 40°C using a water pump and a rotary evaporator. The resulting concentrate was frozen into a solid with liquid nitrogen, and then freeze-dried in a freeze dryer for 48 hours to obtain the target compound. The molecular weight was correct ([M-2] 2- =1137.3).

[0236] 38. Preparation of BX-229 sodium salt

[0237] Dissolve the BX-229 solid obtained in the previous step in a 0.1N sodium hydroxide aqueous solution (containing 10 equivalents of sodium hydroxide) and use a preparative liquid phase to transfer the salt. Preparative column: C18-10-100, 30×250mm. Flow rate: 25mL / min. Phase A: water, phase B: acetonitrile. After loading, rinse with water for 15 minutes, and then elute the product from the preparative column by gradient elution. Combine the target compound components with a purity of >95%, use a water pump and a rotary evaporator to reduce the pressure at about 40°C to rotate out the acetonitrile, and the resulting concentrated solution is frozen into a solid with liquid nitrogen, and then freeze-dried in a freeze dryer for 48 hours to obtain the target compound.

[0238] 39. Synthesis of BX-238

[0239] 0.50 g (0.25 mmol) of Fmoc-Trp(Boc)-Wang Resin was placed in a reactor and swelled with 10 mL of DCM for 10 minutes. The mixture was filtered and washed twice with DMF. 25% 4-methylpiperidine / DMF (volume ratio) was added and reacted for 30 minutes to remove the Fmoc group. The mixture was filtered and washed four times with DMF and twice with DCM. The resin solution was blue when tested with ninhydrin. Fmoc-Ile-OH (1 mmol, 4 eq.) and HBTU (1 mmol, 4 eq.) were weighed and dissolved in DMF. DIEA (2 mmol, 8 eq.) was added and mixed thoroughly. The resin was added to the resin and reacted with magnetic stirring at room temperature (25°C ± 5°C) for 1 hour. The mixture was filtered and washed three times with DMF, MeOH, and DCM. The reaction was complete when tested with ninhydrin. The resin was colorless and transparent, and the solution was light yellow. The Fmoc group was removed by adding 25% 4-methylpiperidine / DMF (volume ratio). The coupling of Val, Asp(OtBu), Lys(Dde), His(Trt), Ala, Phe, Tyr(tBu), Val, Ala, Glu(OtBu), Glu(OtBu), Asp(OtBu), and OtBu-Fum was completed in a sequential cycle. Subsequently, 2% hydrazine hydrate / DMF was added to the peptide resin for 10 min three times to remove the Dde from the Lys side chain. AEEA, Glu(OtBu), and C12-OH were then condensed in sequence. The peptide resin was drained and 15 mL of pre-chilled lysis buffer (TFA:TIS:H2O = 95:2.5:2.5) was added. The reaction was stirred at room temperature (25°C ± 5°C) for 3 h. The reaction solution was then withdrawn and the resin washed twice with a small amount of TFA and withdrawn. The reaction mixture was combined and about 200 mL of pre-cooled methyl tert-butyl ether was added to precipitate a white precipitate. The mixture was centrifuged (5000 rpm × 5 min), the supernatant was discarded, and the precipitate was added with methyl tert-butyl ether again, vortexed, centrifuged, and the supernatant discarded. The precipitate was placed in a vacuum desiccator and dried for 12 hours to obtain a crude product. The crude peptide was dissolved in about 1% ammonia water, filtered, and the filtrate was purified by preparative liquid phase. Preparative column: C18-10-100, 30 × 250 mm. Flow rate: 25 mL / min. Phase A: 0.1% TFA / water, Phase B: 0.1% TFA / 90% acetonitrile / water. Gradient elution was performed to obtain the pure target compound. The target compound fractions with a purity > 95% were combined, and the acetonitrile was removed by vacuum pump and rotary evaporator at about 40°C. The resulting concentrate was frozen into a solid with liquid nitrogen and then freeze-dried in a freeze dryer for 48 hours to obtain the target compound. The molecular weight was correct ([M-2] 2- =1136.8).

[0240] 40. Synthesis of BX-76

[0241] The synthesis of the main chain was completed by referring to the synthesis method of BX-34. Under nitrogen protection, 0.3 equivalents of tetrakistriphenylphosphine palladium and 12 equivalents of phenylsilane in DCM were added to the peptide resin and reacted at room temperature (25°C ± 5°C) for 2 hours to remove the allyl ester of the Glu side chain and the allyloxycarbonyl of the Lys side chain. Filter, wash with DCM solvent 5 times, wash with DMF 3 times, add 2 equivalents of PYBOP, 2 equivalents of HOBt and 4 equivalents of DIEA, dissolve in DMF, and react at room temperature (25°C ± 5°C) for 5 hours. Filter, wash with DMF 3 times, wash with DCM 3 times, and detect with ninhydrin. If the reaction is incomplete, repeat the condensation once. Then, refer to the method of BX-34 for cleavage and purification to obtain the pure target compound. The molecular weight is correct ([M+2] 2+ =910).

[0242] 41. Synthesis of BX-48

[0243] The target compound was obtained by referring to the synthesis method of BX-76. The molecular weight was correct ([M+2] 2+ =917.5).

[0244] 42. Synthesis of BX-49

[0245] The target compound was obtained by referring to the synthesis method of BX-76. The molecular weight was correct ([M+2] 2+ =903.2).

[0246] 43. Synthesis of BX-50

[0247] The target compound was obtained by referring to the synthesis method of BX-76. The molecular weight was correct ([M+2] 2+ =939).

[0248] 44. Synthesis of BX-62

[0249] The target compound was obtained by referring to the synthesis method of BX-76. The molecular weight was correct ([M+2] 2+ =952.9).

[0250] 45. Synthesis of BX-63

[0251] The target compound was obtained by referring to the synthesis method of BX-76. The molecular weight was correct ([M+2] 2+ =902.9).

[0252] 46. ​​Synthesis of BX-64

[0253] The target compound was obtained by referring to the synthesis method of BX-76. The molecular weight was correct ([M+2]2+ =901.9).

[0254] 47. Synthesis of BX-65

[0255] The target compound was obtained by referring to the synthesis method of BX-76. The molecular weight was correct ([M+2] 2+ =908.2).

[0256] 48. Synthesis of BX-77

[0257] The target compound was obtained by referring to the synthesis method of BX-76. The molecular weight was correct ([M+2] 2+ =1282.8).

[0258] 49. Synthesis of BX-79

[0259] The target compound was obtained by referring to the synthesis method of BX-76. The molecular weight was correct ([M+2] 2+ =1276.4).

[0260] 50. Synthesis of BX-100

[0261] The target compound was obtained by referring to the synthesis method of BX-76. The molecular weight was correct ([M+2] 2+ =1274.7).

[0262] 51. Synthesis of BX-103

[0263] The target compound was obtained by referring to the synthesis method of BX-76. The molecular weight was correct ([M+2] 2+ =1281.6).

[0264] 52. Synthesis of BX-104

[0265] The target compound was obtained by referring to the synthesis method of BX-76. The molecular weight was correct ([M+2] 2+ =1268.4).

[0266] 53. Synthesis of BX-99

[0267] The target compound was obtained by referring to the synthesis method of BX-76. The molecular weight was correct ([M+2] 2+ =959.3).

[0268] 54. Synthesis of BX-102

[0269] The target compound was obtained by referring to the synthesis method of BX-76. The molecular weight was correct ([M+2]2+ =915.5).

[0270] 55. Synthesis of BX-56

[0271] The main chain was synthesized by referring to the synthesis method of BX-34. 5 equivalents of iodine in DMF solution were added to the peptide resin and reacted at room temperature (25°C ± 5°C) for 6 hours to remove the protecting group Trt of the Cys side chain and simultaneously oxidize it. Filter, wash with DMF 6 times, wash with DCM 3 times, and dry. Referring to the synthesis method of BX-34, cleavage and purification were performed to obtain the pure target compound. The molecular weight was correct ([M+2] 2+ =993.2).

[0272] 56. Synthesis of BX-57

[0273] The target compound was obtained by referring to the synthesis method of BX-56. The molecular weight was correct ([M+2] 2+ =1000.4).

[0274] 57. Synthesis of BX-95

[0275] The main chain pure peptide was obtained by referring to the synthesis method of BX-34. Similarly, the CPAK pure peptide was obtained by referring to the synthesis method of BX-34. Equivalent amounts of the two were dissolved in 50% acetonitrile aqueous solution, the pH value was adjusted to 8-9 with triethylamine, and the reaction was completed at room temperature (25℃±5℃). Purification was performed according to the method of BX-34 to obtain the pure target compound, which was identified by mass spectrometry and had the correct molecular weight ([M+3] 3+ =850.5).

[0276] Example 1: Screening of polypeptide activity against human ETB receptor

[0277] 293T-17 cells were plated at 650,000 per well in a 6-well plate and cultured overnight at 37°C in a 5% CO2 incubator. The cells were then transfected with the expression vector pcDNA3.1-hETB, containing the human ETB receptor gene, at a rate of 2.5 μg per well. The cells were observed for green fluorescence under a fluorescence microscope, then trypsinized and counted. The activity of the compounds against the human ETB receptor was assayed using the Cisbio Bioassays IP-One Gq kit. Cells were suspended in the Stim Buffer provided in the kit and added to a 96-well plate. 7 μL of the cell suspension (containing 30,000 cells) was added to each well. The peptide to be screened (prepared in the Preparation Example of the present invention) and the positive control peptide IRL1620 were dissolved in Stim B to a 2x concentration and added to the cells. In the negative control well, 7 μL of PBS was added to the cells. IRL1620 served as the positive control peptide. The plates were sealed and incubated in a 37°C cell culture incubator for 1 hour. Add 3 μL of IP1d2 working solution and 3 μL of IP1Tb cryptotate antibody working solution to each test well, positive control well, and PBS control well. Seal the plate and incubate in the dark at room temperature (25°C ± 5°C) for 1 hour. Remove the plate seal and measure the absorbance at 665 nm and 620 nm on an ID5 microplate reader. Calculate the ratio of the acceptor and donor emission signals for each well as (665 nm signal / 620 nm signal) × 10 4 Numerical values. The average of the 665nm signal / 620nm signal values ​​for the test sample at a specific concentration is F1, the average of the 665nm signal / 620nm signal values ​​for the positive control IRL1620 at the same concentration is F2, and the average of the 665nm signal / 620nm signal values ​​for the PBS negative control is T1. The percentage activity of the test peptide sample relative to the positive control IRL1620 is (F1 - T1) × 100 / (F2 - T1). hETB sequence (NCBI accession number: NM_000115.5).

[0278] Example 2: Selectivity of polypeptides for human ETA receptor

[0279] 293T-17 cells were plated at 650,000 per well in a 6-well plate and cultured overnight in a 37°C, 5% CO2 incubator. The cells were then transfected with the expression vector PCDNA3.1-hETA, containing the human ETA receptor gene, at a rate of 2.5 μg per well. The cells were cultured for an additional 48 hours. After observing the cells for green fluorescence under a fluorescence microscope, the cells were trypsinized and counted. The activity of the compounds against the human ETA receptor was assayed using the Cisbio Bioassays IP-One Gq kit. Cells were suspended in the Stim Buffer provided in the kit and added to a 96-well assay plate. 7 μL of the cell suspension (containing 30,000 cells) was added to each well. The peptide to be screened (prepared in the Preparation Example of the present invention) and the positive control peptide, human endothelin ET1, were dissolved in Stim B to a 2x concentration and added to the test cells (7 μL). In the negative control well, 7 μL of PBS was added to the cells. Human endothelin ET1 (ET1: CSCSSLMDKECVYFCHLDIIW (C1-C15, C3-C11)) SEQ ID No. 85 was used as a positive control peptide. The plates were sealed and incubated in a cell culture incubator at 37°C for 1 hour. 3 μL of IP1 d2 working solution and 3 μL of IP1 Tb cryptotate Antibody working solution were added to each of the test wells, positive control wells, and PBS control wells. The plates were sealed and incubated in the dark at room temperature (25°C ± 5°C) for 1 hour. The plate seal was removed and the absorbance at 665 nm and 620 nm was measured on an ID5 microplate reader. The ratio of the acceptor and donor emission signals in each well was calculated as (665 nm signal / 620 nm signal) × 10 4 The average value of the 665nm signal / 620nm signal value measured for the test sample at a specific concentration is F3, the average value of the 665nm signal / 620nm signal value measured for the positive control human endothelin ET1 at the same concentration is F5, and the average value of the 665nm signal / 620nm signal value measured for the PBS negative control is T3. The percentage activity of the test peptide sample relative to the positive control sample ET1 = (F3-T3) × 100 / (F5-T3) (see Table 6). hETA sequence (NCBI accession number: L06622.1)

[0280] Example 3: EC50 determination of peptide activity on human ETB receptor (see Table 1, Figures 1 to 7)

[0281] 293T-17 cells were plated at 650,000 per well in a 6-well plate and cultured overnight at 37°C in a 5% CO2 incubator. The cells were then transfected with the expression vector PCDNA3.1-hETB, containing the human ETB receptor gene, at a rate of 2.5 μg per well. The cells were observed for green fluorescence under a fluorescence microscope, then trypsinized and counted. The activity of the compounds against the human ETB receptor was assayed using the Cisbio Bioassays IP-One Gq kit. Cells were suspended in the Stim Buffer provided in the kit and added to a 96-well plate. 7 μL of the cell suspension (containing 30,000 cells) was added to each well. The peptide to be assayed (prepared in the preparation example of the present invention) was diluted with Stim B to 11 concentration gradients. Each concentration gradient of the peptide to be assayed was dissolved in Stim B to a 2x concentration and 7 μL was added to the cells. In the negative control well, 7 μL of PBS was added to the cells. The plates were sealed and incubated in a 37°C cell culture incubator for 1 hour. Add 3 μL of IP1 d2 working solution and 3 μL of IP1 Tb cryptotate antibody working solution to each test well, positive control well, and PBS control well. Seal the plate and incubate in the dark at room temperature (25°C ± 5°C) for 1 hour. Remove the plate seal and measure absorbance at 665 nm and 620 nm on an ID5 microplate reader. Calculate the ratio of the acceptor and donor emission signals for each well as (665 nm signal / 620 nm signal) × 10. 4 The average value of the 665nm signal / 620nm signal values ​​measured for the test sample at different concentrations is F. A dot plot is plotted with the F value as the ordinate and the logarithmic value of the polypeptide concentration as the abscissa. A curve is generated using a suitable fitting method to calculate the EC50 value of the polypeptide for the human ETB receptor. Another curve fitting method is to first calculate the activation rate of each concentration of the test polypeptide relative to the positive control sample, human endothelin ET1, using the method in Example 1. A dot plot is plotted with the activation rate as the ordinate and the logarithmic value of the polypeptide concentration as the abscissa. A curve is generated using a suitable fitting method to calculate the EC50 value of the polypeptide for the human ETB receptor.

[0282] Example 4: EC50 determination of peptide activity on rat ETB receptor (see Table 8)

[0283] The method was the same as in Example 3, except that the transfection plasmid was the expression vector PCDNA3.1-RETB containing the rat ETB receptor gene. The EC50 values ​​of the peptides for the rat ETB receptor were calculated (Figures 11-13). Rat ETB receptor sequence (NCBI accession number: X57764.1)

[0284] Example 5: EC50 determination of peptide activity on mouse ETB receptor (see Table 9)

[0285] The method was the same as in Example 3, except that the transfection plasmid was the expression vector PCDNA3.1-mETB containing the mouse ETB receptor gene. The EC50 values ​​of the peptides for the mouse ETB receptor were calculated (Figures 14 and 15). Mouse ETB sequence (NCBI accession number: NM_001276296)

[0286] Example 6: EC50 determination of polypeptide activity on canine ETB receptor (see Table 7)

[0287] The method was the same as in Example 3, except that the transfection plasmid was the expression vector PCDNA3.1-DETB containing the canine ETB receptor gene. The EC50 values ​​of the peptides for the canine ETB receptor were calculated (Figures 8-10). Canine endothelin receptor B sequence: (NCBI accession number: NM_001010943.2)

[0288] Example 7: Effect of polypeptide BX-34 on cerebral blood perfusion in dMACO model mice

[0289] Test drug: BX-34 prepared in the preparation example of the present invention.

[0290] Eight-week-old male C57BL / 6 mice were used and acclimated for at least 3 days. On the day of the experiment, a 2-cm midline incision was made in the cerebral cortex under isoflurane anesthesia. The mice were placed under laser speckle flowmetry and video recordings of cerebral blood flow were taken from both sides of the brain to serve as pre-ischemic perfusion images. Subsequently, a skull drill (3 mm × 1.5 mm) was used to create a 3 mm × 1.5 mm incision just posterior to the intersection of the sagittal and coronal sutures. The distal end of the right middle cerebral artery was located under a microscope and ligated with a No. 11 suture. Simultaneously, the right common carotid artery was isolated and ligated. Video recordings of cerebral blood flow were again taken from both sides of the brain to serve as post-ischemic perfusion images. The scalp was sutured, and the animals were rewarmed in a rewarming incubator. Mice with successfully established dMACO were randomly assigned to receive single doses of 7, 14, or 42 μg / kg of BX-34, single doses of 5 or 10 μg / kg of IRL1620, and three doses of 10 μg / kg of IRL1620, as well as a saline control group. The single-dose group received the drug via the tail vein 1 hour after modeling, while the triple-dose group received the drug 1, 3, and 5 hours after modeling. Blood flow videos of the right and left cerebral regions of each animal were collected again 24 hours, 48 ​​hours, and 7 days after drug administration. For data analysis, import the ROI or select the region for blood flow calculation; double-click the blood flow image to calculate the mean blood flow value for the selected region. Statistical analysis for each group was performed by comparing the mean right cerebral blood flow perfusion values ​​after modeling, 24 hours, 48 ​​hours, and 7 days after drug administration to the mean right cerebral blood flow value before modeling. The results are shown in Figure 16.

[0291] The results showed that after a single dose of 42μg / kg of BX-34, cerebral blood perfusion increased significantly compared to the saline group at 24h, 48h, and 7d after administration of a single dose of 42μg / kg of BX-34 to dMCAO model mice. However, no significant difference was observed between IRL1620 and the saline group, whether administered as a single dose of 5μg / kg, a single dose of 10μg / kg, or three doses of 10μg / kg. A single dose of 42μg / kg of BX-34 (the same molar dose as the three doses of 10μg / kg of IRL1620) significantly increased cerebral blood perfusion in dMCAO mice, and this effect was dose-dependent.

[0292] Example 8: Determination of polypeptide half-life

[0293] The polypeptides in this example (F-BX-5, BX-34, BX-48, BX-76, BX-86, BX-69, BX-70, BX-71, BX-56, BX-225, BX-238, BX-229, BX-253) were prepared according to the preparation examples of the present invention.

[0294] Plasma Sample Dosing and Processing: Three C57BL / 6 mice were injected with the compounds listed in Table 12 (using BX-34 as an example) at 60 μg / mL, with 200 μL injected into each mouse. Blood was collected 1, 5, 15, and 30 minutes after drug injection. Protease inhibitors were immediately added and the blood was centrifuged at 3200 rpm for 10 minutes at 4°C. Plasma was collected and protein was precipitated with 2 volumes of acetonitrile. The plasma was then centrifuged at 10,000 rpm for 5 minutes, filtered through a 0.22 μm filter, and analyzed by liquid chromatography-mass spectrometry. Plasma concentrations were calculated based on the integrated area of ​​the control and test drugs. The three values ​​measured at each time point for each drug were averaged. A blank mouse plasma sample was also prepared after protein precipitation as a control.

[0295] Table 12 Half-life data of polypeptides

[0296] LC-MS / MS detection conditions:

[0297] Instrument model: Agilent InfinityLab LC / MSD 1260-G6125C

[0298] Chromatographic column: Poroshell 120 SB-C18 4.6*100mm, 2.7μm

[0299] Mobile phase A: 0.1% formic acid-ultrapure water Mobile phase B: 0.1% formic acid-acetonitrile

[0300] Gradient settings:

[0301] Column temperature: 45°C; injection volume: 20 μl;

[0302] Ion source: ESI; dryer temperature: 350°C; dryer flow rate: 12 L / min; nebulizer pressure: 45 psi; capillary voltage: 4000 V.

[0303] Scan type: SIM; fragmentor voltage: 135 V; scan / dwell time: 200 ms.

[0304] Example 9: In vivo efficacy of peptides BX34 and BX48 in pMACO model rats

[0305] Test drugs: BX-34 and BX-48 prepared in the preparation examples of the present invention (due to different molecular weights, the same molar dose was used for administration).

[0306] Male Sprague-Dawley rats, weighing 280-300 g, were used and acclimated for at least 3 days. Focal cerebral ischemia was induced by middle cerebral artery occlusion using a suture method under isoflurane anesthesia. The animals were placed in a secure supine position, and a midline incision was made to expose the right common carotid artery, external carotid artery, and internal carotid artery. A 4.0 monofilament nylon suture was advanced from the external carotid artery into the internal carotid artery lumen until resistance was felt. Indicates occlusion of the middle cerebral artery. The nylon filament was allowed to remain in place to create a permanent model of focal cerebral ischemia. The rats with successful modeling were randomly divided into 4 groups: 10 rats in the solvent (normal saline) group, 10 rats in the 7μg / Kg BX-3410 group, 10 rats in the 5μg / Kg BX-48 group, and 10 rats in the 5μg / Kg IRL1620 group. The rats were given once 2h, 4h, and 6h after modeling, for a total of three doses. The grip strength of each group was measured before administration and 24h, 3D, and 7D after administration. Rotarod test and foot error test were performed before administration and 24h and 7D after administration. The rotarod test recorded time, distance, and the comparison of the measured values ​​at 24h and 7D with the pre-measured values ​​was used as the analysis index; the specific method of foot error measurement was as follows: the rat was placed on an elevated grid floor (29×29cm, grid opening 3cm 2 ) for 2 minutes. The number of foot errors resulting from each forelimb falling off the grid and the total number of steps were recorded. % Foot Error = (Number of Forelimb Error Steps / Total Forelimb Steps) × 100. Behavioral test results are shown in Tables 13, 14, and 15.

[0307] The results showed that all treatment groups performed better than the saline group in the 3D and 7D rotarod tests and grip strength tests, showing significant improvement compared to the saline group. Seven days after treatment, the percentage of foot errors in the treatment groups decreased compared to the saline group. The percentage of foot errors in the BX-34 and BX-48 groups was lower, lower than that in the saline and IRL1620 groups.

[0308] Table 13 Grip strength measurement results

[0309] Table 14 Rotarod assay results

[0310] Table 15 Foot error measurement results

[0311] Example 10: Preliminary study on acute toxicity of IRL1620, BX225, BX229, BX238, BX253, BX254, BX255, and BX256 peptides after intravenous administration

[0312] In the intravenous acute toxicity study, male SD rats weighing 200-230g were administered doses ranging from 480μg / kg to 30μg / kg, from high to low doses, via tail vein injection. The rats were observed for 24 hours after administration. If a rat died at a certain dose, the dose was reduced and the rats were observed again. The toxic reactions and mortality of the rats were initially observed, and the time of death and the dose at which no death occurred were recorded. The dose at which no death occurred was used to preliminarily determine the acute toxicity of the peptide. Acute toxicity studies showed that the maximum tolerated doses of IRL1620, BX225, BX229, BX238, BX253, BX254, and BX255 that rats could tolerate were 30 μg / Kg, 30 μg / Kg, 60 μg / Kg, 30 μg / Kg, 480 μg / Kg, 120 μg / Kg, and 120 μg / kg, respectively. The maximum tolerated dose of BX225, BX229, BX238, BX253, BX254, and BX255 polypeptides injected into SD rats was better than that of IRL1620, and the acute toxicity of intravenous injection of BX253 was the lowest (Table 16).

[0313] Table 16: Acute toxicity of IRL1620, BX225, BX229, BX238, BX253, BX254, BX255, and BX256 administered intravenously

[0314] Example 11: Preliminary study on acute toxicity of IRL1620, BX229, and BX238 polypeptides after subcutaneous administration

[0315] In the subcutaneous acute toxicity study, male SD rats weighing 200-230g were administered doses ranging from 60 μg / kg to 960 μg / kg and observed for 48 hours. Preliminary observations were made regarding toxic reactions and mortality in the rats, with the time of death and the dose at which no mortality occurred being recorded. The dose at which no mortality occurred was used to preliminarily determine the acute toxicity of the peptide. The experimental results are shown in Table 17 below. As can be seen, the acute toxicity of the BX229 peptide following subcutaneous administration was significantly less than that of IRL1620.

[0316] Table 17: Preliminary study on acute toxicity of IRL1620, BX229, and BX238 peptides administered subcutaneously

[0317] Example 12: Effects of intravenous administration of IRL1620, BX225, and BX229 peptides on blood pressure in SD rats

[0318] Test drugs: IRL1620, BX225, and BX229 prepared in the preparation examples of the present invention

[0319] After SD male rats were anesthetized with isoflurane, the neck hair was prepared, and an opening was made along the middle of the neck to expose the trachea; the small animal ventilator and anesthesia machine were turned on, and the anesthesia concentration of the anesthesia machine was set to 1.2; the outlet channel was connected to the ventilator inlet; the ventilator tidal volume was set to 2 mL, the respiratory rate was 90 times / min, and the respiratory ratio was 1:2; the ventilator outlet was connected to a plastic hose, and the plastic hose was inserted into the rat's airway.

[0320] Turn on the biosignal data acquisition and analysis system machine and software, connect one end of the data acquisition board to the plastic hose, and the other end to the connector valve connected to a 1 mL syringe, and fill the syringe with heparinized saline; inject heparinized saline into the data acquisition board, and expel the air in the entire space until the heparinized saline flows out from the other end of the plastic hose; free the right common carotid artery of the rat, tie a knot at the distal end of the common carotid artery, tie a loose knot at the proximal end, use microscissors to cut a 1 / 2-sized hole in the artery in the middle of the knot, insert the plastic hose with heparinized saline into the cut hole and fix it so that the hose cannot move back and forth at will; use the zeroing valve of the biosignal data acquisition and analysis system to reset the initial blood pressure to zero; open the loose knot at the proximal end of the common carotid artery, and the real-time blood pressure data of the animal will appear on the software. After the animal's blood pressure stabilizes, administer the drug through the tail vein and observe and record the changes in the animal's blood pressure in real time. The dosage of IRL1620 was 5 μg / Kg, the dosage of BX34 was 42 μg / kg, and the dosage of BX229 was 6.6 μg / Kg.

[0321] The results showed that 5μg / Kg IRL620, 42μg / Kg BX34, and 6.6μg / Kg BX229 peptides had a certain effect on rat blood pressure and could cause a decrease in blood pressure. After administration of 5μg / Kg IRL620, the rat blood pressure changed rapidly instantaneously, reaching the lowest blood pressure within 2 minutes, with the maximum blood pressure reduction being 16.1%±2.4% of the baseline blood pressure. The effects of 42μg / Kg BX34 and 6.6μg / Kg BX229 on rat blood pressure were relatively gentle, with blood pressure decreasing slowly, reaching the lowest blood pressure within 20-35 minutes, with the maximum blood pressure reductions being 9.5%±1.7% and 17.0%±2.0% of the baseline blood pressure, respectively.

[0322] (Table 18)

[0323] Table 18 Effects of intravenous administration of IRL1620, BX34, and BX229 peptides on blood pressure in SD rats

[0324] Example 13: Efficacy of BX229-Na administered three times a week on the mouse tMCAO model

[0325] Test drug: BX-229 prepared in the preparation example of the present invention

[0326] Fifty-one male C57 / 6J mice, 10-12 weeks old, weighing 25-30 g, were housed for at least 2 days to acclimate to the environment (Speiffer) before the experiment. A tMCAO mouse model was established, and mice were anesthetized with isoflurane. Body temperature was monitored and maintained at 37±0.5°C using a thermostatic blanket. A midline incision was made in the skin over the mouse skull, and the skin was retracted laterally. A flexible microneedle tip was fixed to the surface of the left parietal bone of the mouse skull. A midline incision was made in the neck to isolate the right common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA). According to the Longa method (Longa et al., 1989), a silicone rubber-coated suture (Rayward, suture tip diameter 0.22 mm-0.23 mm, MSMC23B120PK50) was introduced through the external carotid artery and slowly advanced through the internal carotid artery to the origin of the middle cerebral artery. To ensure sustained and successful middle cerebral artery occlusion, we monitored regional cerebral blood flow in all stroke animals using laser speckle flowmetry (typically, blood flow can decrease by 40-60% after stabilization). The occlusion was removed 45 minutes after modeling. After suturing the incision, 1 mL of 37°C warm saline was injected subcutaneously, and the mice were placed on a heating pad until they recovered. To alleviate pain, after recovery from anesthesia, the mice were given a subcutaneous injection of analgesia.

[0327] The C57tMCAO mice with successful modeling were randomly divided into three groups: normal saline group, 30μg / Kg BX229-Na administration group, and 100μg / Kg BX229-Na administration group. Each group was administered subcutaneously in the neck 4.5 hours after embolization (D1), and again at the same time point and site on D3 and D6. The grouping and administration of this experiment are shown in Table 19. There were 6 mice in the sham operation group. The drug was administered by subcutaneous injection in the neck at 2mL / kg. Garcia JH scores were completed 7D and 14D after administration. After the scoring was completed on D14, the mice were euthanized, and the brain tissue on the infarct side was obtained to extract total protein. The changes in VEGF-A and BDNF protein markers in each experimental group were detected by ELISA.

[0328] Table 19: Experimental groups and dosing regimens

[0329] The experimental results showed that, based on the 7D Garcia JH-15 score analysis, there was a significant difference in the 7D score between the sham surgery group and the saline control group. The scores of the BX229-30μg / Kg and BX229-100μg / Kg treatment groups were better than those of the saline group, with a certain dose-dependent trend. The total Garcia JH-15 score of the BX229-100μg / Kg treatment group was 1 point higher than that of the saline group. The 14D Garcia JH-15 score analysis showed a significant difference in the 14D score between the sham surgery group and the saline control group. The scores of the BX229-30μg / Kg and BX229-100μg / Kg treatment groups were better than those of the saline group, with a dose-dependent relationship. The Garcia JH-15 score of the BX229-100μg / Kg treatment group was significantly different from that of the saline group (P<0.05). Figures 32 and 33

[0330] The results showed that the expression of VEGF-A and BDNF proteins in the BX229Na-30 μg / Kg three-times-administered group and the BX229Na-100 μg / Kg three-times-administered group increased compared to the saline group. The VEGF-A protein expression in the BX229Na-100 μg / Kg three-times-administered group was higher than that in the sham-operated group, and there was a significant difference compared to the saline group ( Figures 34 , 35 , Tables 20 , and 21 ). The BDNF protein expression in the BX229Na-100 μg / Kg three-times-administered group was higher than that in the sham-operated group.

[0331] Table 20: Changes in VEGF-A protein expression

[0332] Table 21: Changes in BDNF protein expression

[0333] Example 14: In vivo efficacy of peptides BX225 and IRL1620 in pMACO model rats

[0334] Test drugs: BX-225 and IRL1620 prepared in the preparation examples of the present invention

[0335] Male Sprague-Dawley rats, weighing 280-300 g, were used and acclimated for at least 3 days. Focal cerebral ischemia was induced by middle cerebral artery occlusion using a suture method under isoflurane anesthesia. The animals were placed in a secure supine position, and a midline incision was made to expose the right common carotid artery, external carotid artery, and internal carotid artery. A 4.0 monofilament nylon suture was advanced from the external carotid artery into the internal carotid artery lumen until resistance was felt. Indicates occlusion of the middle cerebral artery. The nylon filament is allowed to remain in place to create a permanent model of focal cerebral ischemia. The day of modeling is defined as D1, the next day as D2, and so on. Because the experiment cannot be completed in the same batch and to prevent the occurrence of accidental errors, all operations in this experiment must follow the principle of parallel operation, that is, animals of each group must be included in the experiment every day. The surgical modeling sequence number is obtained by arranging the animals according to their weight before the experiment. After the operation, the pMCAO rats with successful modeling were randomly divided into the model group and different treatment groups. The detailed experimental groupings are as follows: (1) 15 rats in the group of 5μg / Kg IRL1620 administered 9 times over 3 days (2) 15 rats in the group of 6.6μg / Kg BX225 administered 9 times over 3 days (3) 15 rats in the group of 19.8μg / Kg BX225 administered 3 times over 3 days (4) 15 rats in the model (normal saline) group (5) 6 rats in the sham operation group (the sham operation group used the same method without ligating the blood vessels or inserting sutures). Administration was via tail vein (iv) injection (2 mL / kg), with the first dose administered 2 hours after surgery. Dosing frequency was divided into single and multiple administrations per day, administered on Days 1, 3, and 6, respectively, according to the experimental design. Details are shown in Table 22. The model and sham groups were administered the same volume of vehicle saline.

[0336] The mRS-6 score results showed that the mRS-6 score results of the BX225-6.6 μg / Kg three-times-dosing group continued to improve on Day 7, Day 14, Day 21, and Day 30 (Table 23). The results on Day 14 were nearly significantly different from those of the saline group and the IRL1620 three-times-dosing group, and the results on Day 7, Day 21, and Day 30 were significantly different from those of the saline and IRL1620 three-times-dosing groups (P < 0.05, Table 23). The Garcia JH score results showed that the Garcia JH scores of the BX225-6.6 μg / Kg three-times-dosing group showed a trend of continuous improvement on Day 7, Day 14, Day 21, and Day 30, and the results on Day 21 and Day 30 were statistically different from those of the saline group (Tables 24 and 25).

[0337] Table 22 IRL620 and BX225 experimental groups and dosing regimens

[0338] Table 23 mRS-6 scores of animals in each experimental group at 4 weeks (*P<0.05&saline)

[0339] Table 24 Garcia JH score of each experimental group on day 21 (*P<0.05&saline)

[0340] Table 25 Garcia JH scores of each experimental group at day 30 (*P<0.05&saline)

[0341] The polypeptides and their applications provided by the present invention are described in detail above. The principles and implementation methods of the present invention are described herein using specific examples. The descriptions of the above examples are intended only to facilitate understanding of the methods and core concepts of the present invention. It should be noted that those skilled in the art may make various improvements and modifications to the present invention without departing from the principles of the present invention, and such improvements and modifications also fall within the scope of protection of the claims of the present invention.

[0342] Sequence Listing

Claims

1. Peptides, It is characterized in that include: (I), having a structure as shown in Formula I; or X1-X2-X3-(A5)-X4-X5-(A1)-X6-(A2)-X7-X8-X9-(A3)-X10-X11-(A4) -X12-X13-X14-X15-X16 Formula I (II) A sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the structure shown in (I); or (III) a sequence with a structural similarity of more than 90% to that shown in (I); or (IV), a pharmaceutically acceptable salt, solvate, chelate or non-covalent complex having a structure as shown in Formula I; and / or (V), a prodrug based on the structure shown in Formula I; and / or (VI), any mixture comprising (IV) and / or (V); X1 is selected from Suc, Fum, MeO-Fum, Ac, SA, MA, FA, A1-D or A1-NH-C 2 H 4 -NH-Suc; Said X2 is selected from D, E or K (A1); The X1 and X2 as a whole can be: Said X3 is selected from E, D or K (A1); X4 is selected from E, E(C16) or D; X5 is selected from A, K, R, H, E or D; X6 is selected from V, I, K, Dab, Orn, L, F, W or C; Said X7 is selected from Y, F or W; Said X8 is selected from F, Y or W; X9 is selected from A, K, R, H, D, E, Dab or Orn; X10 is selected from H, K or R; The X11 is selected from K, L, L(N-Me), I, V, Cit, H, R, L-2-amino-4-guanidinobutyric acid or Orn; Said X12 is selected from D or E; Said X13 is selected from I, I(N-Me), 2-Abu, L, P or V; Said X14 is selected from I, 2-Abu, L or V; X15 is selected from W, F, Y or X16 is selected from OH or NH 2 ; A5 is selected from C or does not exist; Said A1, A2, A3 or A4 independently include (Y1-Y2-Y3-Y4); The Y1 is selected from -NH-(CH 2 -CH 2 -O)m1-(CH 2 )m2-CO-, L / D-Glu, -CPAK- or not present, Among them, L / D-Glu is connected to the side chain amino group of the main chain amino acid of the polypeptide through the α- or γ-carboxyl group, and is connected to the Y2 carboxyl group through the amino group; The Y2 is selected from -NH-(CH 2 -CH 2 -O)m3-(CH 2 )m4-CO-, or does not exist; The Y3 is selected from -NH-(CH 2 -CH 2 -O)m5-(CH 2 )m6-CO-, L / D-Glu or not present, wherein L / D-Glu is connected to the amino group of Y2 through an α- or γ-carboxyl group, and is connected to the carboxyl group of Y4 through an amino group; The Y4 is selected from D-Biotin, a structure as shown in Formula 2, HOOC-(CH 2 ) n -CO, CH 3 -(CH 2 ) n -CO or not present; wherein m1, m2, m3, m4, m5 or m6 is an integer from 1 to 10; wherein n is selected from an integer from 10 to 25; A1, A2, A3 or A4 can only exist alone or not at all; and The polypeptide of formula I does not include Suc-DEEAVYFAHLDIIW.

2. The polypeptide according to claim 1, It is characterized in that The X1 is selected from Suc, Fum, MeO-Fum, Ac, SA, MA, FA, (ODDA-γE-AEEA-AEEA-)D or (ODDA-γE-AEEA-AEEA-)NH-C 2 H 4 -NH-Suc; Said X2 is D or K(A1); The X1 and X2 as a whole can be: X3 is E or K (A1); Said X4 is selected from E or E(C16); X5 is selected from A, K or E; X6 is selected from V, K, Dab, L, F or C; X7 is Y; X8 is F; Said X9 is selected from A, K or E; X10 is H; The X11 is selected from K, L, L(N-Me), R, Cit, L-2-amino-4-guanidinobutyric acid or Orn; X12 is D; Said X13 is selected from I, I(N-Me), 2-Abu, P or V; X14 is 1; X15 is selected from W or The A1 is selected from -γE-AEEA-AEEA-ODDA, ODDA-γE-AEEA-AEEA-, Biotin, -AEEA-AEEA-γE-ODDA or does not exist; A2 is selected from -γE-AEEA-AEEA-ODDA or is absent; The A3 is selected from -γE-AEEA-AEEA-ODDA or is absent; Described A4 is selected from -AEEA-AEEA-γE-ODDA, -γE-AEEA-AEEA-C18, -γE-AEEA-AEEA-C16, -γE-AEEA-AEEA-C12, -γE-AEEA-A EEA-B, -γE-AEEA-AEEA-ODDA, CPAK-MPA-, -AEEA-γE-C18, -AEEA-γE-C16, -AEEA-γE-C12, -AEEA-E-C12 or absent.

3. The polypeptide according to claim 1 or 2, It is characterized in that The structure shown in Formula I is cyclized by any of the following: (I), introducing C to form a disulfide bond; and / or (II) The carboxyl group of the D or E side chain forms an amide bond with the amino group of the K or Dab side chain.

4. The polypeptide according to claim 3, It is characterized in that The cyclization includes any of the following: (I), inserting C between X3 and X4, and replacing X6 with C, to form a disulfide bond cyclization through the two Cs; and / or (II), E of X3 and K or Dab of X6 form an amide bond cyclization; and / or (III) E of X5 and K of X11 form an amide bond for cyclization.

5. The polypeptide according to claim 1, It is characterized in that The polypeptide sequence is as follows:

6. A method for preparing a polypeptide according to any one of claims 1 to 5, It is characterized in that The steps include: Step 1: obtaining a peptide resin of the polypeptide by solid phase peptide synthesis; Step 2: cleavage and purification to obtain the polypeptide.

7. The polypeptide obtained by the preparation method according to claim 6.

8. Use of any of the following in the preparation of a drug for treating ETBR receptor-related diseases: (I), a polypeptide according to any one of claims 1 to 5; and / or (II) The polypeptide according to claim 7.

9. The use according to claim 8, It is characterized in that The ETBR receptor-related diseases include one or more of stroke, Alzheimer's disease, spinal cord injury, aortic valve stenosis or neonatal hypoxic-ischemic encephalopathy; The stroke includes ischemic stroke and / or hemorrhagic stroke.

10. Medication, It is characterized in that Includes any of the following items and pharmaceutically acceptable excipients or adjuvants: (I), a polypeptide according to any one of claims 1 to 5; and / or (II) The polypeptide according to claim 7.

11. Pharmaceutical composition, It is characterized in that The method comprises the drug as claimed in claim 10 and any other active ingredients.

12. Methods for treating diseases associated with ETBR receptors, It is characterized in that It comprises administering to a subject any of the following: (I), a polypeptide according to any one of claims 1 to 5; and / or (II), the polypeptide according to claim 7; and / or (III), the medicament according to claim 10; and / or (IV) The pharmaceutical composition according to claim 11.

13. The method according to claim 12, It is characterized in that The ETBR receptor-related diseases include one or more of stroke, Alzheimer's disease, spinal cord injury, aortic valve stenosis or neonatal hypoxic-ischemic encephalopathy; The stroke includes ischemic stroke and / or hemorrhagic stroke.

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