Polypeptide pharmaceutical composition for treating nervous system diseases and use thereof

Abstract

Provided in the present application are a polypeptide pharmaceutical composition for treating nervous system diseases and the use thereof, wherein by means of performing structural modification on a polypeptide, the half-life of the drug is extended and the side effects thereof are reduced, and the use comprises the use in the preparation of a drug for treating ischemic strokes.

Description

Peptide drug compositions for the treatment of nervous system diseases and their applications

[0001] This application claims priority and benefit to Chinese Patent Application No. CN 202411499112.6, filed on October 25, 2024, entitled “Polypeptide Pharmaceutical Composition for the Treatment of Nervous System Diseases and Its Application Thereof,” which is incorporated herein by reference in its entirety for all purposes. Technical Field

[0002] This application relates to the field of biotechnology. Specifically, this application provides polypeptide pharmaceutical compositions for treating nervous system diseases and their applications. Background Technology

[0003] Currently, the main treatments and medications for stroke include interventional vascular procedures, thrombolysis, antiplatelet therapy, anticoagulation, fibrinolysis, and neuroprotective agents. Interventional vascular procedures and thrombolysis are generally required to be performed in the very early stages after a stroke, and these procedures must be completed in hospitals with sufficient capabilities. Antiplatelet and anticoagulant drugs are generally used in conjunction with thrombolytic drugs in the later stages, with limited effectiveness and a certain risk of bleeding. While neuroprotective agents show good potential, they are generally used for acute-phase treatment. Currently, there are no marketed drugs specifically designed to promote rehabilitation in the subacute and recovery phases of stroke patients.

[0004] Previous studies have shown that endothelin receptor B agonists have great potential in promoting long-term recovery and rehabilitation in stroke patients. Endothelin (ET) is a 21-amino acid polypeptide that can be divided into three subtypes: ET-1, ET-2, and ET-3, which share structural homology with sarafotoxin b and c (S6b, S6c) from snake venom. Endothelin is currently the most potent and longest-lasting vasoactive peptide known, exhibiting strong vasoconstrictive effects and promoting smooth muscle cell migration and proliferation. It acts extensively on multiple systems in the body and is closely associated with diseases such as hypertension, congestive heart failure, diabetes, cancer, and fibrosis.

[0005] Endothelin exerts its biological effects primarily by binding to endothelin receptors (ETRs) on target cell membranes, playing a crucial role in the development and progression of numerous diseases, including cardiovascular and cerebrovascular diseases, kidney diseases, 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 their functions differ. ETA is considered a fundamental vasoconstrictor and growth-promoting receptor, while ETB inhibits cell growth and vasoconstriction in the vascular system and is known as a "clearance receptor" due to its involvement in the clearance of ET-1. Endothelin and its receptors are widely distributed in various tissues, mediating complex biological effects through different subtypes and signal transduction systems, and are closely related to a variety of diseases, with ETA and ETB receptors playing particularly important roles.

[0006] Studies have shown that endothelin B receptor (ETBR) agonists have demonstrated great potential in promoting neurovascular unit regeneration, improving blood and oxygen supply to the nervous system, and promoting long-term recovery and rehabilitation of stroke patients. Among the currently discovered ETBR agonist compounds, sovateltide (IRL-1620, Suc-DEEAVYFAHLDIIW) is the only ETBR agonist to have advanced to the clinical research stage. However, IRL-1620 has a very short half-life (less than 2 minutes) and very low exposure, requiring repeated dosing in preclinical and clinical studies, which significantly reduces its duration of efficacy and patient compliance.

[0007] For peptide drugs, their ability to withstand environmental stress is limited by storage conditions. Peptides may undergo pH changes and degradation to some extent during high-temperature and long-term storage, resulting in decreased purity, drastic changes in appearance, and short shelf life, which in turn affects efficacy. Furthermore, high demands are placed on transportation, which limits the large-scale commercial application of peptide drugs. Therefore, technological improvements are urgently needed. Summary of the Invention

[0008] In view of this, this application provides a novel endothelin receptor agonist polypeptide pharmaceutical composition and its application.

[0009] The peptide provided in this application is based on mechanistic studies of ETBR agonists and belongs to the category of highly selective ETBR agonists. Through structure-activity relationship studies and structural modifications, the peptide provided in this application prolongs the drug's half-life and in vivo exposure, reduces the frequency of dosing, and exhibits lower side effects in animal experiments compared to IRL-1620, the only ETBR agonist currently in clinical trials, thus providing a wider safety window. The peptide pharmaceutical composition provided in this application can be used to treat diseases related to the ETBR receptor, such as rehabilitation issues in the recovery period of ischemic stroke patients, promoting better recovery for stroke patients.

[0010] Specifically, this application provides the following technical solutions:

[0011] In a first aspect, this application provides a pharmaceutical composition comprising a polypeptide, a pharmaceutically acceptable salt thereof, a solvate, a conjugate or a non-covalent complex thereof, and a filler, wherein the polypeptide comprises the amino acid sequence Fum-DEEAVYFAHK(-AEEA-γE-C12)DVIW(SEQ ID NO:1) or a functional variant thereof.

[0012] In some embodiments, the pharmaceutical composition comprises a polypeptide, a pharmaceutically acceptable salt thereof, a solvate, a conjugate or a non-covalent complex thereof, a filler and a pH adjuster, wherein the polypeptide comprises the amino acid sequence Fum-DEEAVYFAHK(-AEEA-γE-C12)DVIW (SEQ ID NO:1) or a functional variant thereof.

[0013] In some implementations, the functional variant is a variant resulting from one or more conservative substitutions of SEQ ID NO:1.

[0014] 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, and substitutions between H, K and R.

[0015] In some embodiments, the functional variant has the same ETBR receptor activating activity as SEQ ID NO:1.

[0016] In some implementations, the polypeptide is a pharmaceutically acceptable salt of itself.

[0017] In some embodiments, the polypeptide is a pharmaceutically acceptable sodium, potassium, ammonium, trifluoroacetic acid, acetate, hydrochloride, sulfate, or phosphate salt.

[0018] In some embodiments, the polypeptide is a pharmaceutically acceptable sodium, potassium, or ammonium salt.

[0019] In some embodiments, the polypeptide is a pharmaceutically acceptable sodium salt of itself. In some embodiments, the pH adjuster is selected from glycine buffer, histidine buffer, arginine buffer, sodium succinate buffer, potassium succinate buffer, sodium citrate buffer, gluconate buffer, acetate buffer, phosphate buffer, disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, Tris buffer, or any combination thereof.

[0020] In some implementations, the pH adjuster is a phosphate buffer solution.

[0021] In some embodiments, the pH adjuster is a disodium hydrogen phosphate / sodium dihydrogen phosphate buffer. In some embodiments, the pH of the pharmaceutical composition is about 6 to 9.

[0022] In some embodiments, the pH of the pharmaceutical composition is about 7 to 8.

[0023] In some embodiments, the pH of the pharmaceutical composition is about 7.0.

[0024] In some embodiments, the filler is selected from trehalose, mannitol, glucose, lactose, cyclodextrin, hydroxypropyl-β-cyclodextrin, dextran-40, sorbitol, sucrose, glycine, or any combination thereof.

[0025] In some embodiments, the filler is selected from trehalose and hydroxypropyl-β-cyclodextrin or any combination thereof.

[0026] In some embodiments, the filler is trehalose.

[0027] In some embodiments, the mass ratio of the polypeptide or its pharmaceutically acceptable salt to the filler is about 1:200 to 1:0.1.

[0028] In some embodiments, the mass ratio of the polypeptide or its pharmaceutically acceptable salt to the filler is about 1:100 to 1:5.

[0029] In some embodiments, the mass ratio of the polypeptide or its pharmaceutically acceptable salt to the filler is about 1:50 to 1:12.5.

[0030] In some embodiments, the mass ratio of the polypeptide or its pharmaceutically acceptable salt to the filler is about 1:25.

[0031] In some embodiments, the pharmaceutical composition comprises a sodium salt of the amino acid sequence Fum-DEEAVYFAHK(-AEEA-γE-C12)DVIW (SEQ ID NO:1) or a functional variant thereof, trehalose, and disodium hydrogen phosphate-sodium dihydrogen phosphate buffer.

[0032] In some embodiments, the pH of the pharmaceutical composition is 7.0.

[0033] In some embodiments, the mass ratio of the sodium salt of the polypeptide to trehalose is about 1:50.

[0034] In some embodiments, the mass ratio of the sodium salt of the polypeptide to trehalose is about 1:25.

[0035] In some embodiments, the mass ratio of the sodium salt of the polypeptide to trehalose is about 1:12.5.

[0036] In some embodiments, the pharmaceutical composition further comprises a cryoprotectant and / or a surfactant.

[0037] In some embodiments, the cryoprotectant is polyethylene glycol and / or the surfactant is polysorbate, for example, polysorbate 20 or polysorbate 80.

[0038] In some embodiments, the pharmaceutical composition further comprises a deamidation inhibitor.

[0039] In some embodiments, the pharmaceutical composition is in the form of a pre-lyophilized formulation, or a lyophilized formulation, or a reconstituted formulation obtained by combining a lyophilized formulation with an aqueous solution.

[0040] In some embodiments, the pharmaceutical composition is used to treat, improve, or prevent diseases in mammals such as: nervous system injury and related diseases caused by such injury, neurodegenerative diseases, anxiety, epilepsy, aortic stenosis or hypoxic-ischemic encephalopathy of the newborn, or other diseases requiring improvement of neurovascular unit regeneration and improvement of blood and oxygen supply to the nervous system.

[0041] In a second aspect, this application provides a method for promoting the regeneration of an individual's neurovascular unit, improving blood and oxygen supply to the individual's nervous system, or treating, improving, or preventing damage to the individual's nervous system and related diseases caused by such damage, neurodegenerative diseases, anxiety, epilepsy, spinal cord injury, aortic stenosis, or hypoxic-ischemic encephalopathy of the newborn, or other diseases requiring improved neurovascular unit regeneration and improved blood and oxygen supply to the nervous system, said method comprising administering the pharmaceutical composition described in the first aspect to the individual.

[0042] Thirdly, this application provides the pharmaceutical composition described in the first aspect for use in treating, improving, or preventing the following diseases in an individual: nervous system injury and related diseases caused by such injury, neurodegenerative diseases, anxiety, epilepsy, aortic stenosis or hypoxic-ischemic encephalopathy in newborns, or other diseases requiring improvement in neurovascular unit regeneration and improvement in blood and oxygen supply to the nervous system.

[0043] Accordingly, this application also provides the use of the pharmaceutical composition described in the first aspect in the preparation of a medicament for treating, improving or preventing the following diseases in an individual: nervous system injury and diseases related to such injury, neurodegenerative diseases, anxiety, epilepsy, aortic stenosis or hypoxic-ischemic encephalopathy in newborns.

[0044] Fourthly, this application provides the use of the pharmaceutical composition described in the first aspect in a medicament for improving the regeneration of an individual's neurovascular unit or for improving blood and oxygen supply to the nervous system.

[0045] Accordingly, this application also provides the use of the pharmaceutical composition described in the first aspect in the preparation of a medicament for improving the regeneration of neurovascular units or the blood and oxygen supply to the nervous system in an individual.

[0046] Fifthly, this application provides the pharmaceutical composition described in the first aspect for use in individual ETBR receptor activation.

[0047] Accordingly, this application also provides the use of the pharmaceutical composition described in the first aspect in the preparation of a medicament for individual ETBR receptor activation.

[0048] In some embodiments of any of the foregoing aspects, the disease is stroke or neurological damage resulting from stroke, such as ischemic stroke, hemorrhagic stroke, or hemorrhagic stroke that develops from ischemic stroke.

[0049] In some embodiments of any of the foregoing aspects, neurological injury and related diseases resulting from such injury include stroke, spinal cord injury, ischemic or traumatic injury to the brain or spinal cord, and injury to neurons of the central nervous system (CNS).

[0050] In some implementations of any of the above aspects, neurodegenerative diseases include Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Parkinson's disease, or Huntington's disease.

[0051] In some embodiments of any of the foregoing aspects, the individual is a mammal, such as a non-primate or a primate, such as a human.

[0052] The inventors of this application have developed a peptide for treating central nervous system injuries. To better apply this peptide in practice, the inventors have conducted extensive research to provide a pharmaceutical composition comprising the peptide, a filler, and a pH buffer. Such a pharmaceutical composition has at least one of the following advantages:

[0053] 1. Provides an aesthetically pleasing white, loose, lyophilized block for pharmaceutical use. The filler will have beneficial effects in improving the collapse temperature of the (white, loose, lyophilized block), providing lyophilization protection, and enhancing the long-term storage stability of the protein.

[0054] 2. Improve peptide stability by providing an amorphous, glassy matrix and binding to proteins via hydrogen bonds, replacing water molecules removed during the drying process. This helps maintain the peptide's conformation, minimizing degradation during the freeze-drying cycle and improving the long-term stability of the product.

[0055] 3. It resists environmental stress and can maintain its product without degradation during a long storage period. Its appearance, purity and impurity content can meet the requirements of clinical applications. Attached Figure Description

[0056] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0057] Figure 1 shows the EC50 assay results of the peptide's activity against the human ETB receptor in Example 2.

[0058] Figure 2 shows the EC50 assay results of the peptide's activity against the canine ETB receptor in Example 2.

[0059] Figure 3 shows the EC50 assay results of the peptide's effect on rat ETB receptor activity in Example 2.

[0060] Figure 4 shows the EC50 assay results of the peptide's activity against mouse ETB receptor in Example 2.

[0061] Figure 5 shows the Garcia JH score results on day 7 after three-week administration in Example 8, where 1 is the saline group, 2 and 3 are the BX-229-Na subcutaneous administration groups of 30 μg / Kg and 100 μg / Kg, respectively, and 4 is the sham surgery group.

[0062] Figure 6 shows the Garcia JH score results on day 14 after three-week administration in Example 8, where 1 is the saline group, 2 and 3 are the BX-229-Na subcutaneous administration groups of 30 μg / Kg and 100 μg / Kg, respectively, and 4 is the sham surgery group.

[0063] Figure 7 shows the changes in VEGF-A protein expression after three-week administration in Example 8, where 1 is the saline group, 2 and 3 are the BX-229-Na subcutaneous administration groups of 30 μg / Kg and 100 μg / Kg, respectively, and 4 is the sham surgery group.

[0064] Figure 8 shows the changes in BDNF protein expression after three-week administration in Example 8, where 1 is the saline group, 2 and 3 are the BX-229-Na subcutaneous administration groups of 30 μg / Kg and 100 μg / Kg, respectively, and 4 is the sham surgery group.

[0065] Figure 9 shows the scores on days 8 and 15 after modeling in Example 9 for the BX-229 subcutaneous administration group and the IRL-1620 intravenous administration group (* indicates significant difference P<0.05), where 1 and 3 are the BX-229-Na subcutaneous administration groups of 30 μg / Kg, and 2 and 4 are the IRL-1620 intravenous administration groups of 3×2.7 μg / Kg. Detailed Implementation

[0066] This application discloses polypeptide pharmaceutical compositions and their applications. Those skilled in the art can appropriately modify the process parameters based on the disclosure of this application to achieve these compositions. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0067] Unless otherwise specified, the terms used in this application have the meanings commonly understood by those skilled in the art.

[0068] The use of single-letter or three-letter abbreviations for amino acids in this application follows international conventions.

[0069] In this specification and claims, the terms “comprising,” “including,” and “containing” mean “including but not limited to” and are not intended to exclude other parts, additives, components, or steps.

[0070] Statistically significant means p < 0.05, preferably < 0.01, and most preferably < 0.001.

[0071] In a first aspect, this application provides a pharmaceutical composition comprising a polypeptide or a pharmaceutically acceptable salt, solvate, conjugate or non-covalent complex thereof, as well as a pH adjuster and a filler, wherein the polypeptide comprises the amino acid sequence Fum-DEEAVYFAHK(-AEEA-γE-C12)DVIW (SEQ ID NO:1) or a functional variant thereof.

[0072] The term "functional variant" refers to a variant that has the same or similar biological functions and properties as the parent organism. As a non-limiting example, a "functional variant" can be obtained by performing one or more conserved substitutions in the parent organism.

[0073] In some implementations, the functional variant is a variant resulting from one or more conservative substitutions of SEQ ID NO:1.

[0074] 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, and substitutions between H, K and R.

[0075] In some embodiments, the functional variant has the same ETBR receptor activating activity as SEQ ID NO:1.

[0076] The term "solvate of polypeptide" refers to the form in which a polypeptide exists in a solvent. It usually refers to a compound formed by the combination of a polypeptide and solvent molecules, which retains the biological functions and properties of the polypeptide.

[0077] The term "peptide conjugate" refers to a compound formed by covalently linking a peptide with another type of compound, retaining the biological functions and properties of the peptide. For example, peptides can be linked with biomolecules such as carbohydrates, lipids, nucleic acids, and antibodies, as well as other substances such as nanoparticles, drugs, and molecular probes, to form peptide conjugates with various functions.

[0078] The term "non-covalent complex of a polypeptide" refers to a compound formed by a polypeptide and another type of compound through non-covalent bonding, which retains the biological functions and properties of the polypeptide.

[0079] 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, and substitutions between H, K and R.

[0080] In some embodiments, the functional variants disclosed herein also include amino acid sequences having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even higher identity with the aforementioned polypeptides. It is known in the art that “identity” between two proteins is determined by comparing the amino acid sequence of one protein with the sequence of a second protein whose conserved amino acid substitutions. The degree of identity between two proteins is determined using computer algorithms and methods known to those skilled in the art. The identity between two amino acid sequences is preferably determined using the BLASTP algorithm.

[0081] In some embodiments, the functional variants disclosed herein include peptides that differ from the specific peptides disclosed above by having substitutions, deletions, additions, and / or insertions of amino acid residues at positions 1, 2, 3, 4, 5, or more.

[0082] As described above, functional variants can be distinguished from the specific peptides disclosed herein by one or more substitutions, deletions, additions, and / or insertions. These variants can be naturally occurring or synthetically produced, for example, by modifying one or more of the peptide sequences disclosed herein and assessing their biological activity using any of a variety of techniques known in the art as described herein.

[0083] The active peptides of this application can be synthesized by solid-phase synthesis or recombination methods. The peptides can be synthesized using various schemes and methods described in scientific and patent literature, such as Organic Syntheses Collective Volumes, Gilman et al. (eds.), 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.

[0084] In some embodiments, the polypeptide is a pharmaceutically acceptable salt, preferably a sodium salt, potassium salt, ammonium salt, trifluoroacetate, acetate, hydrochloride, sulfate, or phosphate, more preferably a sodium salt, potassium salt, or ammonium salt, and most preferably a sodium salt.

[0085] This application also provides a method for preparing the polypeptide and its salt, comprising the following steps:

[0086] Step 1: Obtain the peptide resin of the peptide through solid-phase peptide synthesis;

[0087] Step 2: The polypeptide is obtained by lysis and purification.

[0088] Step 3: Salt preparation.

[0089] In some embodiments, the method for preparing the salt of the polypeptide (SEQ ID NO:1) provided in this application includes the following steps:

[0090] Step 1: Weigh 0.50 g (0.25 mmol) of Fmoc-Trp(Boc)-Wang Resin into a reactor, add 10 mL of DCM to swell for 10 min, filter, wash twice with DMF, add 25% 4-methylpiperidine / DMF (volume ratio) and react for 30 min to remove the Fmoc groups. Filter, wash the resin 4 times with DMF and 2 times with DCM, and test with ninhydrin; the resin solution is blue. Weigh 1 mmol (4 eq.) of Fmoc-Ile-OH and 1 mmol (4 eq.) of HBTU, dissolve in DMF, add 2 mmol (8 eq.) of DIEA, mix well, add to the resin, and react magnetically at room temperature (25℃±5℃) for 1 h. Filter, wash 3 times sequentially with DMF, MeOH, and DCM, and test with ninhydrin; the resin is colorless and transparent, the solution is pale yellow, indicating the reaction is complete. Add 25% 4-methylpiperidine / DMF (volume ratio) to remove the Fmoc groups. 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 sequentially. Subsequently, 2% hydrazine hydrate / DMF was added to the peptide resin and reacted three times for 10 min each time to remove the Dde from the Lys side chain. Then, AEEA, Glu-α-OtBu, and C12-OH were condensed sequentially.

[0091] Step 2: The peptide resin was dried under vacuum. 15 mL of pre-cooled lysis buffer (TFA:TIS:H2O = 95:2.5:2.5) was added. The mixture was stirred at room temperature (25℃±5℃) for 3 h. The reaction solution was then removed. The resin was washed twice with a small amount of TFA and removed. The reaction solutions were combined, and approximately 200 mL of pre-cooled methyl tert-butyl ether was added. A white precipitate formed. The precipitate was centrifuged (5000 rpm × 5 min), and the supernatant was discarded. Methyl tert-butyl ether was added back to the precipitate, vortexed, centrifuged, and 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% ammonia water, 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 used to obtain the pure target compound. The target compound components with a purity >95% were combined, and acetonitrile was removed under reduced pressure at approximately 40°C using a water pump and rotary evaporator. The resulting concentrate was frozen into a solid using liquid nitrogen and then freeze-dried for 48 hours to obtain the target compound BX-229. Mass spectrometry analysis confirmed that the molecular weight was correct ([M-2]2-=1137.3).

[0092] Step 3: Preparation of Sodium Polypeptide Salt (BX-229-Na)

[0093] The polypeptide solid obtained in step 2 was dissolved in a 0.1N sodium hydroxide aqueous solution (containing 10 equivalents of sodium hydroxide) and transferred to a preparative liquid phase using a salt transfer method. The preparative column was a C18-10-100, 30×250mm. The flow rate was 25mL / min. Phase A was water, and phase B was acetonitrile. After loading the sample, the column was rinsed with water for 15 min, followed by gradient elution to elute the product from the preparative column. Target compound fractions with a purity >95% were combined, and acetonitrile was removed under reduced pressure at approximately 40℃ using a water pump and rotary evaporator. The resulting concentrate was frozen into a solid using liquid nitrogen and then freeze-dried for 48 h to obtain the target compound.

[0094] In some embodiments, the purification uses a C18-10-100 column, 30×250 mm, with a flow rate of 25 mL / min; the preparative liquid phase includes phase A: 0.1% TFA / water and phase B: 0.1% TFA / 90% acetonitrile / water.

[0095] In some embodiments, the pH adjuster is selected from histidine buffer, arginine buffer, sodium succinate buffer, potassium succinate buffer, sodium citrate buffer, gluconate buffer, acetate buffer, phosphate buffer, Tris buffer, or any combination thereof, preferably phosphate buffer, and more preferably disodium hydrogen phosphate / sodium dihydrogen phosphate buffer.

[0096] In some embodiments, the pH of the pharmaceutical composition is about 6 to 9, preferably about 7 to 8, and more preferably about 7.0.

[0097] In some embodiments, the filler is selected from trehalose, mannitol, glucose, lactose, cyclodextrin, hydroxypropyl-β-cyclodextrin, dextran-40, sorbitol, sucrose, glycine, or any combination thereof.

[0098] In some embodiments, the filler is selected from any combination of trehalose and hydroxypropyl-β-cyclodextrin, more preferably trehalose.

[0099] In some embodiments, the mass ratio of the polypeptide or its pharmaceutically acceptable salt to trehalose is about 1:200 to 1:0.1, preferably about 1:100 to 1:5, more preferably about 1:50 to 1:12.5, and most preferably about 1:25.

[0100] In some embodiments, the polypeptide is a sodium salt, the filler is trehalose, and the pH adjuster is disodium hydrogen phosphate / sodium dihydrogen phosphate buffer.

[0101] In some embodiments, the pH value of the pharmaceutical composition is 7.

[0102] In some implementations, the mass ratio of the sodium polypeptide salt to trehalose is approximately 1:50.

[0103] In some implementations, the mass ratio of sodium polypeptide salt to trehalose is approximately 1:25.

[0104] In some implementations, the mass ratio of the sodium polypeptide salt to trehalose is approximately 1:12.5.

[0105] In some embodiments, the pharmaceutical composition further comprises a cryoprotectant and / or a surfactant, preferably polyethylene glycol and / or a polysorbate, preferably polysorbate 20 or polysorbate 80.

[0106] In some embodiments, the pharmaceutical composition further comprises a deamidation inhibitor.

[0107] In some embodiments, the pharmaceutical composition is in the form of a pre-lyophilized formulation, or a lyophilized formulation, or a reconstituted formulation obtained by combining a lyophilized formulation with an aqueous solution.

[0108] In some embodiments, the composition can be administered parenterally, intravenously, subcutaneously, intraarterially, intracranially, intrathecally, intraperitoneally, locally, intranasally, or intramuscularly. Intravenous administration is preferred.

[0109] In some embodiments, the pharmaceutical composition for parenteral administration is preferably sterile and substantially isotonic. For injection, the pharmaceutical composition comprising peptides may be formulated in an aqueous solution, preferably in a physiologically compatible buffer such as Hank's solution, Ringer's solution, or physiological saline or acetate buffer (to reduce discomfort at the injection site). The solution may contain formulations such as suspending agents, stabilizers, and / or dispersants.

[0110] In addition to the formulations previously described, pharmaceutical compositions comprising peptides can also be formulated as storage formulations. Such long-acting formulations can be administered by implantation (e.g., subcutaneous or intramuscular) or by intramuscular injection. Thus, peptides can be formulated, for example, with suitable polymeric or hydrophobic materials (e.g., emulsions formulated in acceptable oils) or ion exchange resins, or as slightly soluble derivatives, such as slightly soluble salts.

[0111] The peptide is used in an amount that is effective in achieving the intended purpose (e.g., reducing the damage effect of traumatic stroke and related conditions). A therapeutically effective amount is defined as the amount of peptide sufficient to significantly reduce stroke-related damage in a control population of patients (or animal models) treated with the peptide disclosed herein, relative to central nervous system damage in a control population of patients (or animal models) not treated with the peptide disclosed herein. The amount is also considered therapeutically effective if the treated individual achieves a better output (measured by infarct volume or disability index) compared to the mean output (measured by infarct volume or disability index) in a comparable control population not treated with the methods disclosed herein. The amount is also considered therapeutically effective if the treated individual shows a disability of 2 or less on the Rankin scale and 75 or more on the Barthel scale. The dose is also considered therapeutically effective if the treated patient population shows a significantly improved (i.e., less disability) score distribution on the disability scale 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 the frequency of administration required to achieve the desired therapeutic effect. Often, a single administration is sufficient.

[0112] In some implementations, the amount of peptide administered depends on the treated subject, the subject's weight, the severity of pain, the method of administration, and adjustments made by the prescribing physician. Treatment can be repeated, whether or not symptoms are detectable. Treatment can be provided alone or in combination with other medications.

[0113] In some embodiments, therapeutically effective doses of the disclosed peptides can provide therapeutic benefit without causing significant toxicity. The toxicity of the peptides can be determined in cell cultures or laboratory animals using standard pharmaceutical procedures, for example, by determining the LD50 (the dose that causes 50% of the population to be lethal) or LD100 (the 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. Peptides exhibiting a high therapeutic index are preferred (see, for example, Fingl et al., 1975, In: The Pharmacological Basis of Therapeutics, Chapter 1, page 1).

[0114] In some embodiments, because the polypeptides disclosed herein may contain charged side chains or terminals, they may be included in any of the above formulations as free acids or bases or as pharmaceutically acceptable salts. Pharmaceutically acceptable salts are salts prepared by reacting with inorganic acids, essentially retaining the biological activity of the free base. Pharmaceutical salts tend to be more soluble in water and other protic solvents than their corresponding free base forms.

[0115] In some embodiments, the pharmaceutical composition is used to treat, improve, or prevent diseases in mammals such as: nervous system injury and related diseases caused by such injury, neurodegenerative diseases, anxiety or epilepsy, spinal cord injury, aortic stenosis or hypoxic-ischemic encephalopathy of the newborn, or other diseases requiring improvement of neurovascular unit regeneration and improvement of blood and oxygen supply to the nervous system.

[0116] In a second aspect, this application provides a method for promoting the regeneration of neurovascular units, improving blood and oxygen supply to the nervous system, and treating, improving, or preventing nervous system damage in an individual and related diseases, neurodegenerative diseases, anxiety, epilepsy, aortic stenosis, or hypoxic-ischemic encephalopathy in newborns, the method comprising administering the pharmaceutical composition described in the first aspect to the individual.

[0117] Thirdly, this application provides the use of the pharmaceutical composition described in the first aspect in the preparation of a medicament or neurovascular unit regeneration for the treatment, improvement or prevention of the following diseases in an individual, and for improving blood and oxygen supply to the nervous system: nervous system injury and diseases related to such injury, neurodegenerative diseases, anxiety, epilepsy, aortic stenosis or hypoxic-ischemic encephalopathy in newborns.

[0118] In some implementations of any of the above aspects, the disease is stroke or neurological damage caused by stroke.

[0119] In some embodiments of any of the foregoing aspects, stroke includes ischemic stroke, hemorrhagic stroke, and hemorrhagic stroke transformed from ischemic stroke, preferably, the stroke is ischemic stroke.

[0120] In some embodiments of any of the foregoing aspects, there is damage to the nervous system and related diseases resulting from such damage, including stroke, spinal cord injury, ischemic or traumatic injury to the brain or spinal cord, and damage to neurons of the central nervous system (CNS).

[0121] In some implementations of any of the above aspects, neurodegenerative diseases include Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Parkinson's disease, or Huntington's disease.

[0122] In some embodiments of the second or third aspect, the individual is a mammal, such as a non-primate or a primate, such as a human.

[0123] Stroke is a condition caused by impaired blood flow in the CNS. Possible causes include embolism, hemorrhage, and thrombosis. The death of CNS tissue is called infarction. Infarct volume (i.e., the volume of neurons in the brain that die due to stroke) can be used as an indicator of the extent of pathological damage caused by stroke. Symptomatic effects depend on both infarct volume and location within the brain. Disability indices can be used as measures of symptomatic impairment, such as the Rankin Stroke Outcome Scale (Rankin, Scott MedJ; 2:200-15 (1957)) and the Barthel Index. The Rankin scale is based on the following direct assessment of a patient's global condition:

[0124] 0. No symptoms at all.

[0125] 1. Despite having symptoms, there is no significant disability; able to perform all daily work and activities.

[0126] 2. Mild disability; unable to perform all previous activities, but able to take care of their own affairs without assistance.

[0127] 3. Moderate disability requiring some assistance, but able to walk without assistance.

[0128] 4. Moderate to severe disability, unable to walk without assistance, and unable to take care of their own physical needs without assistance.

[0129] 5. Severe disability; bedridden, incontinent, and requiring ongoing care and attention.

[0130] The Barthel Index is based on a series of questions about a patient’s ability to perform 10 basic activities of daily living, with scores ranging from 0 to 100, and lower scores indicating more disability (Mahoney et al., Maryland State Medical Journal 14:56-61 (1965)).

[0131] Alternatively, stroke severity / output can be measured using the NIH Stroke Scale, which is available at ninds.nih.gov / doctors / NIH_Stroke_Scale_Booklet.pdf. This scale is based on a patient's ability to perform 11 functional groups, including assessments of consciousness, motor, sensory, and language function.

[0132] Ischemic stroke is more specifically defined as a type of stroke caused by obstruction of blood flow to the brain. The most common underlying condition for this type of obstruction is the formation of fatty deposits along the vessel walls, a condition known as atherosclerosis. These fatty deposits can cause two types of obstruction. Cerebral thrombosis refers to the formation of a blood clot (thrombus) at the site of an obstruction in a blood vessel. "Cerebral embolism" usually refers to various emboli (such as mural thrombi in the heart, atherosclerotic plaques, fat, tumor cells, fibrocartilage, or air) entering the cerebral arteries and obstructing the vessels. When collateral circulation cannot compensate, it causes ischemic necrosis of brain tissue in the area supplied by that artery, resulting in focal neurological deficits. A second important cause of embolism is irregular heartbeats, called arterial myofascitis. This causes conditions in which blood clots can form in the heart, move, and migrate to the brain. Other potential causes of ischemic stroke include hemorrhage, thrombosis, severance of an artery or vein, cardiac arrest, shock from any cause (including hemorrhage), and iatrogenic causes such as direct surgical damage to cerebral blood vessels or vessels leading to the brain, or cardiac surgery. Ischemic stroke accounts for approximately 83% of all stroke cases.

[0133] The term "individual" as used in this application 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, cattle, horses, pigs, sheep, goats, dogs, cats, and rodents such as rats and mice.

[0134] It should be understood that the above detailed description is only intended to provide a clearer understanding of the contents of this application to those skilled in the art, and is not intended to limit in any way. Those skilled in the art can make various modifications and variations to the described embodiments.

[0135] In this application, the Chinese explanations of abbreviations or full English names are as follows:

[0136] The present invention will be further illustrated below with reference to the embodiments.

[0137] Example

[0138] The following embodiments are used to illustrate this application, but are not intended to limit the scope of this application. Any modifications or substitutions made to the methods, steps, or conditions of this application without departing from the spirit and substance of this application shall fall within the scope of this application.

[0139] Unless otherwise specified, all reagents used in the examples are commercially available and all technical means used in the examples are conventional means well known to those skilled in the art.

[0140] Unless otherwise specified, the main reagents and solvents used in the following examples are commercially available, for example, from Gir Biochemical (Shanghai) Co., Ltd. or Sangon Biotech (Shanghai) Co., Ltd. The control IRL-1620 was synthesized according to the sequence and synthetic method reported in the literature (Michihiro Takai, et al., BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, Vol. 184, No. 2, 1992, Pages 953-959).

[0141] Example 1: Preparation of BX-229 and its sodium salt

[0142] In this embodiment, BX-229 (SEQ ID NO.:1, Fum-DEEAVYFAHK(-AEEA-γE-C12)DVIW) and its sodium salt (BX-229-Na) were prepared and synthesized according to the following method.

[0143] 1) Synthesis of BX-229

[0144] Weigh 0.50 g (0.25 mmol) of Fmoc-Trp(Boc)-Wang Resin into a reactor, add 10 mL of DCM to swell for 10 min, filter, wash twice with DMF, add 25% 4-methylpiperidine / DMF (volume ratio) and react for 30 min to remove the Fmoc groups. Filter, wash the resin four times with DMF and twice with DCM, and test with ninhydrin; the resin solution is blue. Weigh 1 mmol (4 eq.) of Fmoc-Ile-OH and 1 mmol (4 eq.) of HBTU, dissolve in DMF, add 2 mmol (8 eq.) of DIEA, mix well, add to the resin, and react magnetically at room temperature (25℃±5℃) for 1 h. Filter, wash three times sequentially with DMF, MeOH, and DCM, and test with ninhydrin; the resin is colorless and transparent, the solution is pale yellow, indicating complete reaction. Add 25% 4-methylpiperidine / DMF (volume ratio) to remove the Fmoc groups. 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 sequentially. Subsequently, 2% hydrazine hydrate / DMF was added to the peptide resin and reacted three times for 10 min each time to remove the Dde from the Lys side chain. Then, AEEA, Glu-α-OtBu, and C12-OH were condensed sequentially. The peptide resin was dried under vacuum, and 15 mL of pre-cooled lysis buffer (TFA:TIS:H2O = 95:2.5:2.5) was added. The reaction was stirred at room temperature (25℃±5℃) for 3 h. The reaction solution was then removed, and the resin was washed twice with a small amount of TFA and removed. Combine the reaction solutions, add approximately 200 mL of pre-cooled methyl tert-butyl ether, and a white precipitate forms. Centrifuge (5000 rpm × 5 min), discard the supernatant, add methyl tert-butyl ether back to the precipitate, vortex, centrifuge, and discard the supernatant. Dry the precipitate in a vacuum desiccator for 12 h to obtain the crude product. Dissolve the crude peptide in approximately 1% ammonia water, filter, and purify the filtrate 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 is used to obtain the pure target compound. Combine the target compound fractions with a purity >95%, and evaporate the acetonitrile under reduced pressure at approximately 40 °C using a water pump and rotary evaporator. Freeze the resulting concentrate in liquid nitrogen to form a solid, and then freeze-dry it for 48 h to obtain the target compound BX-229. Mass spectrometry analysis confirmed the molecular weight was correct ([M-2]). 2- =1137.3).

[0145] 2) Preparation of sodium salt of BX-229

[0146] The obtained BX-229 solid was dissolved in a 0.1N aqueous solution of sodium hydroxide (containing 10 equivalents of sodium hydroxide) and transferred to a preparative liquid phase. The preparative column was a C18-10-100, 30×250 mm. The flow rate was 25 mL / min. Phase A was water, and phase B was acetonitrile. After loading the sample, the column was rinsed with water for 15 min, followed by gradient elution to elute the product from the preparative column. Target compound fractions with a purity >95% were combined, and acetonitrile was removed under reduced pressure at approximately 40°C using a water pump and rotary evaporator. The resulting concentrate was frozen into a solid using liquid nitrogen and then freeze-dried for 48 h to obtain the sodium salt of the target compound BX-229 (BX-229-Na).

[0147] Example 2: EC50 determination of BX-229-Na activity against ETB receptors in different species

[0148] This embodiment tested the agonistic activity of BX-229-Na prepared in Example 1 on ETB receptors of different species and compared it with IRL-1620.

[0149] 293T-17 cells were seeded at 650,000 per well in 6-well plates and cultured overnight at 37°C in a 5% CO2 incubator. Then, they were transfected with the expression vector PCDNA3.1-hETB (human hETB sequence, CBI accession number: NM_000115.5) containing the human ETB receptor gene. 2.5 μg of plasmid was transfected into each well and cultured for another 48 h. After observing the expression of green fluorescence under a fluorescence microscope, the cells were digested with trypsin and counted. The activity of BX-229-Na against the human ETB receptor was determined using the Cisbio Bioassays IP-One Gq kit. Cells were suspended in the kit's Stim Buffer and added to a 96-well plate, with 7 μL of cell suspension (containing 30,000 cells) added to each well. The peptides BX-229-Na and IRL-1620 prepared in Example 1 were diluted to 11 concentration gradients using Stim B, and each concentration gradient was dissolved to a 2× concentration using Stim B. 7 μL of each gradient was added to the cells in the test wells. 7 μL of PBS solution was added to the negative control wells. The plates were sealed and incubated at 37°C for 1 h. 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 (human endothelin ET1 (ET1, SEQ ID NO.:2, CSCSSLMDKECVYFCHLDIIW(C1-C15,C3-C11)), and PBS control wells. Seal the plate and incubate at room temperature (25℃±5℃) for 1 hour, protected from light. Remove the seal and measure the absorbance at 665nm and 620nm using an ID5 microplate reader. Calculate the ratio of acceptor to donor emission signals for each well, i.e., (665nm signal / 620nm signal) × 10⁻⁶.4 Numerical values. The average value of the 665nm / 620nm signal measured at different concentrations of the test sample is F. First, the activation rate of each concentration of the test peptide relative to the positive control sample human endothelin ET1 is calculated. With this activation rate as the ordinate and the logarithm of the peptide concentration as the abscissa, a dot plot is drawn. The curve is fitted using the four-parameter fitting method in Prism software, and the EC50 value of the peptide on the human ETB receptor is calculated.

[0150] The EC50 assay for ETB receptor activity in dogs, rats, and mice was performed in the same manner as above, except that the plasmids were transfected as follows: expression vector PCDNA3.1-DETB containing the canine ETB receptor gene (canine ETB sequence, NCBI accession number: NM_001010943.2), expression vector PCDNA3.1-RETB containing the rat ETB receptor gene (rat ETB receptor sequence, NCBI accession number: X57764.1), and expression vector PCDNA3.1-mETB containing the mouse ETB receptor gene (mouse ETB sequence, NCBI accession number: NM_001276296).

[0151] To obtain more reliable data, comparative tests were conducted in the same experiment and repeated multiple times. The three data points with the best curve fit were selected and averaged. The mean value was used as the final cell activity result, as shown in Table 1 and Figures 1 to 4.

[0152] Table 1. Agonistaltic activity of BX-229-Na and IRL-1620 on ETB receptors (EC50, nM)

[0153] Example 3: Activity of BX-229-Na on human ETA receptors

[0154] This embodiment tested the activity of BX-229-Na prepared in Example 1 against the human ETA receptor.

[0155] 293T-17 cells were seeded at 650,000 per well in 6-well plates and cultured overnight at 37°C in a 5% CO2 incubator. Then, they were transfected with the expression vector PCDNA3.1-hETA (hETA sequence, NCBI accession number: L06622.1) containing the human ETA receptor gene, with 2.5 μg of plasmid per well. After culturing for another 48 h, the cells were observed to express green fluorescence under a fluorescence microscope, and then digested with trypsin and counted. The activity of BX-229-Na against human ETA receptor was determined using the Cisbio Bioassays IP-One Gq kit. Cells were suspended in the kit's Stim Buffer and added to a 96-well plate, with 7 μL of cell suspension (containing 30,000 cells) 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 dissolved in Stim B to a 2× concentration and added to 7 μL of each solution in the test cells. For the negative control wells, 7 μL of PBS solution was added to the cells. The plate was sealed and incubated at 37°C for 1 h. 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 plate was sealed and incubated at room temperature (25°C ± 5°C) for 1 h in the dark. The plate was then removed from the seal, and the absorbance was measured at 665 nm and 620 nm using an ID5 microplate reader. Calculate the ratio of the signal emitted by the acceptor to the signal emitted by the donor in each pore, i.e., (665nm signal / 620nm signal) × 10. 4 Numerical values. The average 665nm / 620nm signal ratio of the test sample at a specific concentration was F3; the average 665nm / 620nm signal ratio of the positive control ET1 at the same concentration was F5; and the average 665nm / 620nm signal ratio of the PBS negative control was T3. The percentage of ET1 activity of the test peptide sample relative to the positive control sample was calculated as (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.

[0156] Table 2. Activity of BX-229-Na on human ETA receptors

[0157] Example 4: Determination of the half-life of BX-229 peptide

[0158] This embodiment tested the half-life of the polypeptide BX-229 prepared in Example 1 and compared it with the control IRL-1620.

[0159] Plasma sample administration and processing: Three C57 BL / 6 mice were injected with BX-229 (60 μg / mL) and the control IRL-1620 (200 μL per mouse). Blood samples were collected at 1 min, 5 min, 15 min, and 30 min after injection. Immediately after blood collection, a protease inhibitor was added, and the samples were centrifuged at 3200 rpm for 10 min at 4 degrees Celsius. After collecting the plasma, twice the volume of acetonitrile was added to precipitate the protein, followed by centrifugation at 10000 rpm for 5 min. The plasma was then filtered through a 0.22 μm filter and analyzed using liquid chromatography-mass spectrometry (LC-MS). The blood drug concentration was calculated based on the integral area of ​​the control and test drugs, and the average value of the three detection values ​​at each time point for each drug was calculated. Blank mouse plasma samples after protein precipitation were also prepared as a control. The results are shown in Table 3.

[0160] Table 3 shows the half-life data for BX-229 and IRL-1620.

[0161] LC-MS detection conditions:

[0162] Instrument Model: Agilent InfinityLab LC / MSD 1260-G6125C

[0163] Column: Poroshell 120 SB-C18 4.6*100mm, 2.7μm

[0164] Mobile phase A: 0.1% formic acid-ultrapure water; Mobile phase B: 0.1% formic acid-acetonitrile.

[0165] Gradient settings:

[0166] Column temperature: 45℃; Injection volume: 20μl;

[0167] Ion source: ESI; Dryer temperature: 350℃; Dryer flow rate: 12L / min; Atomization pressure: 45psi; Capillary voltage: 4000V.

[0168] Scan type: SIM; Fragmentation voltage: 135V; Scan / dwell time: 200ms.

[0169] Example 5: Preliminary study of acute toxicity of BX-229 polypeptide administered intravenously

[0170] This embodiment presents a preliminary study on the acute toxicity of the BX-229 polypeptide prepared in Example 1 after intravenous administration, and compares it with IRL-1620.

[0171] In the acute toxicity experiment after intravenous administration, male SD rats weighing 200-230g were administered BX-229 and the control IRL-1620 at doses ranging from 120μg / kg to 30μg / kg via tail vein injection, from high to low doses. Rats were observed for 24 hours after administration. If any dose resulted in death, the dose was reduced and observation continued. Preliminary observations of toxic reactions and mortality were made, and the time to death and the dose at which no death occurred were recorded. The acute toxicity of the peptide was preliminarily determined based on the dose at which no death occurred. The results are shown in Table 4. The acute toxicity study showed that the maximum tolerated doses of IRL-1620 and BX-229 in rats were 30μg / kg and 60μg / kg, respectively. The maximum tolerated dose of BX-229 peptide injected into SD rats was significantly better than that of IRL-1620.

[0172] Table 4. Results of acute toxicity tests of intravenous administration of BX-229 and IRL-1620

[0173] Example 6: Preliminary study of acute toxicity of BX-229 polypeptide administered subcutaneously

[0174] This embodiment presents a preliminary study on the acute toxicity of the BX-229 polypeptide prepared in Example 1 after subcutaneous administration, and compares it with that of IRL-1620.

[0175] In the acute toxicity experiment using subcutaneous administration, male SD rats weighing 200-230g were administered BX-229 and the control IRL-1620 subcutaneously at doses ranging from 60 μg / kg to 960 μg / kg, and observed for 48 hours afterward. The toxic reactions and mortality of the rats were preliminarily observed, and the time to death and the dose at which no death occurred were recorded. The acute toxicity of the peptide was preliminarily determined based on the dose at which no death occurred. The experimental results are shown in Table 5. The results in the table show that the acute toxicity of BX-229 peptide administered subcutaneously is significantly lower than that of IRL-1620.

[0176] Table 5. Results of acute toxicity tests of subcutaneous administration of BX-229 and IRL-1620.

[0177] Example 7: Effect of intravenous administration of BX-229 peptide on blood pressure in SD rats

[0178] This example tested the effect of intravenous administration of the BX-229 polypeptide prepared in Example 1 on the blood pressure of SD rats and compared it with IRL-1620.

[0179] After anesthetizing male SD rats with isoflurane, the neck was shaved and an opening was made along the midline of the neck to expose the trachea. The small animal ventilator and anesthesia machine were turned on, and the gas concentration of the anesthesia machine was set to 1.2. The outlet channel was connected to the air inlet of the ventilator. The tidal volume of the ventilator was set to 2 mL, the respiratory rate was 90 breaths / min, and the respiratory ratio was 1:2. A plastic tubing was connected to the air outlet of the ventilator and inserted into the rat's airway.

[0180] Turn on the biosignal data acquisition and analysis system and software. Connect one end of the data acquisition board to a plastic tubing and the other end to a connector valve connected to a 1mL syringe. Fill the syringe with heparinized saline solution. Inject the heparinized saline solution into the data acquisition board, expelling all air from the space until the heparinized saline solution flows out from the other end of the plastic tubing. Isolate the rat's right common carotid artery. Tie a knotted knot at the distal end and a slipknot at the proximal end. Use microscissors to cut a 1 / 2-inch opening in the artery at the knot. Insert the plastic tubing filled with heparinized saline solution into the opening and fix it in place, preventing it from moving freely. Use the zeroing valve of the biosignal data acquisition and analysis system to zero the initial blood pressure value. Open the slipknot at the proximal end of the common carotid artery. The software will display the animal's real-time blood pressure data. After the animal's blood pressure stabilizes, administer the drug via the tail vein and observe and record the changes in blood pressure in real time. The dosage of IRL-1620 is 5μg / Kg, and the dosage of BX-229 is 6.6μg / Kg.

[0181] The results are shown in Table 6. It can be seen that 5 μg / Kg IRL620 and 6.6 μg / Kg BX-229 peptides have a certain effect on rat blood pressure, causing a decrease in blood pressure. After administration of 5 μg / Kg IRL620, rat blood pressure showed a rapid and immediate change, reaching its lowest point within 2 minutes, with a maximum decrease of 16.1% ± 2.4% of baseline blood pressure. The effect of 6.6 μg / Kg BX-229 on rat blood pressure was more gradual, with a slow decrease reaching its lowest point within 20-35 minutes, and a maximum decrease of 17.0% ± 2.0% of baseline blood pressure.

[0182] Table 6. Effects of intravenous administration of BX-229 and IRL-1620 on blood pressure in SD rats.

[0183] Example 8: Efficacy of BX-229-Na administered subcutaneously three times a week in a mouse tMCAO model

[0184] This embodiment tested the efficacy of BX-229-Na prepared in Example 1 on a mouse tMCAO model.

[0185] Fifty-one male C57 / 6J mice, 10-12 weeks old and weighing 25-30g, were used. Prior to the experiment, they were housed for at least 2 days to acclimatize (Spefer). A tMCAO mouse model was established, and mice were anesthetized with isoflurane. Body temperature was controlled using a thermostat and maintained at 37±0.5℃. A midline incision was made in the skin of the mouse skull, and the skin was pulled laterally to fix a flexible microneedle tip to the surface of the left parietal bone. The right common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA) were separated through a midline incision in the neck. Following Longa's method (Longa et al., 1989), a silicone-coated suture plug (Reward, 0.22mm-0.23mm diameter, MSMC23B120PK50) was introduced via external carotid artery transection and slowly advanced through the internal carotid artery towards the origin of the middle cerebral artery. To ensure sustained and successful occlusion of the middle cerebral artery, local cerebral blood flow was monitored in all stroke animals using a laser speckle flowmeter (blood flow typically decreases by 40-60% after stabilization). The thrombus was removed 45 minutes after occlusion, the incision was sutured, and 1 mL of 37°C warm saline was injected subcutaneously. Mice were placed on a heating pad until recovery. To alleviate pain, mice were injected subcutaneously with pain reliever after anesthesia recovery. The day of occlusion was defined as D1, the second day as D2, and so on.

[0186] C57 tMCAO mice that successfully modeled the disease were randomly divided into three groups: a saline group, a 30 μg / kg BX-229-Na administration group, and a 100 μg / kg BX-229-Na administration group. Each group received the drug subcutaneously in the neck once 4.5 h after embolization (D1), followed by two more administrations at the same time points on D3 and D6. The grouping and administration regimens for this experiment are shown in Table 7. Six mice were in the sham-operated group. The drug was administered subcutaneously in the neck at a dose of 2 mL / kg. Garcia JH scores were calculated at 7 and 14 days after administration. After the 14-day scoring, the mice were euthanized, and brain tissue from the infarcted side was collected. Total protein was extracted, and changes in VEGF-A and BDNF protein markers in each experimental group were detected using ELISA.

[0187] Table 7 Experimental Groups and Dosing Regimens

[0188] The experimental results showed that, based on the 7-day Garcia JH-15 score analysis, there was a significant difference in the 7-day scores between the sham surgery group and the saline-based control group. The scores of the BX-229-30μg / Kg and BX-229-100μg / Kg administration groups were better than those of the saline group, showing a dose-dependent trend. The total Garcia JH-15 score of the BX-229-100μg / Kg administration group was 1 point higher than that of the saline group. Based on the 14-day Garcia JH-15 score analysis, there was a significant difference in the 14-day scores between the sham surgery group and the saline-based control group. The scores of the BX-229-30μg / Kg and BX-229-100μg / Kg administration groups were better than those of the saline group, showing a dose-dependent relationship. The Garcia JH-15 score of the BX-229-100μg / Kg administration group was significantly different from that of the saline group (P<0.05). The results are shown in Figures 5 and 6.

[0189] The results showed that the expression levels of VEGF-A and BDNF proteins were significantly increased in the BX-229-Na-30 μg / Kg and BX-229-Na-100 μg / Kg three-dose groups compared to the saline group. The expression level of VEGF-A protein in the BX-229-Na-100 μg / Kg three-dose groups was also significantly higher than that in the sham-operated group. The expression level of BDNF protein in the BX-229-Na-100 μg / Kg three-dose groups was also significantly higher than that in the sham-operated group. See Figures 7 and 8, and Tables 8 and 9 for the results.

[0190] Table 8. Changes in VEGF-A protein expression levels

[0191] Table 9. Changes in BDNF protein expression levels

[0192] Example 9: Efficacy of subcutaneous administration of BX-229-Na and intravenous administration of IRL-1620 in a tMCAO mouse model

[0193] This embodiment tested the efficacy of BX-229-Na subcutaneously administered in a mouse tMCAO model, as prepared in Example 1, and compared it with intravenous administration of IRL-1620 (the administration method and dosage of IRL-1620 were derived from an animal equivalence method based on clinical dosing information). The tMCAO mouse modeling method is as described in Example 8.

[0194] After surgery, the successfully modeled tMCAO mice were randomly divided into a model group and different treatment groups. The detailed groupings are as follows: (1) The 2.7 μg / Kg IRL-1620 administration group was administered three times a day on days D1, D3, and D6 after modeling. On day D1, the medication was administered intravenously 2 hours after modeling, and then every 2 hours thereafter. The administration times on days D3 and D6 were the same as on day D1; (2) The 30 μg / Kg BX-229 administration group was administered for 7 consecutive days from day D1 to day D7. On day D1, the medication was administered subcutaneously 4.5 hours after modeling. The administration times on days D2 to D7 were the same as on day D1; (3) The model (saline) group; and (4) The sham operation group (the sham operation group used the same method without ligating blood vessels or inserting sutures). The model group and the sham operation group were given the same dose of saline solution.

[0195] The results showed that the neurological behavioral score on day 7 of the 30 μg / Kg BX-229-Na subcutaneous administration group (days 1-7) was better than that of the 3×2.7 μg / Kg IRL-1620 intravenous administration group (days 1, 3, and 6). Mice showed significant improvement in various functions compared to the IRL-1620 group, with a greater recovery in neurological behavioral scores (see Figure 9). Therefore, BX-229-Na, administered subcutaneously, is more convenient due to its lower frequency of administration and better patient compliance.

[0196] Example 10: Excipient Screening Experiment

[0197] This embodiment describes the process of converting the BX-229-Na prepared in Example 1 into a lyophilized powder injection that is easy to store and transport. Different excipients were selected for experiments, and excipients with good shape and stability of the lyophilized powder injection were screened out.

[0198] The specific screening experimental prescriptions are shown in Table 10.

[0199] Table 10 Screening Excipient Formulations

[0200] The specific preparation method is as follows:

[0201] Weigh the excipients, add 80% water for injection, and stir to dissolve;

[0202] Add the ingredients and stir until dissolved;

[0203] Add water for injection to the total volume and stir well;

[0204] Filtered using a 0.22μm filter membrane;

[0205] Fill vials with 0.5 ml of solution per vial, and stopper halfway.

[0206] Place in a freeze dryer for vacuum freeze drying: pre-freeze at -40℃ for 4 hours, maintain at -20℃ for 10 hours, maintain at -10℃ for 10 hours, maintain at 0℃ for 10 hours, and maintain at 30℃ for 4 hours.

[0207] The detection methods for related substances are as follows, and the results are shown in Table 11.

[0208] Chromatographic conditions for the determination of related substances in BX-229:

[0209] Column: Kromasil 100-3.5-C8 4.6×250mm

[0210] Potassium dihydrogen phosphate buffer: 100 mmol / L potassium dihydrogen phosphate solution (adjust pH to 4.6 ± 0.1 with 1 mol / L sodium hydroxide or dilute phosphoric acid; if the initial pH is 4.6 ± 0.1, pH adjustment is not necessary).

[0211] Mobile phase A: Potassium dihydrogen phosphate buffer-acetonitrile (90:10)

[0212] Mobile phase B: Potassium dihydrogen phosphate buffer-acetonitrile (40:60)

[0213] Flow rate: 1.0 ml / min; Detection wavelength: 210 nm

[0214] Gradient settings:

[0215] Table 11 Results of detection of related substances in screened excipients

[0216] It is evident that trehalose exhibits the best stability as an excipient when stored at 40℃ for 4 weeks; and hydroxypropyl-β-cyclodextrin shows the best stability when stored at 5℃ for 2 months. Mannitol, dextran, and sodium chloride, when used as excipients, show significantly worse stability than trehalose and hydroxypropyl-β-cyclodextrin.

[0217] Example 11: pH control range screening experiment

[0218] In this embodiment, BX-229-Na prepared in Example 1 was converted into a lyophilized powder injection that is easy to store and transport. Different amounts of pH buffer solution containing disodium hydrogen phosphate and citric acid (pH values ​​of 5.0, 6.0, 7.0, 8.0, and 9.0, respectively) were used to screen out the pH value that provides the best stability for BX-229 lyophilized powder injection.

[0219] The specific screening experimental prescriptions are shown in Table 12.

[0220] Table 12 Screening pH Value Prescriptions

[0221] The preparation method is the same as in Example 10. The results of related substance detection are shown in Table 13.

[0222] Table 13 Results of pH-related substance screening.

[0223] It is evident that the BX-229 lyophilized powder injection exhibits the best stability at a pH of around 7.0.

[0224] Example 12: pH Buffer Screening Experiment

[0225] In this embodiment, the BX-229-Na prepared in Example 1 is converted into a lyophilized powder injection that is easy to store and transport. Based on the results of the pH range screening experiment, different pH buffers with a pH value of around 7.0 are selected for comparative experiments to screen out the pH buffer with the best stability for the lyophilized powder injection.

[0226] The specific screening experimental prescriptions are shown in Table 14.

[0227] Table 14 Screening Buffer Prescriptions

[0228] The preparation method is the same as in Example 10. The results of related substance detection are shown in Table 15.

[0229] Table 15 Results of detection of related substances for screening buffers

[0230] It is evident that disodium hydrogen phosphate-sodium dihydrogen phosphate, when used as a pH buffer, provides significantly better formulation stability than citrate, glycine, and proline. However, the total impurities in the lyophilized powder injection at 40°C for 4 weeks exceed 2.0%, requiring further optimization.

[0231] Example 13: Comparative Experiment of Trehalose and Hydroxypropyl-β-Cyclodextrin as Excipients

[0232] Based on the results of the pH buffer screening experiment, we tried to conduct an experiment without adding a pH buffer, using only trehalose and / or hydroxypropyl-β-cyclodextrin as excipients, to determine whether the results of the related substances test of the lyophilized powder injection after being placed at 40°C for 4 weeks met the requirement of ≤2.0%.

[0233] The specific experimental formulation is shown in Table 16, and the formulation specification is 1 mg / bottle.

[0234] Table 16 Experimental Formulation Prescriptions

[0235] The preparation method is the same as in Example 10, but the rubber stopper is pressed under vacuum, and after being removed, an aluminum-plastic composite cap is rolled.

[0236] Take three vials of lyophilized powder for injection and visually inspect their properties. They should be white, loose, lyophilized lumps or powder, plump and not shrunken. Dissolve each vial in 2 ml of pure water and observe the clarity and color of the solution under a clarity tester. The solution should be colorless and clear. Take a solution from the clarity and color section and test its pH value on a pH meter. It should be between 7.0 and 8.0. Take a solution from the clarity and color section and test for related substances using a high-performance liquid chromatograph. The total impurities should be ≤2.0%. See Table 17 for specific results.

[0237] The specific results are shown in Tables 17 to 20.

[0238] Table 17 Results of Physical Properties Inspection of Lyophilized Powder Injection

[0239] Table 18 Results of Clarity and Color Inspection of Lyophilized Powder Injection Solutions

[0240] Table 19: pH Value Test Results of Lyophilized Powder Injection

[0241] Table 20 Results of Related Substances Tests for Lyophilized Powder Injections

[0242] It is evident that without excipients, the BX-229 lyophilized powder injection does not form a full shape and will shrink significantly after long-term storage. At room temperature (25℃), using trehalose, hydroxypropyl-β-cyclodextrin, or trehalose and hydroxypropyl-β-cyclodextrin (1:1) as excipients, there are no significant differences in various test indicators, all meeting the quality requirements of lyophilized powder injection. When using hydroxypropyl-β-cyclodextrin alone as an excipient, the impurities in the lyophilized powder injection increased significantly (1.59%) after being stored at 40℃ for 4 weeks, indicating poor high-temperature stability. When using trehalose alone or a combination of trehalose and hydroxypropyl-β-cyclodextrin (1:1) as excipients, all test indicators meet the quality requirements of lyophilized powder injection. Therefore, trehalose is the preferred excipient for BX-229 lyophilized powder injection, but a combination of trehalose and hydroxypropyl-β-cyclodextrin (1:1) can also be used as an excipient.

[0243] The application has been described in detail above with general descriptions and specific implementation schemes. Any modifications or improvements made based on this application without departing from its spirit shall fall within the scope of protection claimed in this application.

Claims

1. A pharmaceutical composition comprising: The polypeptide, a pharmaceutically acceptable salt, solvate, conjugate or non-covalent complex of the polypeptide, wherein the polypeptide comprises the amino acid sequence Fum-DEEAVYFAHK(-AEEA-γE-C12)DVIW (SEQ ID NO:1) or a functional variant thereof. Filler; and, Optional pH adjuster.

2. The pharmaceutical composition of claim 1, wherein the functional variant of the polypeptide is a substitution between D and E, 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, as described in SEQ ID NO:1, and the functional variant of the polypeptide has the same ETBR receptor activating activity as SEQ ID NO:

1.

3. The pharmaceutical composition of claim 1 or 2, wherein the polypeptide is a pharmaceutically acceptable salt thereof, preferably a sodium salt, potassium salt, ammonium salt, trifluoroacetate, acetate, hydrochloride, sulfate or phosphate, more preferably a sodium salt, potassium salt or ammonium salt, and even more preferably a sodium salt.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the filler is selected from trehalose, mannitol, glucose, lactose, cyclodextrin, hydroxypropyl-β-cyclodextrin, dextran-40, sorbitol, sucrose, glycine or any combination thereof, preferably trehalose and hydroxypropyl-β-cyclodextrin or any combination thereof, more preferably trehalose; Optionally, the mass ratio of the polypeptide, pharmaceutically acceptable salt of the polypeptide, solvate, conjugate or non-covalent complex of the polypeptide to the filler is 1:200 to 1:0.1, preferably about 1:100 to 1:5, more preferably about 1:50 to 1:12.5, and most preferably about 1:

25.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the pH adjuster is selected from glycine buffer, histidine buffer, arginine buffer, sodium succinate buffer, potassium succinate buffer, sodium citrate buffer, gluconate buffer, acetate buffer, phosphate buffer, disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, and Tris buffer or any combination thereof, preferably disodium hydrogen phosphate-sodium dihydrogen phosphate buffer or glycine buffer, more preferably disodium hydrogen phosphate-sodium dihydrogen phosphate buffer.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the pH value of the pharmaceutical composition is 6 to 9, preferably 7 to 8, and more preferably 7.

7. The pharmaceutical composition of claim 1, wherein the polypeptide is its pharmaceutically acceptable sodium salt, the filler is trehalose, and the pH adjuster is disodium hydrogen phosphate-sodium dihydrogen phosphate buffer. Optionally, the pH of the pharmaceutical composition is 7.0; Optionally, the mass ratio of the sodium salt of the polypeptide to trehalose is about 1:

25.

8. The pharmaceutical composition of claim 1, wherein the filler is trehalose, hydroxypropyl-β-cyclodextrin or a combination thereof, and the pharmaceutical composition is free of pH adjuster; preferably, when the filler is trehalose and the hydroxypropyl-β-cyclodextrin, the mass ratio of the two is 1:

1.

9. The pharmaceutical composition according to any one of claims 1 to 7, further comprising a cryoprotectant, a surfactant, and / or an inhibitor; preferably, the cryoprotectant is polyethylene glycol, the surfactant is polysorbate, preferably polysorbate 20 or polysorbate 80, and / or the inhibitor is a deamidating agent.

10. The pharmaceutical composition of any one of claims 1 to 9, wherein the pharmaceutical composition is in the form of a pre-lyophilized formulation, or in the form of a lyophilized formulation, or in the form of a reconstituted formulation obtained by combining a lyophilized formulation with an aqueous solution.

11. Use of the pharmaceutical composition of any one of claims 1 to 10 in the preparation of a medicament for improving the regeneration of neurovascular units or the blood and oxygen supply to the nervous system in an individual.

12. Use of the pharmaceutical composition of any one of claims 1 to 10 in the preparation of a medicament for treating diseases associated with the ETBR receptor in an individual.

13. Use of the pharmaceutical composition of any one of claims 1 to 10 in the preparation of a medicament for treating, improving or preventing in an individual any of the following diseases: nervous system injury and related diseases caused by such injury, neurodegenerative diseases, anxiety, epilepsy, aortic stenosis or hypoxic-ischemic encephalopathy of the newborn, or other diseases requiring improvement of neurovascular unit regeneration and improvement of blood and oxygen supply to the nervous system.

14. The pharmaceutical composition of any one of claims 1 to 10, for treating, improving or preventing the following diseases in mammals: nervous system injury and related diseases caused by such injury, neurodegenerative diseases, anxiety, epilepsy, aortic stenosis or hypoxic-ischemic encephalopathy of the newborn, or other diseases requiring improvement of neurovascular unit regeneration and improvement of blood and oxygen supply to the nervous system.

15. A method for treating, improving, or preventing diseases in mammals including: nervous system injury and related diseases caused by such injury, neurodegenerative diseases, anxiety, epilepsy, aortic stenosis or hypoxic-ischemic encephalopathy of the newborn, or other diseases requiring improvement of neurovascular unit regeneration and improvement of blood and oxygen supply to the nervous system, including administering to an individual in need the pharmaceutical composition of any one of claims 1 to 10.

16. The pharmaceutical composition of claim 13, the pharmaceutical composition of claim 14, or the method of claim 15, wherein the disease is selected from: nervous system injury and related diseases caused by such injury, neurodegenerative diseases, anxiety, epilepsy, aortic stenosis, or hypoxic-ischemic encephalopathy of the newborn; preferably, the nervous system injury and related diseases caused by such injury include stroke, spinal cord injury, ischemic or traumatic injury to the brain or spinal cord, and injury to neurons of the central nervous system (CNS); preferably, the neurodegenerative diseases include Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Parkinson's disease, or Huntington's disease; preferably, the stroke includes ischemic stroke, hemorrhagic stroke, and hemorrhagic stroke converted from ischemic stroke, preferably ischemic stroke.

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