Pharmaceutically acceptable salt of polypeptide and use thereof

Abstract

NOT VISIBLE DUE TO STATUS OF PATENT

Description

Pharmaceutically acceptable salts of polypeptides and their applications Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to pharmaceutically acceptable salts of polypeptides and applications thereof. Background Art

[0002] 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 a stroke and must be performed in a hospital with sufficient capacity. Antiplatelet and anticoagulant medications are generally used in conjunction with thrombolytics, but their effectiveness is limited and carries a certain risk of bleeding. While neuroprotective agents have great potential, they are generally used for acute treatment. Currently, there are no marketed treatments designed to promote recovery in the subacute and recovery phases of stroke patients.

[0003] Previous studies have shown that endothelin receptor B agonists have great potential in promoting long-term recovery and post-stroke rehabilitation in stroke patients. 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 snake venom sarafotoxins b and c (S6b and S6c). Endothelin is the most potent and long-lasting vasoactive peptide known, exhibiting 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.

[0004] 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.

[0005] Studies have shown that endothelin B receptor (ETBR) agonists have great potential in promoting neurovascular unit regeneration, improving blood and oxygen supply to the nervous system, and promoting long-term recovery and post-stroke 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 (less than 2 minutes) and low exposure, IRL-1620 requires repeated dosing in preclinical and clinical studies, which greatly reduces its sustained efficacy and patient compliance.

[0006] Therefore, there is an urgent need to develop new endothelin receptor agonists.

[0007] Summary of the Invention

[0008] In view of this, the present application provides a novel pharmaceutically acceptable salt of a polypeptide and its application.

[0009] The polypeptides and pharmaceutically acceptable salts thereof provided in this application are based on the mechanism research of ETBR agonists and are highly selective agonists of ETBR. The polypeptides and pharmaceutically acceptable salts provided in this application extend the half-life and in vivo exposure of the drug through structure-activity relationship research and structural modification, reduce the number of administrations, and have lower side effects in animal experiments than the only ETBR agonist IRL-1620 that has entered the clinical research stage, and have a larger safety window for the drug. The polypeptides and pharmaceutically acceptable salts provided in this application can be used to treat diseases related to ETBR receptors, such as rehabilitation problems in the recovery period of ischemic stroke patients, to promote better recovery of stroke patients. Specifically, this application provides the following technical solutions:

[0010] In a first aspect, the present application provides a pharmaceutically acceptable salt of a polypeptide, wherein the polypeptide comprises the amino acid sequence Fum-DEEAVYFAHK(-AEEA-γE-C12)DVIW (SEQ ID NO: 1) or a functional variant thereof.

[0011] In some embodiments, the functional variant is a variant of SEQ ID NO: 1 resulting from one or more conservative substitutions.

[0012] In some embodiments, the conservative substitution is selected from substitutions between D and E, substitutions between V, L and I, substitutions between Y, F and W, substitutions between H, K and R.

[0013] In some embodiments, the functional variant has the same or similar ETBR receptor activating activity as SEQ ID NO:1.

[0014] In some embodiments, the pharmaceutically acceptable salt is selected from sodium salt, potassium salt, ammonium salt, trifluoroacetate salt, acetate salt, hydrochloride salt, sulfate salt, and phosphate salt.

[0015] In some embodiments, the pharmaceutically acceptable salt is selected from sodium salt, potassium salt, and ammonium salt.

[0016] In some embodiments, the pharmaceutically acceptable salt is a sodium salt.

[0017] In a second aspect, the present application provides a pharmaceutical composition comprising a pharmaceutically acceptable salt of the polypeptide according to the first aspect, and a pharmaceutically acceptable carrier, excipient and / or diluent.

[0018] In some embodiments, the pharmaceutical composition is a pre-lyophilized formulation.

[0019] In some embodiments, the pharmaceutical composition comprises sodium chloride and / or optionally trehalose and / or cyclodextrin.

[0020] In some embodiments, the pharmaceutical composition is a lyophilized formulation, preferably prepared by lyophilizing the pre-lyophilized formulation described above.

[0021] In some embodiments, the pharmaceutical composition is a reconstituted formulation, preferably prepared by combining the lyophilized formulation described above with an aqueous solution.

[0022] In some embodiments, the pharmaceutical composition is used to treat a disease associated with the ETBR receptor.

[0023] In some embodiments, the pharmaceutical composition is used to treat, improve or prevent nervous system damage and related diseases caused by the damage, neurodegenerative diseases, anxiety, epilepsy, aortic valve stenosis or neonatal hypoxic-ischemic encephalopathy.

[0024] In a third aspect, the present application provides a method for promoting the regeneration of neurovascular units in an individual, improving blood and oxygen supply to the nervous system, treating, improving or preventing nervous system damage in an individual and related diseases caused by the damage, neurodegenerative diseases, anxiety, epilepsy, aortic valve stenosis or neonatal hypoxic-ischemic encephalopathy, the method comprising administering to the individual a pharmaceutically acceptable salt of the polypeptide described in the first aspect or the pharmaceutical composition described in the second aspect.

[0025] In a fourth aspect, the present application provides the use of a pharmaceutically acceptable salt of the polypeptide described in the first aspect or the pharmaceutical composition described in the second aspect in the preparation of a drug for promoting regeneration of neurovascular units, improving blood and oxygen supply to the nervous system, treating, improving or preventing nervous system damage in an individual and related diseases caused by the damage, neurodegenerative diseases, anxiety, epilepsy, aortic valve stenosis or neonatal hypoxic-ischemic encephalopathy.

[0026] In some embodiments of the second, third or fourth aspects, the nervous system injury and related diseases caused by the injury include cerebral stroke, spinal cord injury, ischemic or traumatic injury to the brain or spinal cord, and damage to central nervous system (CNS) neurons, the cerebral stroke includes ischemic stroke, hemorrhagic stroke and hemorrhagic stroke converted from ischemic stroke; the neurodegenerative diseases include Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Parkinson's disease or Huntington's disease.

[0027] In some embodiments of the second, third, or fourth aspect, the individual is a mammal, eg, a non-primate or a primate, eg, a human.

[0028] In a fifth aspect, the present application further provides a drug comprising a pharmaceutically acceptable salt of the polypeptide described in the first aspect or the pharmaceutical composition described in the second aspect, and optionally any other active ingredients.

[0029] The present application also provides a method for preparing a pharmaceutically acceptable salt of the polypeptide, comprising the following steps:

[0030] Step 1: obtaining a peptide resin of the polypeptide by solid phase peptide synthesis;

[0031] Step 2: cleavage and purification to obtain the polypeptide; and

[0032] Step 3: Preparation of salt from the polypeptide.

[0033] In addition, the present application also provides the use of the polypeptide (SEQ ID NO: 1) and a pharmaceutically acceptable salt thereof in the preparation of a drug for treating a disease associated with an ETBR receptor in an individual.

[0034] In some embodiments, the ETBR receptor-related diseases include one or more of nervous system damage and diseases caused by the damage, neurodegenerative diseases, anxiety, epilepsy, aortic valve stenosis, or neonatal hypoxic-ischemic encephalopathy.

[0035] In some embodiments, the nervous system injury and related diseases caused by the injury include stroke, spinal cord injury, ischemic or traumatic injury to the brain or spinal cord, and damage to central nervous system (CNS) neurons.

[0036] In some embodiments, the neurodegenerative disease comprises Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Parkinson's disease, or Huntington's disease.

[0037] In some embodiments, the stroke includes ischemic stroke, hemorrhagic stroke, and hemorrhagic stroke converted from ischemic stroke.

[0038] Accordingly, the present application also provides a drug comprising a pharmaceutically acceptable salt of the polypeptide, and a pharmaceutically acceptable excipient or adjuvant.

[0039] In some embodiments, the drug further comprises any other active ingredients.

[0040] In other inventions, the present application also provides a method for treating diseases related to ETBR receptors, which comprises administering a pharmaceutically acceptable salt of the polypeptide; the pharmaceutical composition; or the drug to an individual.

[0041] In some embodiments of the present application, the ETBR receptor-related diseases include one or more of nervous system damage and related diseases caused by the damage, neurodegenerative diseases, anxiety, epilepsy, aortic valve stenosis or neonatal hypoxic-ischemic encephalopathy.

[0042] In some embodiments, the nervous system injury and related diseases caused by the injury include stroke, spinal cord injury, ischemic or traumatic injury to the brain or spinal cord, and damage to central nervous system (CNS) neurons.

[0043] In some embodiments, the neurodegenerative disease comprises Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Parkinson's disease, or Huntington's disease.

[0044] In some embodiments, the stroke includes ischemic stroke, hemorrhagic stroke, and hemorrhagic stroke converted from ischemic stroke. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] 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.

[0046] FIG1 shows the results of the EC50 determination of the activity of the polypeptides on human ETB receptor in Example 2.

[0047] FIG2 shows the results of EC50 determination of the activity of the polypeptides on canine ETB receptor in Example 2.

[0048] FIG3 shows the results of EC50 determination of the activity of the polypeptides in Example 2 on rat ETB receptor.

[0049] FIG4 shows the results of EC50 determination of the activity of the polypeptides in Example 2 on mouse ETB receptor.

[0050] FIG5 shows the Garcia JH score results on the 7th day after three times weekly administration in Example 9, wherein 1 is the normal saline group, 2 and 3 are the 30 μg / Kg and 100 μg / Kg BX-229-Na subcutaneous administration groups, respectively, and 4 is the sham operation group.

[0051] FIG6 shows the Garcia JH score results on the 14th day after three times weekly administration in Example 9, wherein 1 is the normal saline group, 2 and 3 are the 30 μg / Kg and 100 μg / Kg BX-229-Na subcutaneous administration groups, respectively, and 4 is the sham operation group.

[0052] Figure 7 shows the changes in VEGF-A protein expression after three doses of medication per week in Example 9, where 1 is the normal saline group, 2 and 3 are the 30 μg / Kg and 100 μg / Kg BX-229-Na subcutaneous administration groups, respectively, and 4 is the sham operation group.

[0053] FIG8 shows the changes in BDNF protein expression after three doses of medication per week in Example 9, wherein 1 is the normal saline group, 2 and 3 are the 30 μg / Kg and 100 μg / Kg BX-229-Na subcutaneous administration groups, respectively, and 4 is the sham operation group.

[0054] Figure 9 shows the scores of the BX-229 subcutaneous administration group and the IRL-1620 intravenous administration group on day 8 and day 15 after modeling in Example 10 (* indicates significant difference P < 0.05), where 1 and 3 are the 30 μg / Kg BX-229-Na subcutaneous administration groups, and 2 and 4 are the 3×2.7 μg / Kg IRL-1620 intravenous administration groups. DETAILED DESCRIPTION

[0055] The present invention discloses pharmaceutically acceptable salts of polypeptides and their uses. 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.

[0056] Unless otherwise specified, the terms used in this application have the meanings that are commonly understood by those skilled in the art.

[0057] The single-letter or three-letter abbreviations used for amino acids in this application follow international conventions.

[0058] Throughout the specification and claims, the words “comprises,” “comprising,” and “including” mean “including but not limited to,” and are not intended to exclude other parts, additives, components, or steps.

[0059] In a first aspect, the present application provides a pharmaceutically acceptable salt of a polypeptide, wherein the polypeptide comprises the amino acid sequence Fum-DEEAVYFAHK(-AEEA-γE-C12)DVIW (SEQ ID NO: 1) or a functional variant thereof.

[0060] The term "functional variant" refers to a variant having the same or similar biological functions and properties as the parent. As a non-limiting example, a "functional variant" can be obtained by making one or more conservative substitutions in the parent.

[0061] In some embodiments, the conservative substitution is selected from substitutions between D and E, substitutions between V, L and I, substitutions between Y, F and W, substitutions between H, K and R.

[0062] In some embodiments, the functional variants disclosed herein also include amino acid sequences that are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even higher identical to the above-mentioned peptides. As is known in the art, the "identity" between two proteins is determined by comparing the amino acid sequence of one protein with the sequence of a second protein in which its conservative amino acids are substituted. The degree of identity between two proteins is determined using computer algorithms and methods well known to those skilled in the art. The identity between two amino acid sequences is preferably determined by utilizing the BLASTP algorithm.

[0063] In some embodiments, the functional variants disclosed herein include peptides having 1, 2, 3, 4, 5 or more substitutions, deletions, additions and / or insertions of amino acid residues that differ from the specific peptides disclosed above.

[0064] As described above, functional variants can be distinguished from the specific peptides disclosed above by one or more substitutions, deletions, additions and / or insertions. These variants can be naturally occurring or can be produced synthetically, for example, by modifying one or more of the above-mentioned peptide sequences disclosed herein and evaluating their biological activity as described herein using any of a variety of techniques known in the art.

[0065] The active peptides of the present application can be synthesized by solid phase synthesis or recombinant methods. Peptidomimetics can be synthesized using a variety of protocols and methods described in the scientific literature and patent literature, such as Organic Syntheses Collective Volumes, Gilman et al. (ed.) John Wiley & Sons, Inc., NY, al-Obeidi (1998) Mol. Biotechnol. 9: 205-223; Hruby (1997) Curr. Opin. Chem. Biol. 1: 114-119; Ostergaard (1997) Mol. Divers. 3: 17-27; Ostresh (1996) Methods Enzymol. 267: 220-234.

[0066] In addition, the present invention provides a method for preparing a pharmaceutically acceptable salt of the polypeptide (SEQ ID NO: 1), comprising the following steps:

[0067] Step 1: Weigh 0.50 g (0.25 mmol) of Fmoc-Trp(Boc)-Wang Resin into a reactor, add 10 mL of DCM, and swell for 10 minutes. Filter with suction, wash twice with DMF, and add 25% 4-methylpiperidine / DMF (volume ratio) to react for 30 minutes to remove the Fmoc group. Filter with suction, 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.), 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 hour. Filter with suction, wash three times with DMF, MeOH, and DCM, sequentially. 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 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 × 3 times to remove the Dde from the Lys side chain, followed by the condensation of AEEA, Glu-α-OtBu, and C12-OH.

[0068] Step 2: Drain the peptide resin and add 15 mL of pre-chilled cleavage buffer (TFA:TIS:H2O = 95:2.5:2.5). Stir and react at room temperature (25°C ± 5°C) for 3 h. The reaction mixture is then removed and the resin is washed twice with a small amount of TFA and removed. The combined reaction mixtures are then added with approximately 200 mL of pre-chilled methyl tert-butyl ether to precipitate a white precipitate. The mixture is centrifuged (5000 rpm for 5 min), the supernatant is discarded, and the precipitate is re-added with methyl tert-butyl ether, vortexed, and centrifuged again. The supernatant is discarded. The precipitate is dried in a vacuum desiccator for 12 h to obtain the crude product. The crude peptide is dissolved in approximately 1% aqueous ammonia, filtered, and the filtrate is purified by preparative chromatography. 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 is used 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 using 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).

[0069] Step 3: Preparation of the salt (BX-229-Na) of the polypeptide (SEQ ID NO: 1)

[0070] Here we take the preparation of sodium salt as an example.

[0071] The polypeptide (SEQ ID NO: 1) solid obtained in step 2 was dissolved in a 0.1N sodium hydroxide aqueous solution (containing 10 equivalents of sodium hydroxide) and salt was transferred using a preparative liquid phase. Preparative column: C18-10-100, 30×250 mm. Flow rate: 25 mL / 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 > 95%, use a water pump and a rotary evaporator to reduce the pressure at about 40°C to remove the acetonitrile, and freeze the resulting concentrated solution with liquid nitrogen to form a solid, and then use a freeze dryer to freeze-dry for 48 hours to obtain the target compound.

[0072] 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.

[0073] Without wishing to be bound by any theory, it is expected that combining a drug with a molecule or ion with an opposite charge to the drug to form a salt can improve certain undesirable physicochemical or biopharmaceutical properties of the drug, such as altering solubility or dissolution, reducing hygroscopicity, improving stability, or changing the melting point. Ultimately, determining the ideal salt form requires finding a balance between physicochemical and biopharmaceutical properties. The selection of a pharmaceutically acceptable salt form of a drug prioritizes the following requirements: solubility, hygroscopicity, and stability to environmental factors under various conditions.

[0074] The pharmaceutically acceptable salts of the polypeptides of the present application may be in any suitable pharmaceutically acceptable salt form. In some embodiments, the pharmaceutically acceptable salt of the polypeptide is a sodium salt. In some embodiments, the pharmaceutically acceptable salt of the polypeptide is a potassium salt. In some embodiments, the pharmaceutically acceptable salt of the polypeptide is an ammonium salt. In some embodiments, the pharmaceutically acceptable salt of the polypeptide is a trifluoroacetate or acetate. In some specific embodiments, the pharmaceutically acceptable salt of the polypeptide is a hydrochloride, sulfate, or phosphate.

[0075] In a second aspect, the present application provides a pharmaceutical composition comprising a pharmaceutically acceptable salt of the polypeptide according to the first aspect, and a pharmaceutically acceptable carrier, excipient and / or diluent.

[0076] The pharmaceutically acceptable salts of the polypeptides described herein can be prepared in the form of lyophilized formulations. In some embodiments, the present application provides lyophilized formulations. The lyophilized formulation is prepared by lyophilizing a pre-lyophilized formulation, which comprises at least an active ingredient, a buffer, a filler, and water, wherein the active ingredient is a pharmaceutically acceptable salt of the polypeptide of the present application. The filler provides structure for the lyophilized compound. In some embodiments, the filler is selected from mannitol, trehalose, dextran-40, glycine, lactose, sorbitol, cyclodextrin, and sucrose, among which trehalose and cyclodextrin are preferred. In some embodiments, the lyophilized formulation of the present application comprises the pharmaceutically acceptable salt of the polypeptide described above as well as trehalose and cyclodextrin.

[0077] The lyophilized formulation can be reconstituted, i.e., rehydrated with a solution to form a solution of particles invisible to the naked eye. In some embodiments, the present application provides a reconstituted formulation prepared by combining the lyophilized formulation with an aqueous solution. In some embodiments, the aqueous solution is water for injection. In some embodiments, the aqueous solution is normal saline.

[0078] The term "lyophilization" refers to a process in which the material to be dried is first frozen and then sublimed under vacuum to remove the ice or frozen solvent.

[0079] In some embodiments, the pharmaceutically acceptable salts of the polypeptides disclosed herein can be administered in the form of pharmaceutical compositions. Pharmaceutical compositions can be manufactured by conventional mixing, dissolving, granulating, tableting, grinding, emulsifying, encapsulating, entrapping or lyophilizing methods.

[0080] The pharmaceutical composition can be formulated in a conventional manner using one or more physiologically acceptable carriers, diluents, excipients or adjuvants that facilitate processing of the polypeptide pharmaceutically acceptable salt into a pharmaceutically acceptable preparation. Appropriate formulation depends on the chosen route of administration.

[0081] In some embodiments, administration may be parenteral, intravenous, oral, subcutaneous, intraarterial, intracranial, intrathecal, intraperitoneal, topical, intranasal, or intramuscular, with intravenous administration being preferred.

[0082] In some embodiments, pharmaceutical compositions for parenteral administration are preferably sterile and substantially isotonic. For injection, a pharmaceutically acceptable salt of the polypeptide can be formulated into an aqueous solution, preferably into a physiologically compatible buffer such as Hank's solution, Ringer's solution, or normal saline or acetate buffer (to reduce discomfort at the injection site). The solution may contain preparatons such as suspending agents, stabilizers, and / or dispersants.

[0083] Alternatively, the pharmaceutically acceptable salt of the polypeptide may be in powder form for constitution with a suitable vehicle (eg, sterile pyrogen-free water) before use.

[0084] For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. This route of administration can be used to deliver the compound to the nasal cavity or for sublingual administration.

[0085] In some embodiments, for oral administration, a pharmaceutically acceptable salt of the polypeptide can be formulated with a pharmaceutically acceptable carrier into tablets, pills, lozenges, capsules, liquids, gels, syrups, slurries, suspensions, etc. for oral ingestion by the patient being treated. For oral solid formulations such as powders, capsules, and tablets, suitable excipients include fillers such as sugars, such as lactose, sucrose, mannitol, and sorbitol; cellulose preparations such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, carboxypropyl methylcellulose, sodium carboxymethylcellulose, and / or polyvidone (PVP); granulating agents and binders. If necessary, a disintegrant such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof, such as sodium alginate, can be added. If necessary, the solid dosage form can be sugar-coated or enteric-coated using standard techniques. For oral liquid formulations such as suspensions, elixirs, and solutions, suitable carriers, excipients, or diluents include water, glycerol, oils, and alcohols. In addition, flavoring agents, preservatives, coloring agents and the like may be added.

[0086] In addition to the formulations described above, pharmaceutically acceptable salts of the polypeptides can also be formulated as storage formulations. Such long-acting formulations can be administered by implantation (e.g., subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compound can be formulated with a suitable polymeric material or hydrophobic material (e.g., formulated as an emulsion in an acceptable oil) or an ion exchange resin, or as a sparingly soluble derivative, such as a sparingly soluble salt.

[0087] Alternatively, other drug delivery systems can be used. Chimeric peptides can be delivered using liposomes and emulsions. Certain organic solvents such as dimethyl sulfoxide can also be used. Additionally, sustained-release systems (e.g., semipermeable matrices of solid polymers containing therapeutic agents) can be used to deliver the compound.

[0088] Depending on its chemical nature, sustained-release capsules can release the chimeric peptide for several weeks up to over 100 days. Depending on the chemical nature and biological stability of the therapeutic reagent, other strategies for protein stabilization can be used.

[0089] The pharmaceutically acceptable salt of the polypeptide is used in an amount that effectively achieves the intended purpose (e.g., reducing the damaging effects of traumatic stroke and related conditions). A therapeutically effective amount means that, relative to central nervous system damage in a control group of patients (or animal models) not treated with a pharmaceutically acceptable salt of the polypeptide disclosed herein, the amount of a pharmaceutically acceptable salt of the polypeptide that is sufficient to significantly reduce the damage caused by stroke in a patient (or animal model group) treated with a pharmaceutically acceptable salt of the polypeptide disclosed herein. If the individual treated patient achieves a better output compared to the mean output (measured by infarct volume or disability index) in a comparable patient control group not treated by the methods disclosed herein, the amount is also considered to be therapeutically effective. If the individual treated patient shows a disability of 2 or less on the Rankin scale and 75 or more on the Barthel scale, the amount is also considered to be a therapeutically effective amount. A dose is also considered therapeutically effective if the treated patient population shows a distribution of scores on the disability scale that is significantly improved (i.e., less disabled) compared to a comparable untreated population, see Lees et al., N Engl J Med 2006; 354: 588-600. A therapeutically effective regimen refers to a combination of a therapeutically effective dose and frequency of administration required to achieve the intended purpose as described above.

[0090] In some embodiments, the amount of a pharmaceutically acceptable salt of a polypeptide administered is determined by the subject being treated, the subject's weight, the severity of the affliction, the mode of administration, and the discretion of the prescribing physician. Treatment may be repeated when symptoms are detectable, or even when they are not. Treatment may be provided alone or in combination with other drugs.

[0091] In some embodiments, the therapeutically effective dose of the pharmaceutically acceptable salt of the polypeptide disclosed herein can provide therapeutic benefits without causing significant toxicity. The toxicity of the chimeric peptide can be determined by standard pharmaceutical procedures in cell culture or experimental animals, for example, by determining LD50 (a dose that causes 50% of the population to be lethal) or LD100 (a dose that causes 100% of the population to be lethal). The dose ratio of the toxic effect to the therapeutic effect is the therapeutic index. Pharmaceutically acceptable salts of polypeptides that preferably show a high therapeutic index (see, for example, Fingl et al., 1975, In: The Pharmacological Basis of Therapeutics, Chapter 1, page 1) are preferably used.

[0092] In a third aspect, the present application provides a method for treating, ameliorating or preventing a disease associated with ETBR receptor in an individual.

[0093] In some embodiments, diseases associated with ETBR receptors include one or more of nervous system damage and related diseases caused by the damage, neurodegenerative diseases, anxiety, epilepsy, aortic valve stenosis, or neonatal hypoxic-ischemic encephalopathy; the nervous system damage and related diseases caused by the damage include stroke, spinal cord injury, ischemic or traumatic injury to the brain or spinal cord, and damage to central nervous system (CNS) neurons.

[0094] In some embodiments, the neurodegenerative disease comprises Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Parkinson's disease, or Huntington's disease.

[0095] In some embodiments, stroke includes ischemic stroke, hemorrhagic stroke, and hemorrhagic stroke converted from ischemic stroke.

[0096] In some embodiments, the method comprises administering to the individual a pharmaceutically acceptable salt of the polypeptide of the first aspect or the pharmaceutical composition of the second aspect.

[0097] Cerebral stroke is the disease that is caused by the blood flow that is damaged in CNS. Possible causes include embolism, hemorrhage and thrombosis. The death of CNS tissue is called infarction. Infarct volume (i.e. the volume of the neuronal cells that die in the brain caused by stroke) can be used as an index of the pathological damage degree caused by stroke. The symptomatic effect depends not only on the infarct volume, but also on where the infarction is located in the brain. The disability index can be used as a measure of symptomatic damage, such as the Rankin Stroke Outcome Scale (Rankin, Scott Med J; 2: 200-15 (1957)) and the Barthel Index (Barthel Index). The Rankin scale is based on the following direct evaluation of the patient's global symptoms:

[0098] 0 No symptoms at all.

[0099] 1. No significant disability despite symptoms; able to perform all daily tasks and activities.

[0100] 2 Mild disability; unable to perform all previous activities, but able to take care of own affairs without assistance.

[0101] 3 Moderate disability requiring some assistance but able to walk without assistance.

[0102] 4 Moderate to severe disability, unable to walk without assistance and unable to take care of own physical needs without assistance.

[0103] 5. Severe disability; bedridden, incontinent, and requiring constant care and attention.

[0104] The Barthel Index is based on a series of questions about a patient's ability to perform 10 basic activities of daily living, which yield a score between 0 and 100, with lower scores indicating more disability (Mahoney et al., Maryland State Medical Journal 14:56-61 (1965)).

[0105] Alternatively, stroke severity / outcome can be measured using the NIH Stroke Scale, which is available on the World Wide Web at ninds.nih.gov / doctors / NIH_Stroke_Scale_Booklet.pdf. This scale is based on the patient's ability to perform 11 functions that assess the patient's level of consciousness, movement, perception, and language function.

[0106] Ischemic stroke is more specifically defined as a type of stroke caused by blockage of blood flow to the brain. The most common underlying condition for this type of blockage is the development of fatty deposits along the blood vessel walls. This condition is called atherosclerosis. These fatty deposits can cause two types of obstruction. Cerebral thrombosis refers to a thrombus (blood clot) that forms in the blocked part of a blood vessel. "Cerebral embolism" generally refers to various emboli in the blood (such as mural thrombi in the heart, atherosclerotic plaques, fat, tumor cells, fibrocartilage, or air, etc.) that enter the cerebral artery with the blood flow and block the blood vessel. When the collateral circulation cannot compensate, it causes ischemic necrosis of the brain tissue in the arterial blood supply area, resulting in focal neurological deficits. The second important cause of embolism is irregular heartbeat, called arterial myocardial fibrillation. It causes the following conditions, in which blood clots can form in the heart, move, and metastasize to the brain. Other potential causes of ischemic stroke are hemorrhage, thrombosis, cutting of an artery or vein, cardiac arrest, shock from any cause (including hemorrhage), and iatrogenic causes such as direct surgical injury to a cerebral blood vessel or a blood vessel leading to the brain or heart surgery. Ischemic stroke accounts for approximately 83% of all stroke cases.

[0107] In a fourth aspect, the present application provides the use of a pharmaceutically acceptable salt of the polypeptide described in the first aspect or the pharmaceutical composition described in the second aspect for treating, ameliorating, or preventing stroke, Alzheimer's disease, spinal cord injury, aortic valve stenosis, or neonatal hypoxic-ischemic encephalopathy in an individual. Accordingly, the present application also provides the use of a pharmaceutically acceptable salt of the polypeptide described in the first aspect or the pharmaceutical composition described in the second aspect for preparing a medicament for treating, ameliorating, or preventing stroke, Alzheimer's disease, spinal cord injury, aortic valve stenosis, or neonatal hypoxic-ischemic encephalopathy in an individual.

[0108] Similarly, the present application provides the use of a pharmaceutically acceptable salt of the polypeptide described in the first aspect or the pharmaceutical composition described in the second aspect for treating, ameliorating, or preventing a disease associated with an ETBR receptor in an individual. Accordingly, the present application also provides the use of a pharmaceutically acceptable salt of the polypeptide described in the first aspect or the pharmaceutical composition described in the second aspect for preparing a medicament for treating, ameliorating, or preventing a disease associated with an ETBR receptor in an individual.

[0109] In some embodiments, diseases associated with ETBR receptors include one or more of nervous system damage and related diseases caused by the damage, neurodegenerative diseases, anxiety, epilepsy, aortic valve stenosis, or neonatal hypoxic-ischemic encephalopathy; the nervous system damage and related diseases caused by the damage include stroke, spinal cord injury, ischemic or traumatic injury to the brain or spinal cord, and damage to central nervous system (CNS) neurons.

[0110] In some embodiments, the neurodegenerative disease comprises Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Parkinson's disease, or Huntington's disease.

[0111] In some embodiments, stroke includes ischemic stroke, hemorrhagic stroke, and hemorrhagic stroke converted from ischemic stroke.

[0112] The term "individual" as used herein refers to animals including birds, reptiles, and mammals. In some embodiments, the individual is a mammal, including primates and non-primates, such as humans, chimpanzees, cows, horses, pigs, sheep, goats, dogs, cats, and rodents such as rats and mice.

[0113] It should be understood that the above detailed description is only for the purpose of enabling those skilled in the art to more clearly understand the content of the present application and is not intended to limit the present invention in any respect. Those skilled in the art can make various modifications and variations to the embodiments described.

[0114] In this application, the Chinese meanings of abbreviations or full English names are as follows:

[0115] Example

[0116] The following examples are provided to illustrate the present invention but are not intended to limit the scope of the present invention. Any modification or substitution of the methods, steps, or conditions of the present invention without departing from the spirit and substance of the present invention shall fall within the scope of the present invention.

[0117] Unless otherwise specified, the reagents used in the examples are conventional commercially available reagents, and the technical means used in the examples are conventional means well known to those skilled in the art.

[0118] Unless otherwise specified, the main reagents and solvents used in the following examples are commercially available, for example, from Jier Biochemical (Shanghai) Co., Ltd. or Sangon Biotech (Shanghai) Co., Ltd. The control IRL-1620 was synthesized according to the sequence and synthesis method reported in the literature (Michihiro Takai, et al., BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, Vol. 184, No. 2, 1992, Pages 953-959).

[0119] Example 1: Preparation of BX-229 polypeptide and its salt

[0120] In this example, the BX-229 polypeptide (SEQ ID NO.: 1, Fum-DEEAVYFAHK(-AEEA-γE-C12)DVIW) and its different salts were prepared and synthesized as follows.

[0121] 1) Synthesis of BX-229

[0122] 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 approximately 200 mL of pre-cooled methyl tert-butyl ether was added to a white precipitate. The mixture was centrifuged (5000 rpm for 5 min), the supernatant discarded, and the precipitate was re-added with methyl tert-butyl ether, vortexed, and centrifuged again. The supernatant was discarded. The precipitate was dried in a vacuum desiccator for 12 h to obtain the crude product. The crude peptide was dissolved in approximately 1% aqueous ammonia, filtered, and the filtrate was purified using preparative liquid chromatography. 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. Fractions of the target compound with a purity >95% were combined, and the acetonitrile was removed under reduced pressure using a water pump and a rotary evaporator at approximately 40°C. The resulting concentrate was frozen to a solid using liquid nitrogen and then freeze-dried in a lyophilizer for 48 h to obtain the target compound BX-229. The molecular weight was confirmed by mass spectrometry ([M-2] 2- =1137.3).

[0123] 2) Preparation of BX-229 sodium salt

[0124] The obtained BX-229 solid was dissolved in a 0.1N sodium hydroxide aqueous solution (containing 10 equivalents of sodium hydroxide) and the salt was transferred using a preparative liquid phase. 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 and spin out the acetonitrile at about 40°C, and freeze the resulting concentrated solution with liquid nitrogen to form a solid, and then use a freeze dryer to freeze-dry for 48 hours to obtain the target compound BX-229 sodium salt.

[0125] 3) Preparation of BX-229 potassium salt

[0126] The obtained BX-229 solid was dissolved in a 0.1N potassium hydroxide aqueous solution (containing 10 equivalents of potassium hydroxide) and the salt was transferred using a preparative liquid phase. Preparative column: C18-10-100, 30×250 mm. Flow rate: 25 mL / 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 remove the acetonitrile, and freeze the resulting concentrated solution with liquid nitrogen to form a solid, and then use a freeze dryer to freeze-dry for 48 hours to obtain the target compound BX-229 potassium salt.

[0127] 4) Preparation of BX-229 ammonium salt

[0128] The obtained BX-229 solid was dissolved in a 5% ammonia solution (pH about 9) and the salt was transferred using a preparative liquid phase. Preparative column: C18-10-100, 30×250 mm. Flow rate: 25 mL / 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 and spin out the acetonitrile at about 40°C, and freeze the resulting concentrated solution into a solid with liquid nitrogen, and then use a freeze dryer to freeze-dry for 48 hours to obtain the target compound BX-229 ammonium salt.

[0129] Example 2: EC50 determination of BX-229-Na activity on ETB receptors of different species

[0130] This example tests the agonist activity of BX-229-Na prepared in Example 1 on ETB receptors of different species and compares it with IRL-1620.

[0131] 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 2.5 μg of the human ETB receptor gene expression vector, PCDNA3.1-hETB (human hETB sequence, CBI Accession No.: NM_000115.5), per well. The cells were cultured for 48 hours. After observing the cells for green fluorescence expression under a fluorescence microscope, they were digested with trypsin and counted. The activity of BX-229-Na against the human ETB receptor was measured 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 peptides BX-229-Na and IRL-1620 prepared in Example 1 were diluted with Stim B to 11 concentration gradients, each diluted to a 2x concentration using Stim B. 7 μL of each gradient was added to the test cells. 7 μL of PBS was added to the negative control wells. The plates were sealed and incubated in a 37°C cell culture incubator for 1 hour. 3 μL of IP1 d2 working solution and 3 μL of IP1 Tb cryptotate antibody working solution were added to the test wells, the positive control wells containing human endothelin ET1 (ET1, SEQ ID NO.: 2, CSCSSLMDKECVYFCHLDIIW(C1-C15, C3-C11)), and the PBS control wells. 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 665nm and 620nm on an ID5 microplate reader. Calculate the ratio of the acceptor and donor emission signals in each well, i.e., (665nm signal / 620nm signal) × 10 4 The average value of the 665nm signal / 620nm signal value measured for the test sample at different concentrations is F. First, calculate the activation rate of each concentration of the test peptide relative to the positive control sample, human endothelin ET1. Plot a dot plot with this activation rate as the vertical axis and the logarithm of the peptide concentration as the horizontal axis. Use the four-parameter fitting method in Prism software to fit the generated curve and calculate the EC50 value of the peptide for the human ETB receptor.

[0132] The procedures for determining the EC50 of canine, rat, and mouse ETB receptor activity were the same as above, except that the transfection plasmids were the expression vector PCDNA3.1-DETB containing the canine ETB receptor gene (canine ETB sequence, NCBI accession number: NM_001010943.2), the expression vector PCDNA3.1-RETB containing the rat ETB receptor gene (rat ETB receptor sequence, NCBI accession number: X57764.1), and the expression vector PCDNA3.1-mETB containing the mouse ETB receptor gene (mouse ETB sequence, NCBI accession number: NM_001276296).

[0133] To obtain more reliable data, comparative tests were performed in the same experiment and repeated multiple times. The three data with the best curve fit were averaged, and the obtained mean was used as the final cell activity result, as shown in Table 1 and Figures 1 to 4.

[0134] Table 1 Agonist activity of BX-229-Na and IRL-1620 on ETB receptor (EC50, nM)

[0135] Example 3: Activity of BX-229-Na on human ETA receptor

[0136] This example tests the activity of BX-229-Na prepared in Example 1 on human ETA receptor.

[0137] 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 2.5 μg of the expression vector PCDNA3.1-hETA (hETA sequence, NCBI accession number: L06622.1) containing the human ETA receptor gene. The cells were cultured for 48 hours. After observing the cells for green fluorescence expression under a fluorescence microscope, they were digested with trypsin and counted. The activity of BX-229-Na against the human ETA receptor was measured 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 BX-229-Na prepared in Example 1 and the positive control peptide human endothelin ET1 (SEQ ID NO.: 2) were each dissolved in Stim B to a 2x concentration and 7 μL was added to the test cells. In the negative control wells, 7 μL was added to the cells in PBS. The plates were sealed and incubated in a 37°C cell culture incubator for 1 hour. 3 μL of IP1 d2 working solution and 3 μL of IP1 Tb cryptotate antibody working solution were added to 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. Calculate the ratio of the acceptor and donor emission signals for each well, i.e. (665 nm signal / 620 nm signal) × 10 4The average 665nm signal / 620nm signal value of the test sample at a specific concentration is F3, the average 665nm signal / 620nm signal value of the positive control human endothelin ET1 at the same concentration is F5, and the average 665nm signal / 620nm signal value of 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). The results are shown in Table 2, indicating that BX-229-Na is a selective agonist of the ETB receptor.

[0138] Table 2 Activity of BX-229-Na on human ETA receptor

[0139] Example 4: Determination of half-life of BX-229 polypeptide

[0140] This example tests the half-life of the polypeptide BX-229 prepared in Example 1 and compares it with the control IRL-1620.

[0141] Plasma Sample Administration and Processing: Three C57BL / 6 mice were injected with BX-229 or the control drug, IRL-1620, 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 two 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 ​​at each time point for each drug were averaged. Samples of blank mouse plasma were also prepared as a control after protein precipitation. The results are shown in Table 3.

[0142] Table 3 Half-life data of BX-229 and IRL-1620

[0143] LC-MS / MS detection conditions:

[0144] Instrument model: Agilent InfinityLab LC / MSD 1260-G6125C

[0145] Chromatographic column: Poroshell 120 SB-C18 4.6*100mm, 2.7μm

[0146] Mobile phase A: 0.1% formic acid-ultrapure water Mobile phase B: 0.1% formic acid-acetonitrile

[0147] Gradient settings:

[0148] Column temperature: 45°C; injection volume: 20 μl;

[0149] Ion source: ESI; dryer temperature: 350°C; dryer flow rate: 12 L / min; nebulizer pressure: 45 psi; capillary voltage: 4000 V.

[0150] Scan type: SIM; fragmentor voltage: 135 V; scan / dwell time: 200 ms.

[0151] Example 5: Preliminary study on acute toxicity of BX-229 polypeptide after intravenous administration

[0152] This example conducted a preliminary study on the acute toxicity of intravenous administration of the BX-229 polypeptide prepared in Example 1 and compared it with IRL-1620.

[0153] In the intravenous acute toxicity study, male SD rats weighing 200-230g were administered BX-229 and the control IRL-1620 at doses ranging from 120μg / kg to 30μg / kg, respectively, via tail vein injection. The rats were observed for 24 hours after administration. If a rat died at a given dose, the dose was reduced and the rats were observed again. Preliminary observations were made of toxic reactions and mortality in the rats, and the time of death and the dose at which no mortality occurred were recorded. The dose at which no mortality occurred was used to preliminarily determine the acute toxicity of the peptides. The results are shown in Table 4. Acute toxicity studies showed that rats could tolerate a maximum tolerated dose of 30μg / kg for IRL-1620 and 60μg / kg for BX-229, respectively. The maximum tolerated dose of BX-229 peptide injected into SD rats was significantly higher than that of IRL-1620.

[0154] Table 4 Results of intravenous acute toxicity tests of BX-229 and IRL-1620

[0155] Example 6: Preliminary study on acute toxicity of BX-229 polypeptide after subcutaneous administration

[0156] This example conducted a preliminary study on the acute toxicity of subcutaneous administration of the BX-229 polypeptide prepared in Example 1 and compared it with IRL-1620.

[0157] In the subcutaneous acute toxicity study, male SD rats weighing 200-230g were administered BX-229 or the control IRL-1620 subcutaneously at doses ranging from 60μg / kg to 960μg / kg and then 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 results are shown in Table 5. As can be seen from the results in the table, the acute toxicity of the BX-229 peptide following subcutaneous administration was significantly less than that of IRL-1620.

[0158] Table 5 Results of subcutaneous acute toxicity tests of BX-229 and IRL-1620

[0159] Example 7: Effect of intravenous administration of BX-229 polypeptide on blood pressure in SD rats

[0160] This example tests the effect of intravenous administration of the BX-229 polypeptide prepared in Example 1 on the blood pressure of SD rats and compares it with IRL-1620.

[0161] 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.

[0162] Turn on the biosignal data acquisition and analysis system and software. Connect one end of the data acquisition board to a plastic hose and the other end to a connector valve connected to a 1 mL syringe. Fill the syringe with heparinized saline. Inject the heparinized saline into the data acquisition board, expelling all air until the saline flows out of the other end of the plastic hose. Isolate the rat's right common carotid artery, tie a knot at the distal end and a slipknot at the proximal end. Use microscissors to cut a 1 / 2-inch incision in the artery between the knots. Insert the heparinized saline hose into the incision and secure it to prevent movement. Use the zeroing valve of the biosignal data acquisition and analysis system to reset the initial blood pressure to zero. Open the slipknot at the proximal end of the common carotid artery. Real-time blood pressure data will appear on the software. Wait for the animal's blood pressure to stabilize before administering the drug via the tail vein and recording blood pressure changes in real time. IRL-1620 and BX-229 were administered at a dose of 5 μg / kg and 6.6 μg / kg, respectively.

[0163] The results are shown in Table 6. As can be seen, 5 μg / Kg IRL620 and 6.6 μg / Kg BX-229 peptides had a certain effect on the blood pressure of rats, causing a decrease in blood pressure. After administration of 5 μg / Kg IRL620, the rats' 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. 6.6 μg / Kg BX-229 had a more gradual effect on the rats' blood pressure, with blood pressure decreasing slowly, reaching the lowest blood pressure within 20-35 minutes, with the maximum blood pressure reduction being 17.0% ± 2.0% of the baseline blood pressure.

[0164] Table 6 Effects of intravenous administration of BX-229 and IRL-1620 on blood pressure in SD rats

[0165] Example 8: Comparative Stability Experiment of Different Salt Forms of BX-229 Polypeptide

[0166] 1. Sample preparation:

[0167] Solution preparation: Weigh 1 mg of each raw material into a 1.5 mL centrifuge tube and dissolve it in 1 mL of normal saline; prepare 3 solutions for each raw material and place one tube each at 40°C, 25°C, and 5°C;

[0168] Powder: Weigh 1 mg of each raw material into a 1.5 mL centrifuge tube and prepare 6 solutions for each raw material;

[0169] Place 3 tubes at 40°C and 25°C respectively;

[0170] 2. Detection method: HPLC method

[0171] Column: Poroshell 120 SB-C18 2.7μm 4.6*100mm

[0172] Gradient: 10-100% B 0-10 min Flow rate: 2 ml / min

[0173] Column temperature: 35°C

[0174] Injection volume: 5 μL

[0175] Mobile phase A: 0.05% TFA-2% acetonitrile-98% ultrapure water

[0176] Mobile phase B: 0.05% TFA-10% ultrapure water-90% acetonitrile

[0177] 3. Test results:

[0178] The purity (%) of different salt samples after being placed under different conditions was determined by HPLC, as shown in Table 11:

[0179] Table 7 Purity of BX-229 polypeptide salt samples after storage under different conditions (%)

[0180] As can be seen, after one or two weeks at 40°C, the sodium and potassium salt powders were stable, while the ammonium salt powder was less stable. The sodium and potassium salt solutions were stable, while the ammonium salt solution was unstable. After four weeks at 40°C, the sodium and potassium salt powders were stable, while the ammonium salt powder was less stable. The sodium and potassium salt solutions were less stable, while the ammonium salt solution was very unstable. After one month at 25°C, the sodium and potassium salt powders were stable, while the ammonium salt powder was less stable. The sodium salt solution was stable.

[0181] Example 9: Efficacy of BX-229 sodium salt administered subcutaneously three times a week in the mouse tMCAO model

[0182] This example tests the efficacy of BX-229 sodium salt (BX-229-Na) prepared in Example 1 on the mouse tMCAO model.

[0183] 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, regional cerebral blood flow was monitored 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 tadalafil. The day of modeling was designated Day 1, the next day Day 2, and so on.

[0184] C57 tMCAO mice with successful modeling were randomly divided into three groups: a saline group, a 30 μg / kg BX-229-Na group, and a 100 μg / kg BX-229-Na group. Each group received a single subcutaneous injection of BX-229-Na into the neck 4.5 hours after embolization (D1), followed by additional injections at the same time point and site on D3 and D6. The experimental grouping and dosing schedule are shown in Table 8. Six mice were included in the sham group. BX-229-Na was administered subcutaneously into the neck at 2 mL / kg. Garcia JH scoring was performed 7 and 14 days after administration. After 14 days, the mice were euthanized, and brain tissue from the infarcted side was harvested for total protein extraction. ELISA was used to measure changes in VEGF-A and BDNF protein markers in each experimental group.

[0185] Table 8 Experimental groups and dosing regimens

[0186] The experimental results showed that the 7D Garcia JH-15 score showed significant differences between the sham group and the saline control group. The scores in the BX-229-30 μg / kg and BX-229-100 μg / kg groups were significantly better than those in the saline group, with a dose-dependent trend. The total Garcia JH-15 score in the BX-229-100 μg / kg group was 1 point higher than that in the saline group. The 14D Garcia JH-15 score showed significant differences between the sham group and the saline control group. The scores in the BX-229-30 μg / kg and BX-229-100 μg / kg groups were significantly better than those in the saline group, with a dose-dependent trend. The BX-229-100 μg / kg group had a significant difference in Garcia JH-15 score compared to the saline group (P < 0.05). The results are shown in Figures 5 and 6.

[0187] The results showed that the expression levels of VEGF-A and BDNF proteins in the BX-229-Na-30 μg / Kg and BX-229-Na-100 μg / Kg three-times-administered groups were significantly higher than those in the saline group. The VEGF-A protein expression level in the BX-229-Na-100 μg / Kg three-times-administered group was significantly higher than that in the sham-surgery group, and significantly higher than that in the saline group. The BDNF protein expression level in the BX-229-Na-100 μg / Kg three-times-administered group was also significantly higher than that in the sham-surgery group. For the results, please see Figures 7 and 8, and Tables 9 and 10.

[0188] Table 9 Changes in VEGF-A protein expression

[0189] Table 10 Changes in BDNF protein expression

[0190] Example 10: Efficacy of subcutaneous administration of BX-229 sodium salt and intravenous administration of IRL-1620 in the tMCAO mouse model

[0191] This example tested the efficacy of subcutaneous administration of the sodium salt of BX-229 (BX-229-Na), prepared in Example 1, in a mouse tMCAO model and compared it with intravenous administration of IRL-1620 (the administration route and dosage of IRL-1620 were derived from an animal equivalent method based on clinical dosing information). The tMCAO mouse modeling method is described in Example 9.

[0192] After surgery, the tMCAO mice with successful modeling were randomly divided into the model group and different treatment groups. The detailed experimental groups are as follows: (1) The 2.7 μg / Kg IRL-1620 administration group was administered three times a day on D1, D3, and D6 after modeling. On D1, it was intravenously administered 2 hours after modeling, and then administered once every 2 hours. The administration time on D3 and D6 was the same as D1; ​​(2) The 30 μg / Kg BX-229 administration group was administered for 7 consecutive days from D1 to D7. The administration time on D1 was once subcutaneously 4.5 hours after modeling, and the administration time on D2-D7 was the same as D1; ​​(3) Model (normal saline) group; and (4) Sham operation group (the sham operation group used the same method without ligating the blood vessels or inserting sutures). The model group and the sham operation group were given the same dose of vehicle normal saline.

[0193] Results showed that the neurobehavioral score on day 7 of the group receiving 30 μg / kg of BX-229-Na subcutaneously for seven consecutive days (D1-D7) was better than that of the group receiving 3 × 2.7 μg / kg of IRL-1620 intravenously on D1, D3, and D6. The mice showed significant improvements in various functionalities compared to the IRL-1620 group, and their neurobehavioral scores showed a significant recovery, as shown in Figure 9. This demonstrates that BX-229-Na, administered subcutaneously, offers lower dosing frequency, greater convenience, and improved patient compliance.

[0194] 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.

Claims

1. A pharmaceutically acceptable salt of a polypeptide, wherein the polypeptide comprises the amino acid sequence Fum-DEEAVYFAHK(-AEEA-γE-C12)DVIW (SEQ ID NO:1) or a functional variant thereof, and the pharmaceutically acceptable salt is selected from sodium salts, potassium salts, ammonium salts, trifluoroacetate salts, acetate salts, hydrochloride salts, sulfate salts, and phosphate salts.

2. The pharmaceutically acceptable salt of the polypeptide according to claim 1, wherein the pharmaceutically acceptable salt is selected from sodium salts, potassium salts, and ammonium salts, preferably a sodium salt.

3. The pharmaceutical composition according to claim 1 or 2, wherein the functional variant of the polypeptide is a substitution between D and E in SEQ ID NO:1, a substitution between V, L, and I, a substitution between Y, F, and W, a substitution between H, K, and R, or any combination thereof, and the functional variant of the polypeptide has the same or similar ETBR receptor activation activity as SEQ ID NO:

1.

4. A method for preparing a pharmaceutically acceptable salt of the polypeptide according to any one of claims 1 to 3, comprising the following steps: Obtaining a peptide resin of the polypeptide by solid-phase polypeptide synthesis; Cleaving and purifying to obtain the polypeptide; And Preparing a salt from the polypeptide.

5. A pharmaceutical composition comprising a pharmaceutically acceptable salt of the polypeptide according to any one of claims 1 - 3, and a pharmaceutically acceptable carrier, excipient, and / or diluent.

6. The pharmaceutical composition according to claim 5, wherein the pharmaceutical composition is in the form of a pre-lyophilized preparation, or a lyophilized preparation, or a reconstituted preparation obtained by combining a lyophilized preparation with an aqueous solution.

7. The pharmaceutical composition according to claim 6, wherein the pharmaceutical composition comprises sodium chloride; and / or optionally, the pharmaceutical composition comprises trehalose and / or cyclodextrin.

8. Use of a pharmaceutically acceptable salt of the polypeptide according to any one of claims 1 to 3, or the pharmaceutical composition according to any one of claims 5 to 7, in the preparation of a drug for treating a disease related to the ETBR receptor; Preferably, wherein the disease related to the ETBR receptor includes one or more of nervous system injury and related diseases caused by the injury, neurodegenerative diseases, anxiety, epilepsy, aortic stenosis, or neonatal hypoxic-ischemic encephalopathy; More preferably, the nervous system injury and related diseases caused by the injury include stroke, spinal cord injury, ischemic or traumatic injury of the brain or spinal cord, and injury of central nervous system (CNS) neurons; the neurodegenerative diseases include Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Parkinson's disease, or Huntington's disease; More preferably, the stroke includes ischemic stroke, hemorrhagic stroke, and hemorrhagic stroke converted from ischemic stroke.

9. A drug, characterized in that, It includes: A pharmaceutically acceptable salt of the polypeptide according to any one of claims 1 to 3, or the pharmaceutical composition according to any one of claims 5 to 7, Pharmaceutically acceptable excipients or adjuvants, and Optional other active ingredients.

10. A method for treating a disease associated with the ETBR receptor, characterized in that, It includes administering to a subject a pharmaceutically acceptable salt of the polypeptide according to any one of claims 1 to 3; the pharmaceutical composition according to any one of claims 5 to 7, or the medicament according to claim 9; Preferably, the diseases associated with the ETBR receptor include one or more of nervous system injury and related diseases caused by such injury, neurodegenerative diseases, anxiety, epilepsy, aortic stenosis or neonatal hypoxic-ischemic encephalopathy; More preferably, the nervous system injury and related diseases caused by such injury include stroke, spinal cord injury, ischemic or traumatic injury of the brain or spinal cord, and injury of central nervous system (CNS) neurons; More preferably, the neurodegenerative diseases include Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Parkinson's disease or Huntington's disease; More preferably, the stroke includes ischemic stroke, hemorrhagic stroke, and hemorrhagic stroke converted from ischemic stroke.