Pharmaceutical composition containing PSD-95 polypeptide inhibitor and preparation method therefor

Abstract

Provided are a pharmaceutical composition comprising a PSD-95 polypeptide inhibitor and a preparation method therefor. Specifically, provided is a composition, comprising the PSD-95 polypeptide inhibitor or a pharmaceutically acceptable salt thereof. The composition has good stability.

Description

A pharmaceutical composition comprising a PSD-95 peptide inhibitor and its preparation method thereof. Technical Field

[0001] This disclosure pertains to the field of pharmaceutical formulations, specifically relating to a pharmaceutical composition comprising a neuroprotective PSD-95 peptide inhibitor and a method for its preparation. Background Technology

[0002] Postsynaptic density protein-95 (PSD-95) is an important scaffold protein located on the postsynaptic membrane of the central nervous system. It consists of three repeating PDZ domains at the N-terminus: PDZ1, PDZ2, and PDZ3, an intermediate SH3 domain, and a C-terminal guanylate kinase domain. PDZ domains are common protein-protein interaction domains. PSD-95 binds to the tSXV-COOH of NMDARs (N-methyl-D-aspartate receptors) via PDZ1 and PDZ2, and to the PDZ of nNOS (neuronal nitric oxide synthase) via PDZ2 (Acta Pharmacol. Sin., 2018, 39:661–668).

[0003] Following ischemic stroke, impaired cerebral blood flow leads to the accumulation of glutamate in the extracellular space. Excessive release of excitatory glutamate continues to act on glutamate receptors, causing neuronal depolarization and calcium influx. Overloaded calcium... 2+ Calmodulin activates nNOS, leading to an abnormal increase in nitric oxide (NO) production, which ultimately causes neuronal damage.

[0004] PSD-95 inhibitors can effectively interfere with the intracellular interaction between NMDA receptors and PSD-95. Disruption of the GluN2B-PSD95-nNOS complex can inhibit NMDA-mediated NO production and protect neurons. Nerinetide (also known as TAT-NR2B9c or NA-1) is a PSD-95 inhibitor developed by NoNO Inc. of Canada (Science, 2002, 298:846-50), belonging to the class of neuroexcitotoxic inhibitors. Nerinetide consists of 20 amino acid residues, where TAT is derived from the human (HIV-1) transcription transactivator and can cross multiple cell membranes, and NR2B9c is selected from the 9 amino acids at the C-terminus of the NR2B subunit.

[0005] In preclinical animal models (rats and cynomolgus monkeys), administration of Nerinetide after ischemic stroke significantly reduced infarct size and improved neurobehavioral function (Sci. Transl. Med., 2021, 13, eabb1498). Phase II clinical trials in patients undergoing endovascular aneurysm repair (ENACT) and Phase III clinical trials in patients with severe acute ischemic stroke (AIS) undergoing endovascular thrombectomy (EVT) both showed no drug-related serious adverse events, and the safety profile of Nerinetide was comparable to placebo.

[0006] Current research on PSD-95 inhibitors includes WO2015078477A, ​​WO2015181756A, and WO2022150655A. To meet clinical needs, there is still a need to develop PSD-95 inhibitors with high stability and good pharmacokinetic properties, and to formulate suitable formulations of PSD-95 inhibitors or their pharmaceutically acceptable salts, requiring good formulation stability to meet the treatment needs of diseases such as ischemic stroke. Summary of the Invention

[0007] This disclosure provides a pharmaceutical composition comprising an active ingredient, a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein the compound of formula (I) or a pharmaceutically acceptable salt thereof is selected from the following amino acid sequence: rKKRrQRRrG-Aib-Dab-T-Tle-Tle-TDV (SEQ ID NO.01) (I);

[0008] The pH of the pharmaceutical composition is 5.0-9.0;

[0009] Note: Lowercase single letters represent D-type amino acids.

[0010] In some embodiments, the pH value of the pharmaceutical composition is 6.0-8.0, and the optional pH values ​​include 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, or any value between any two points.

[0011] In some embodiments, the pH of the pharmaceutical composition is 6.5-7.5, and the optional pH includes 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5 or any value between two points.

[0012] In some specific embodiments, the pH of the pharmaceutical composition is 7.0.

[0013] In some embodiments, the concentration (as a free base) of the active ingredient or its pharmaceutically acceptable salt is 10 mg / mL to 500 mg / mL, with selectable concentrations including 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, 50 mg / mL, 55 mg / mL, 60 mg / mL, 65 mg / mL, 70 mg / mL, 75 mg / mL, 80 mg / mL, 85 mg / mL, 90 mg / mL, 95 mg / mL, 100 mg / mL, 105 mg / mL, 110 mg / mL, and 115 mg / mL. L, 120mg / mL, 125mg / mL, 130mg / mL, 135mg / mL, 140mg / mL, 145mg / mL, 150mg / mL, 155mg / mL, 160mg / mL, 165mg / mL, 170mg / mL, 175mg / mL, 180mg / mL , 185mg / mL, 190mg / mL, 195mg / mL, 200mg / mL, 205mg / mL, 210mg / mL, 215mg / mL, 220mg / mL, 225mg / mL, 230mg / mL, 235mg / mL, 240mg / mL, 245mg / mL, 250mg / mL, 255mg / mL, 260mg / mL, 265mg / mL, 270mg / mL, 275mg / mL, 280mg / mL, 285mg / mL, 290mg / mL, 295mg / mL, 300mg / mL, 305mg / mL, 310mg / mL, 315mg / mL, 320mg / mL, 325mg / mL, 330mg / mL, 335mg / mL, 340mg / mL, 345mg / mL, 350mg / mL, 355mg / mL, 360mg / mL, 365mg / mL, 370mg / mL, 375mg / mL, 3 80 mg / mL, 385 mg / mL, 390 mg / mL, 395 mg / mL, 400 mg / mL, 405 mg / mL, 410 mg / mL, 415 mg / mL, 420 mg / mL, 425 mg / mL, 430 mg / mL, 435 mg / mL, 440 mg / mL, 445 mg / mL, 450 mg / mL, 455 mg / mL, 460 mg / mL, 465 mg / mL, 470 mg / mL, 475 mg / mL, 480 mg / mL, 485 mg / mL, 490 mg / mL, 495 mg / mL, 500 mg / mL, or any value between two points.

[0014] In some embodiments, the concentration of the active ingredient or its pharmaceutically acceptable salt (in the form of free base) is 20 mg / mL to 90 mg / mL, and the optional concentrations include 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, 50 mg / mL, 55 mg / mL, 60 mg / mL, 65 mg / mL, 70 mg / mL, 75 mg / mL, 80 mg / mL, 85 mg / mL, 90 mg / mL, or any value between any two points.

[0015] In some embodiments, the concentration of the active ingredient or its pharmaceutically acceptable salt (in the form of free base) is 55 mg / mL to 65 mg / mL, and the optional concentrations include 55 mg / mL, 56 mg / mL, 57 mg / mL, 58 mg / mL, 59 mg / mL, 60 mg / mL, 61 mg / mL, 62 mg / mL, 63 mg / mL, 64 mg / mL, 65 mg / mL, or any value between any two points.

[0016] In some embodiments, the pharmaceutically acceptable salt of the active ingredient is selected from chloride salts, acetates, or bicarbonates.

[0017] In some specific embodiments, the pharmaceutically acceptable salt of the active ingredient is a chloride salt.

[0018] In some embodiments, the pharmaceutical composition further comprises a buffer.

[0019] In some embodiments, the buffer is selected from one or more of acetate buffers, histidine buffers, glycine buffers, phosphate buffers, succinate buffers, citrate buffers, and tris(hydroxymethyl)aminomethane buffers.

[0020] In some embodiments, the buffer is selected from one or more of acetate buffers, histidine buffers, phosphate buffers, succinate buffers, and citrate buffers.

[0021] In some specific implementations, the buffer is selected from histidine buffers.

[0022] In some embodiments, the concentration of the buffer is from 5.0 mM to 50.0 mM.

[0023] In some embodiments, the concentration of the buffer is from 10.0 mM to 50.0 mM, with selectable concentrations including 10.0 mM, 15.0 mM, 20.0 mM, 25.0 mM, 30.0 mM, 35.0 mM, 40.0 mM, 45.0 mM, 50.0 mM, or any value between any two points.

[0024] In some specific embodiments, the concentration of the buffer is from 10.0 mM to 20.0 mM, and the selectable concentrations include 10.0 mM, 11.0 mM, 12.0 mM, 13.0 mM, 14.0 mM, 15.0 mM, 16.0 mM, 17.0 mM, 18.0 mM, 19.0 mM, 20.0 mM, or any value between any two points.

[0025] In some embodiments, the pharmaceutical composition further comprises a filler.

[0026] In some embodiments, the filler is selected from one or more of amino acids and sugars (including sugar alcohols).

[0027] In some embodiments, the sugars (including sugar alcohols) are selected from one or more of mannitol, sorbitol, xylitol, trehalose, glucose, sucrose, maltose, and dextran.

[0028] In some embodiments, the sugars (including sugar alcohols) are selected from one or more of mannitol, trehalose, glucose, sucrose, and maltose.

[0029] In some embodiments, the sugar (including sugar alcohols) is selected from mannitol, with a concentration of 40mM-400mM.

[0030] In some embodiments, the sugar (including sugar alcohols) is selected from mannitol, with a concentration of 200mM-400mM.

[0031] In some specific embodiments, the sugar (including sugar alcohols) is selected from mannitol, with a concentration of 240mM-360mM. Selectable concentrations include 240mM, 250mM, 260mM, 270mM, 280mM, 290mM, 300mM, 310mM, 320mM, 330mM, 340mM, 350mM, 360mM, or any value between any two points.

[0032] In some embodiments, the sugar (including sugar alcohols) is selected from trehalose and has a concentration of 20mM-200mM.

[0033] In some specific embodiments, the sugar (including sugar alcohols) is selected from trehalose, and its concentration is 60mM-120mM. Selectable concentrations include 60mM, 65mM, 70mM, 75mM, 80mM, 85mM, 90mM, 95mM, 100mM, 105mM, 110mM, 115mM, 120mM, or any value between any two points.

[0034] In some embodiments, the amino acid is selected from one or more of glycine, alanine, arginine, aspartic acid, asparagine, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, methyllysine, ornithine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

[0035] In some embodiments, the amino acid is selected from glycine and has a concentration of 100mM-400mM.

[0036] In some specific embodiments, the concentration of glycine is 120mM-360mM, and the selectable concentrations include 120mM, 130mM, 140mM, 150mM, 160mM, 170mM, 180mM, 190mM, 200mM, 210mM, 220mM, 230mM, 240mM, 250mM, 260mM, 270mM, 280mM, 290mM, 300mM, 310mM, 320mM, 330mM, 340mM, 350mM, 360mM, or any value between any two points.

[0037] In some embodiments, the pharmaceutical composition further comprises a pH adjuster.

[0038] In some specific embodiments, the pH adjuster comprises sodium hydroxide and / or hydrochloric acid.

[0039] In some embodiments, the pharmaceutical composition has a pH of 6.5 to 7.5 and comprises the following components:

[0040] 55 mg / mL to 65 mg / mL (as free base) of the compound represented by formula (I) or its pharmaceutically acceptable salt;

[0041] 10.0 mM to 20.0 mM histidine buffer, and

[0042] 240mM to 360mM mannitol;

[0043] The pharmaceutical composition optionally includes a pH adjuster, which comprises sodium hydroxide and / or hydrochloric acid.

[0044] In some embodiments, the pharmaceutical composition has a pH of 6.5 to 7.5 and comprises the following components:

[0045] 55 mg / mL to 65 mg / mL (as free base) of the compound represented by formula (I) or its pharmaceutically acceptable salt;

[0046] 10.0 mM to 20.0 mM citrate buffer, and

[0047] 240mM to 360mM mannitol;

[0048] The pharmaceutical composition optionally includes a pH adjuster, which comprises sodium hydroxide and / or hydrochloric acid.

[0049] In some embodiments, the pharmaceutical composition has a pH of 6.5 to 7.5 and comprises the following components:

[0050] 55 mg / mL to 65 mg / mL (as free base) of the compound represented by formula (I) or its pharmaceutically acceptable salt;

[0051] 10.0 mM to 20.0 mM phosphate buffer, and

[0052] 240mM to 360mM mannitol;

[0053] The pharmaceutical composition optionally includes a pH adjuster, which comprises sodium hydroxide and / or hydrochloric acid.

[0054] In some embodiments, the pharmaceutical composition has a pH of 6.5 to 7.5 and comprises the following components:

[0055] 55 mg / mL to 65 mg / mL (as free base) of the compound represented by formula (I) or its pharmaceutically acceptable salt;

[0056] 10.0 mM to 20.0 mM histidine buffer, and

[0057] 60mM to 120mM trehalose;

[0058] The pharmaceutical composition optionally includes a pH adjuster, which comprises sodium hydroxide and / or hydrochloric acid.

[0059] In some embodiments, the pharmaceutical composition has a pH of 6.5 to 7.5 and comprises the following components:

[0060] 55 mg / mL to 65 mg / mL (as free base) of the compound represented by formula (I) or its pharmaceutically acceptable salt;

[0061] 10.0 mM to 20.0 mM citrate buffer, and

[0062] 60mM to 120mM trehalose;

[0063] The pharmaceutical composition optionally includes a pH adjuster, which comprises sodium hydroxide and / or hydrochloric acid.

[0064] In some embodiments, the pharmaceutical composition has a pH of 6.5 to 7.5 and comprises the following components:

[0065] 55 mg / mL to 65 mg / mL (as free base) of the compound represented by formula (I) or its pharmaceutically acceptable salt;

[0066] 10.0 mM to 20.0 mM phosphate buffer, and

[0067] 60mM to 120mM trehalose;

[0068] The pharmaceutical composition optionally includes a pH adjuster, which comprises sodium hydroxide and / or hydrochloric acid.

[0069] In some embodiments, the pharmaceutical composition has a pH of 6.5 to 7.5 and comprises the following components:

[0070] 55 mg / mL to 65 mg / mL (as free base) of the compound represented by formula (I) or its pharmaceutically acceptable salt;

[0071] 10.0 mM to 20.0 mM histidine buffer, and

[0072] 120mM to 360mM glycine;

[0073] The pharmaceutical composition optionally includes a pH adjuster, which comprises sodium hydroxide and / or hydrochloric acid.

[0074] In some embodiments, the pharmaceutical composition has a pH of 6.5 to 7.5 and comprises the following components:

[0075] 55 mg / mL to 65 mg / mL (as free base) of the compound represented by formula (I) or its pharmaceutically acceptable salt;

[0076] 10.0 mM to 20.0 mM citrate buffer, and

[0077] 120mM to 360mM glycine;

[0078] The pharmaceutical composition optionally includes a pH adjuster, which comprises sodium hydroxide and / or hydrochloric acid.

[0079] In some embodiments, the pharmaceutical composition has a pH of 6.5 to 7.5 and comprises the following components:

[0080] 55 mg / mL to 65 mg / mL (as free base) of the compound represented by formula (I) or its pharmaceutically acceptable salt;

[0081] 10.0 mM to 20.0 mM phosphate buffer, and

[0082] 120mM to 360mM glycine;

[0083] The pharmaceutical composition optionally includes a pH adjuster, which comprises sodium hydroxide and / or hydrochloric acid.

[0084] In some specific embodiments, the pharmaceutical composition has a pH of 6.5 to 7.5 and comprises the following components:

[0085] 55 mg / mL to 65 mg / mL (as free base) of the compound represented by formula (I) or its pharmaceutically acceptable salt;

[0086] 10.0 mM to 20.0 mM histidine buffer, and

[0087] 240mM to 360mM mannitol;

[0088] The pharmaceutical composition optionally includes a pH adjuster, which comprises sodium hydroxide and / or hydrochloric acid.

[0089] In some embodiments, the pharmaceutical composition is stored stably at 2-8°C for at least 3 months, at least 6 months, at least 1 year, at least 2 years, or at least 3 years.

[0090] In some embodiments, the pharmaceutical composition remains stable at 25°C for at least 3 months, at least 6 months, at least 1 year, at least 2 years, or at least 3 years.

[0091] In some embodiments, the composition is a liquid formulation, obtained by reconstitution of a lyophilized formulation or a liquid formulation prior to lyophilization.

[0092] In some embodiments, the purity of the liquid formulation, after being stored at 25°C for one month or at 40°C for one month, varies by no more than ±10% as measured by HPLC (high performance liquid chromatography).

[0093] In some embodiments, the purity of the liquid formulation, after being stored at 25°C for one month or at 40°C for one month, varies by no more than ±5% as measured by HPLC (high performance liquid chromatography).

[0094] In some embodiments, the pH of the liquid formulation changes by no more than ±10% after being stored at 25°C for one month or at 40°C for one month.

[0095] In some embodiments, the pH of the liquid formulation changes by no more than ±5% after being stored at 25°C for one month or at 40°C for one month.

[0096] This disclosure also provides a method for preparing the aforementioned pharmaceutical composition, comprising the step of dissolving the compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0097] In some implementations, the method optionally includes a step of adjusting the pH.

[0098] This disclosure also provides a lyophilized formulation, which can be reconstituted to form the aforementioned pharmaceutical composition, or the lyophilized formulation is obtained by freeze-drying the aforementioned pharmaceutical composition.

[0099] In some embodiments, the purity of the lyophilized formulation, after being stored at 40°C for one month or at 60°C for one month, varies by no more than ±10% as measured by HPLC (high performance liquid chromatography).

[0100] In some embodiments, the purity of the lyophilized formulation, after being stored at 40°C for one month or at 60°C for one month, varies by no more than ±5% as measured by HPLC (high performance liquid chromatography).

[0101] This disclosure also provides a method for preparing the aforementioned pharmaceutical composition, comprising the step of dissolving the compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0102] This disclosure also provides a method for preparing the aforementioned lyophilized formulation, including the step of lyophilizing the aforementioned pharmaceutical composition.

[0103] This disclosure also provides a reconstitution solution, wherein the reconstitution solution is prepared by reconstitution of the aforementioned lyophilized formulation.

[0104] In some embodiments, the reconstituted solution is prepared by dissolving the aforementioned lyophilized preparation in an aqueous solution selected from water for injection or physiological saline.

[0105] This disclosure also provides an article comprising a container containing the aforementioned pharmaceutical composition, the aforementioned lyophilized preparation, or the aforementioned reconstituted solution.

[0106] This disclosure also provides the use of the pharmaceutical composition, lyophilized formulation, reconstituted solution or article described herein in the preparation of a medicament for inhibiting PSD-95.

[0107] This disclosure also provides the use of the pharmaceutical composition, lyophilized formulation, reconstituted solution or article described herein in the preparation of a medicament for the treatment and / or prevention of stroke, cerebral ischemia, traumatic injury of the central nervous system, reperfusion injury, subarachnoid hemorrhage, concussion, pain, anxiety, epilepsy or neurodegenerative disease (Alzheimer's disease or Parkinson's disease) or diseases with the above risks.

[0108] This disclosure also provides a method for inhibiting PSD-95, the method comprising administering to a desired patient an effective amount of the pharmaceutical composition, lyophilized formulation, reconstituted solution, or product described in this disclosure.

[0109] This disclosure also provides a method for treating and / or preventing stroke, cerebral ischemia, traumatic injury to the central nervous system, reperfusion injury, subarachnoid hemorrhage, concussion, pain, anxiety, epilepsy, or neurodegenerative diseases (Alzheimer's disease or Parkinson's disease) or diseases with the aforementioned risks, the method comprising administering to a desired patient an effective amount of the pharmaceutical composition, lyophilized preparation, reconstituted solution, or product described in this disclosure.

[0110] This disclosure also provides a pharmaceutical composition, lyophilized formulation, reconstituted solution or article described herein, which is used as a PSD-95 inhibitor.

[0111] This disclosure also provides a pharmaceutical composition, lyophilized formulation, reconstituted solution or product described herein for the treatment and / or prevention of stroke, cerebral ischemia, traumatic injury of the central nervous system, reperfusion injury, subarachnoid hemorrhage, concussion, pain, anxiety, epilepsy or neurodegenerative disease (Alzheimer's disease or Parkinson's disease) or diseases with the above risks.

[0112] For an explanation of "reinfusion" in this disclosure, please refer to WO2012176172A.

[0113] In the polypeptide compound sequences provided in this disclosure, lowercase letters represent D-type amino acids. Unless otherwise specified, uppercase letters represent L-type amino acids (of which Gly and Aib have no stereoconfiguration).

[0114] The compound of formula (I) and its pharmaceutically acceptable salt provided in this disclosure are synthesized using a solid-phase method. The synthesis support is Fmoc-Val-Wang resin. The α-amino group of the amino acid derivative used in the synthesis process is protected by the Fmoc group (fluorenylcarbonyl). The side chains of the amino acids are protected by the following groups according to different functional groups: the amino group of the glutamine (L / D) side chain is protected by Trt (triphenylmethyl), the guanidinyl group of the arginine (L / D) side chain is protected by Pbf (2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl), the amino group of the lysine (L / D) side chain and the amino group of the Dab side chain are protected by Boc (tert-butyloxycarbonyl), and the hydroxyl group of the threonine side chain and the carboxyl group of the aspartic acid side chain are protected by t-Bu (tert-butyl). In the synthesis process, Fmoc-Val-Wang resin was first fully swollen in N,N-dimethylformamide (DMF). The Fmoc protecting group on the α-amino group was removed using a DMF solution containing 20% ​​4-methylpiperidine. Then, the carboxyl group of the C-terminal amino acid residue was condensed onto the polymer-insoluble resin via an amide bond. The Fmoc protecting group on the α-amino group was then removed again using a DMF solution containing 20% ​​4-methylpiperidine. Next, the solid support condensed with the next amino acid derivative in the sequence under excess conditions to form an amide bond, thus lengthening the peptide chain. This process of washing → deprotection → washing → next round of amino acid condensation → washing was repeated to achieve the desired peptide chain length. Finally, the peptide was cleaved from the solid support by reacting the resin with a mixed solution of trifluoroacetic acid:water:triisopropylsilane. The solid was then precipitated with frozen methyl tert-butyl ether, centrifuged or filtered to obtain the crude peptide solid, which was then dried overnight. After dissolving the crude polypeptide solid in purified water, the pH was adjusted to 5-7 with ammonia. The product was then purified and separated using a C-18 reversed-phase preparative chromatography column, concentrated, and lyophilized to obtain the pure polypeptide product that has formed salts.

[0115] Terminology Explanation

[0116] To facilitate understanding of this disclosure, certain techniques and scientific methods are specifically defined below. Unless otherwise expressly defined in this disclosure, all other techniques and scientific methods used in this disclosure have the meaning commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0117] The three-letter and single-letter codes for amino acids used in this disclosure are as described in J. Biol. Chem, 243, p3558 (1968).

[0118] The correspondences between some amino acid abbreviations and their structures in this disclosure are as follows:

[0119] In the chemical structure of the compounds described in this disclosure, unless otherwise specified, the bonds are... This indicates that the configuration is not specified; that is, if chiral isomers exist in the chemical structure, the bond... It can be Or simultaneously include Two configurations. Although all the above structural formulas are shown in some isomer forms for simplicity, this disclosure can include all isomers, such as tautomers, rotational isomers, geometric isomers, diastereomers, racemates and enantiomers.

[0120] "Pharmaceutical composition" means a mixture containing one or more of the active ingredients described herein or their physiologically / pharmacologically acceptable salts or prodrugs, along with other chemical components, such as physiologically / pharmacologically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to a living organism, thereby promoting the absorption of the active ingredient and its biological activity.

[0121] The pharmaceutically acceptable salts of the compounds described in this disclosure may be selected from inorganic or organic salts.

[0122] "Buffer" refers to a buffer that is resistant to pH changes through the action of its acid-base conjugate components. Examples of buffers that maintain pH within an appropriate range include acetate, succinate, gluconate, histidine, oxalate, lactate, phosphate, citrate, tartrate, fumarate, glycylglycine, and other organic acid buffers.

[0123] "Histidine buffer" is a buffer containing histidine. Examples of histidine buffers include histidine-histidine hydrochloride, histidine-histidine acetate, histidine-histidine phosphate, and histidine-histidine sulfate buffers, with histidine-histidine hydrochloride buffers being preferred. Histidine-histidine hydrochloride buffers can be prepared by reacting histidine with hydrochloric acid, or by reacting histidine with histidine hydrochloride.

[0124] "Citrate buffer" is a buffer that includes citrate ions. Examples of citrate buffers include sodium citrate, potassium citrate, calcium citrate, magnesium citrate, etc. A preferred citrate buffer is sodium citrate.

[0125] "Succinate buffer" is a buffer containing succinate ions. Examples of succinate buffers include sodium succinate, potassium succinate, and calcium succinate. A preferred succinate buffer is sodium succinate. Exemplarily, the sodium succinate can be prepared from succinic acid and sodium hydroxide, or from succinic acid and sodium succinate.

[0126] A "phosphate buffer" is a buffer that contains phosphate ions. Examples of phosphate buffers include disodium hydrogen phosphate-sodium dihydrogen phosphate, disodium hydrogen phosphate-potassium dihydrogen phosphate, and disodium hydrogen phosphate-citric acid. A preferred phosphate buffer is disodium hydrogen phosphate-sodium dihydrogen phosphate.

[0127] "Acetate buffer" is a buffer that includes acetate ions. Examples of acetate buffers include sodium acetate, histidine-histidine acetate, potassium acetate, calcium acetate, magnesium acetate, etc. The preferred acetate buffer is sodium acetate.

[0128] Unless otherwise specified, the solvent in the solution form of the pharmaceutical compositions described in this disclosure is water.

[0129] "Lyophilized formulation" refers to a pharmaceutical composition or formulation obtained by a vacuum freeze-drying step after the liquid or solution form has been processed.

[0130] In this disclosure, "mixing" means that the order in which the components are added is not limited. For example, mixing A into B can mean that A is added to B or that B is added to A. Mixing A and B can mean that A is added to B or that B is added to A.

[0131] "Treatment" means administering a therapeutic agent, such as a fusion protein or insulin analog comprising any of the present disclosure, to a subject who has, is suspected of having, or is predisposed to having one or more diabetes or hyperglycemia-related diseases or their symptoms, and the therapeutic agent is known to have a therapeutic effect on these symptoms. Typically, in treated subjects or populations, a therapeutic agent is administered in an amount that effectively relieves symptoms of one or more diseases by preventing or delaying the onset of symptoms or complications, reducing symptoms or complications, or eliminating the disease, condition, or symptom to any clinically measurable degree. The amount of a therapeutic agent that effectively relieves symptoms of any specific disease (also referred to as a "therapeuticly effective amount") can vary depending on a variety of factors, such as the subject's disease state, age, and weight, and the drug's ability to produce the desired therapeutic effect in the subject. Whether the disease symptoms have been relieved can be evaluated using any clinical test method commonly used by a physician or other healthcare professional to assess the severity or progression of the symptoms. Although the embodiments of this disclosure (e.g., treatment methods or products) may be ineffective in alleviating the symptoms of the target disease in a particular subject, they should alleviate the symptoms of the target disease in a statistically significant number of subjects, as determined by any statistical test known in the art, such as the Student t-test, chi-square test, U-test according to Mann and Whitney, Kruskal-Wallis test (H-test), Jonckheere-Terpstra test, and Wilcoxon test. The patients to be treated are mammals, and preferably humans.

[0132] "Prevention" means reducing the risk or incidence of one or more conditions, symptoms, complications or symptoms, or eliminating or slowing the progression of one or more conditions, symptoms, complications or symptoms.

[0133] "Optional" or "optionally" means that the event or circumstances described below may, but do not have to, occur, including the circumstances in which the event or circumstances may or may not occur.

[0134] "Subject" and "patient" refer to mammals, especially primates, and particularly humans.

[0135] Unless the context clearly requires otherwise, throughout the specification and claims, the words “comprising,” “having,” “including,” etc., should be understood as having an inclusive meaning, rather than an exclusive or exhaustive meaning; that is, the meaning of “including but not limited to.”

[0136] The concentrations, pH values, etc., described in this disclosure are allowed to have an error of ±5%. For example, if the concentration of the compound of formula (I) or its pharmaceutically acceptable salt in the composition is 10 mg / mL to 100 mg / mL, then this concentration includes schemes where the concentration of the compound of formula (I) or its pharmaceutically acceptable salt is 9.5 mg / mL to 105 mg / mL. Attached Figure Description

[0137] Figure 1. Rabbit whole blood hemolysis risk test;

[0138] Figure 2A. Stability test of polypeptide compounds in human plasma;

[0139] Figure 2B. Rat plasma stability test of polypeptide compounds;

[0140] Figure 3. Pharmacokinetics of the polypeptide compound in beagle dogs;

[0141] Figure 4. The ability of peptide compounds to induce histamine release in beagle dogs;

[0142] Figure 5A. Pharmacodynamic results of compound (I) in rat tMCAO model - cerebral infarction area, * indicates P < 0.05, ** indicates P < 0.01;

[0143] Figure 5B. Efficacy results of compound (I) in rat tMCAO model - neurological function impairment score, * indicates P < 0.05, ** indicates P < 0.01. Detailed Implementation

[0144] The following embodiments are used to further describe this disclosure, but these embodiments are not intended to limit the scope of this disclosure.

[0145] Experimental methods not specifying specific conditions in the embodiments or test examples disclosed herein are generally performed under conventional conditions or as recommended by the raw material or product manufacturer. Reagents not specifying their source are commercially available, conventional reagents.

[0146] Example 1. Preparation of the compound shown in formula (I) and its pharmaceutically acceptable salt.

[0147] rKKRrQRRrG-Aib-Dab-T-Tle-Tle-TDV(SEQ ID NO.01)

[0148] Step 1: Resin swelling and removal of Fmoc protecting groups

[0149] Weigh 221.1 g (74.29 mmol, degree of substitution: 0.336 mmol / g) of Fmoc-Val-Wang resin (solid-phase synthesis support) and place it in a peptide synthesis solid-phase apparatus or glass column. Add 1.6 L of DMF to swell the resin for 1.5 h, filter out the DMF using a vacuum pump, add 1.5 L of DMF to wash the resin, repeat the washing twice, and then dry it for later use. Add 1.5 L of 4-methylpiperidine / DMF (20% v / v) to the above resin, purge with nitrogen at room temperature for 20 min, and then remove the dryness. Add 1.5 L of 4-methylpiperidine / DMF (20% v / v) again, purge with nitrogen at room temperature for 20 min, and then remove the solution. After deprotection, wash the resin 5 times with 1.4 L of DMF.

[0150] Step 2: Coupling of peptide sequences

[0151] The peptide chain sequence of the compound shown in formula (I) was synthesized in order from the carboxyl terminus to the amino terminus. First, Fmoc-Asp(O t Bu)-OH (61.4 g, 2 eq) and HBTU (56.8 g, 2 eq) were dissolved in DMF (1.4 L), and then 4-methylmorpholine (31.1 g, 4 eq) was added. After mixing and dissolving thoroughly, the solution was added to the resin obtained in step 1, and the reaction was carried out under nitrogen at room temperature for 0.5–1 h. The reaction was controlled by the ninhydrin colorimetric method. If the reaction was incomplete, 0.5–1 eq of Fmoc-Asp(O) was added again. t Bu)-OH until the reaction is complete. After the reaction is complete, wash four times with DMF (1.4 L).

[0152] Similar to step 1, 4-methylpiperidine / DMF (20% v / v, 1.5 L) was added to the above resin to remove the Fmoc protecting group at the N-terminus of the amino acid. The reaction was carried out under nitrogen at room temperature for 20 min, the solvent was removed, and then 4-methylpiperidine / DMF (20% v / v, 1.5 L) was added again. The reaction was carried out under nitrogen at room temperature for another 20 min, followed by removal of the solvent. After the reaction was complete, the resin was washed five times with DMF (1.4 L).

[0153] Repeat the condensation process of the above amino acid derivatives, sequentially condensing with the following amino acids in 2-3 eq amounts: Fmoc-Thr( t Bu)-OH, Fmoc-Tle-OH, Fmoc-Tle-OH, Fmoc-Thr( t The condensing agent is changed from HBTU to HATU, following the amino acid sequence (Boc-OH, Fmoc-Dab(Boc)-OH, Fmoc-Aib-OH, Fmoc-Gly-OH, Fmoc-D-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gln(Trt)-OH, Fmoc-D-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-D-Arg(Pbf)-OH), ultimately yielding a complete resin peptide containing the compound shown in formula (I). Following the aforementioned amino acid sequence, starting with the first Fmoc-D-Arg(Pbf)-OH, the condensing agent is replaced by HATU, while the remaining condensation conditions remain unchanged.

[0154] Step 3: Cleavage of resin peptides

[0155] The resin peptide containing the compound shown in formula (I) obtained in the above steps was washed five times with DMF (1.5 L), once with DCM (1.4 L), once with MeOH (1.4 L), and once with DCM (2.0 L). After vacuum drying, 3.47 L of freshly prepared lysis buffer (trifluoroacetic acid: triisopropylsilane: water = 95:2.5:2.5, v:v:v) was added, and the reaction was carried out under nitrogen at room temperature for 20 h. After the reaction, the mixture was filtered, and the resin was washed with a small amount of trifluoroacetic acid. The filtrates were combined and added to 10.5 L of pre-cooled methyl tert-butyl ether / dichloromethane (2:1, v:v) at -10 to 0 °C. The precipitated solid was filtered to obtain the crude peptide, which was then dried.

[0156] Step 4: Reversed-phase liquid chromatography purification of crude peptides

[0157] The crude dry peptides underwent two purification processes: first, a primary purification using a TFA system, followed by a secondary purification using an NH4Cl system and / or a TFA system. The qualified fraction from the secondary purification was used for salt conversion in step 5.

[0158] In the first purification step, the crude dry peptide is dissolved in purified water, the pH is adjusted to 5-7 with ammonia, and then filtered. The product is purified using a DAC-100 or DAC-200 filled with Daisogel SP-100-8-ODS-P packing material (injection volume 20-50 g crude peptide / needle, mobile phase A (0.1% trifluoroacetic acid aqueous solution, v / v) and B (0.1% trifluoroacetic acid acetonitrile solution, v / v)). The chromatograph detection wavelength is set to 210 nm, and the flow rate is 200 mL / min or 500 mL / min. The relevant fractions of the product are collected for the second purification step.

[0159] The preparative solution obtained after primary purification can be diluted with 25 mmol / L NH4Cl solution and pumped into the preparative column for secondary purification using an NH4Cl system. The mobile phases are A (25 mmol / L NH4Cl aqueous solution) and B (100% acetonitrile). The chromatographic column packing material is Daisogel SP-100-8-ODS-P, DAC-200 purification. The injection volume is 14.5–20.5 g of peptide / needle. During purification, the chromatographic detection wavelength is set to 210 nm, and the flow rate is 500 mL / min. The relevant fractions of the product are collected, and the qualified fraction after secondary purification is used for salt conversion in step 5.

[0160] The preparative solution obtained after primary purification can also be diluted with 0.1% TFA solution and pumped into the preparative column for secondary purification using the TFA system. The mobile phase is A (0.1% trifluoroacetic acid aqueous solution, v / v) and B (0.1% trifluoroacetic acid acetonitrile solution, v / v). The chromatographic column packing is Daisogel SP-100-8-ODS-P, DAC-200 purification, and the injection volume is 20-31 g peptide / needle. During the purification process, the chromatograph detection wavelength is set to 210 nm and the flow rate is 500 mL / min. The collected qualified fraction of the secondary purification is used for salt conversion in step 5.

[0161] Step 5: Salting

[0162] The qualified fraction purified from the NH4Cl system was diluted with 100 mmol / L NH4Cl solution or purified water to a volume ratio of 4. After mixing, the product was pumped into the preparative column using a sample pump. By pumping different proportions of NH4Cl aqueous solution and acetonitrile (e.g., 100 mmol / L NH4Cl aqueous solution and acetonitrile) into the preparative column, the product was eluted from the stationary phase. It could be temporarily stored at 2–8 °C. Finally, it was concentrated under reduced pressure and lyophilized to obtain the chloride salt of the compound shown in formula (I). The purity of the chloride salt of the compound in formula (I) was determined by HPLC to be 96.2%. The chloride ion content was 11.1% by weight, as determined by ion chromatography (HPIC).

[0163] or,

[0164] The qualified fraction purified by the TFA system was diluted with 100 mmol / L NH4Cl solution or purified water to a volume of 5. After dilution and mixing, the product was pumped into the preparative column. By pumping different proportions of NH4Cl aqueous solution and acetonitrile (e.g., 100 mmol / L NH4Cl aqueous solution and acetonitrile) into the preparative column, the product was eluted from the stationary phase. It could be temporarily stored at 2–8 °C. Finally, the chloride salt of the compound shown in formula (I) could be obtained by concentration under reduced pressure and lyophilization. The purity of the chloride salt of the compound of formula (I) was determined by HPLC to be 94.7%. The chloride ion content was 10.7% by weight, as determined by ion chromatography (HPIC).

[0165] The ion peak shown in the high-resolution mass spectrometry (HRMS) chromatogram is: 742.4605 [M+3H]. 3+ / 3.

[0166] Example 2: Buffer Screening

[0167] Preparations were made containing the buffer (10 mM) shown in Table 1 and the chloride salt of the compound shown in Formula (I) at a concentration of 60 mg / mL (as free base). Samples were placed at 40 °C under light for 14 days, and samples were taken periodically. The effects of different buffer systems on the stability of the active ingredient were investigated, using pH and the purity of the active ingredient (determined by HPLC) as evaluation indicators.

[0168] The experimental results are shown in Table 1. Stability data showed that the pH value and purity of the formulation did not change significantly during the observation period.

[0169] Table 1. Buffer Screening Results

[0170] Example 3: Screening of fillers for lyophilized formulations

[0171] Table 2. Pharmaceutical Composition Prescriptions

[0172] Weigh the materials according to Table 2, dissolve mannitol and histidine in water for injection, add the active ingredient, and after the active ingredient is completely dissolved, adjust the pH to 7.0 with sodium hydroxide and hydrochloric acid to obtain Formula 1. Prepare Formulas 2-7 according to the same method as shown in Table 2.

[0173] The formulation is filtered and filled. The formulation sample is then lyophilized using a process involving pre-freezing, vacuuming, primary drying, and secondary drying. After the lyophilization process is complete, the sample is vacuum-stopped to obtain the lyophilized formulations of formulations 1-7.

[0174] The samples were placed at 40℃ and 60℃ for one month, and samples were taken periodically. The effects of different fillers on the stability of the lyophilized formulation were investigated using appearance and purity as evaluation indicators.

[0175] The experimental results are shown in Table 3. Under the stability test conditions, the lyophilized formulations of formulations 1-7 all maintained the appearance of white blocky lyophilized products, and the purity of the lyophilized formulations of formulations 1-7 did not change significantly, maintaining good stability.

[0176] Table 3. Screening results of fillers for lyophilized formulations

[0177] Example 4: pH screening

[0178] According to Formulation 4 in Table 2, trehalose (120 mM) and histidine (10 mM) were weighed and dissolved in water for injection. The active ingredient was added, and after the active ingredient was completely dissolved, the pH of the solution was adjusted to 6.0, 6.5, 7.0, 7.5, and 8.0 with sodium hydroxide and hydrochloric acid, respectively. Lyophilized formulations were prepared according to the preparation method described in Example 3.

[0179] After the samples were placed at 40℃ and 60℃ for one month, samples were taken periodically. The effects of different pH values ​​(pH = 6.0, 6.5, 7.0, 7.5, 8.0) on the stability of the formulation were investigated, with pH value and purity as evaluation indicators.

[0180] The experimental results are shown in Table 4. The formulation exhibited good stability within the pH range of 6.0 to 8.0, with no significant changes in pH value or purity during the observation period.

[0181] Table 4. pH value screening results

[0182] Biological test evaluation

[0183] The present disclosure is further described and explained below with reference to test examples, but these embodiments are not intended to limit the scope of the present disclosure.

[0184] 1. Experimental reagents

[0185] Table 5

[0186] 2. Experimental apparatus

[0187] Table 6

[0188] 3. Test Case

[0189] 3.1. Evaluate the affinity of the peptide compound for human PSD-95.

[0190] 3.1.1 Experimental Objective

[0191] The purpose of this test case is to determine the affinity of a peptide compound for human PSD95-PDZ2 using a competitive ELISA method.

[0192] 3.1.2 Expression and purification of human PSD95-PDZ2 protein

[0193] Using human PSD95 (Uniprot Entry: P78352) as a template for PDZ, the amino acid sequence of the PDZ2 protein was designed: PDZ2 (155-249, the italicized part is the Flag-His-Avitag-TEVsite tag, which will be used for purification and biotin labeling later): MDYKDDDDKGSHHHHHHHHGGGGSGGGGSGLNDIFEAQKIEWHEGGGGSENLYFQGGGGGSAEKVMEIKLIKGPKGLGFSIAGGVGNQHIPGDNSIYVTKIIEGGAAHKDGRLQIGDKILAVNSVGLEDVMHEDAVAALKNTYDVVYLKVAKPSNA (SEQ ID NO.02). The PDZ2 gene was constructed into the PET expression vector and induced to express using BL21-DE3 *E. coli* under the following conditions: 37°C, 1 mM IPTG for 4 h. The bacterial pellet was then collected by centrifugation at 10000g for 10 min at 4°C. The pellet was resuspended in 1×PBS, homogenized, and the supernatant was collected by high-speed centrifugation and filtered through a 0.45 μM filter. Five column volumes of a Ni-Sepharose affinity column were equilibrated with 20 mM phosphate buffer (pH 8.0). The sample was centrifuged at high speed to remove impurities and then loaded onto the column for binding. The column was washed with 20 mM phosphate buffer until the A280 reading returned to baseline. Elution was then performed with a 0-500 mM imidazole gradient using 20 mM phosphate buffer. The protein was collected and identified. The purified sample was then transferred to 1×PBS and concentrated to 2 mL. Further purification was performed using a Superdex 200 (GE) gel chromatography system equilibrated with 1×PBS. The target peak was collected and used for separation.

[0194] 3.1.3 Experimental Methods

[0195] Site-directed biotinylation of Biotin-NA-1 (sequence: Biotin–YGRKKRRQRRRKLSSIESDV; SEQ ID NO.03) was used for competitive ELISA binding detection. In this test, the positive controls were NA-1 (sequence: YGRKKRRQRRRKLSSIESDV; SEQ ID NO.04) and NoNO42 (sequence: YGrKKRrQrRRkLSSIESDV; SEQ ID NO.05), and the negative control was the Ala mutant NA-1 at positions 0 and -2. (ADA)(Sequence: YGRKKRRQRRRKLSSIEADA; SEQ ID NO.06), has been shown to lack PDZ2 binding ability (Science, 2002, 298:846-50).

[0196] Dilute PSD95-PDZ2 (tag removed with TEV enzyme) to 1 μg / mL with 1×PBS buffer, and add 100 μL / well to a 96-well microplate (Corning, 9018, 25 / box 96well clear flat bottom plate). Incubate overnight at 4°C for 16-20 hours. After discarding the liquid, wash the plate three times with PBST (pH 7.4, 0.05% Tween-20) buffer, and then add 300 μL / well of 4% BSA blocking buffer diluted with PBST buffer. Incubate at 37°C for 1 hour to block. After blocking, discard the blocking solution and wash the plate three times with PBST buffer. Add a constant 0.3 μM of Biotin-NA-1 and seven serially diluted (100, 10, 1, 0.1, 0.01, 0.001, 0.0001, 0 μM) test compounds initially at 100 μM and serially diluted tenfold with PBS buffer. Incubate at 37°C for 1 hour. After incubation, discard the reaction solution in the microplate, wash the plate three times with PBST, add 100 μL of HRP-SA secondary antibody (1:2000 dilution) to each well, and incubate at 37°C for 1 hour. After washing three times with PBST, add 100 μL of TMB chromogenic substrate, incubate at room temperature for 1-3 min, and terminate the reaction with 100 μL of 1M sulfuric acid.

[0197] 3.1.4 Sample Analysis and Data Processing

[0198] The absorbance was read at 450 nm using a SpectraMax M5 microplate reader, and the IC50 of the compound binding to the PSD95-PDZ2 protein was calculated using a GraphPad Prism 9 nonlinear fitting method. 50 Values, specific data are shown in Table 1.

[0199] Table 7. Binding ability of peptide compounds to human PSD95-PDZ2

[0200] *This test error is within 3 times;**rIC 50 This represents the ratio of the affinity of the polypeptide compound to NA-1.

[0201] 3.1.5 Experimental Conclusions:

[0202] The results show that the compound of formula (I) disclosed herein can effectively bind to the target protein human PSD95-PDZ2. Given the high conservation of the PSD95-PDZ domain across different species, this facilitates subsequent animal evaluation.

[0203] 3.2. Evaluation of the specificity of peptide compounds binding to human PSD95-PDZ2

[0204] 3.2.1 Experimental Objective

[0205] The three PDZ domains in the PSD95 protein have similar structures. Binding to the PDZ1 and PDZ2 domains can effectively block PSD95-mediated neuroexcitotoxicity, but the biological function of binding to the PDZ3 domain is unclear. Therefore, to avoid potential safety issues, this test case examines the selectivity of peptide compounds to the PDZ2 and PDZ3 domains in order to select peptide compounds that specifically bind to the PDZ2 domain.

[0206] 3.2.2 Expression and purification of human PSD95-PDZ3 protein

[0207] The expression and purification of PSD95-PDZ3 protein were performed according to the steps in 3.1.2. The amino acid sequence of PDZ3 protein is as follows: PDZ3 (309-401, the italicized part is the Flag-His-Avitag-TEVsite tag, which will be used for subsequent purification and biotin labeling): MDYKDDDDKGSHHHHHHHHGGGGSGGGGSGLNDIFEAQKIEWHEGGGGSENLYFQGGGGGSREPRRIVIHRGSTGLGFNIVGGEDGEGIFISFILAGGPADLSGELRKGDQILSVNGVDLRNASHEQAAIALKNAGQTVTIIAQYKPEEYSRFE; SEQ ID NO.07.

[0208] 3.2.3 Experimental Methods

[0209] Site-directed biotinylated Biotin-PDZ2 and Biotin-PDZ3 proteins were used for binding ELISA detection. The positive controls in this test were NA-1 and NoNO42 (WO2022150655A), and the negative control was NA-1. (ADA)Dilute the test compound to 2 μM with 1×PBS buffer and add 100 μL / well to a 96-well microplate (Corning, 9018 25 / box 96well clear flat bottom plate). Incubate overnight at 4°C for 16–20 hours. After discarding the liquid, wash the plate three times with PBST (pH 7.4, 0.05% Tween-20) buffer, then add 300 μL / well of 4% BSA blocking buffer diluted with PBST buffer and incubate at 37°C for 1 hour to block. After blocking, discard the blocking solution and wash the plate three times with PBST buffer. Add Biotin-PDZ2 or Biotin-PDZ3 at an initial concentration of 10 μM, diluted 10-fold with 1×PBS buffer in seven gradients (10, 1, 0.1, 0.01, 0.001, 0.0001, 0.00001, 0 μM). Incubate at 37°C for 1 hour. After incubation, discard the reaction solution in the ELISA plate, wash the plate three times with PBST, add 100 μL of HRP-SA secondary antibody (1:2000 dilution) to each well, and incubate at 37°C for 1 hour. After washing three times with PBST, add 100 μL of TMB substrate and incubate at room temperature for 1-3 minutes. Terminate the reaction by adding 100 μL of 1M sulfuric acid.

[0210] 3.2.4 Sample Analysis and Data Processing

[0211] The absorbance was read at 450 nm using a SpectraMax M5 microplate reader, and the binding EC50 of Biotin-PDZ2 or Biotin-PDZ3 to the test compound was calculated using a GraphPad Prism 9. 50 The values ​​are shown in Table 8.

[0212] Table 8. Binding ability of peptide compounds to human PSD95-PDZ2 and human PSD95-PDZ3

[0213] 3.2.5 Experimental Conclusions

[0214] The results show that the compound of formula (I) disclosed herein can selectively bind to the PSD95-PDZ2 domain, and the selectivity of the compound of formula (I) is comparable to that of the positive drug NA-1.

[0215] 3.3 Risk assessment of hemolysis in rabbit whole blood by polypeptide compounds

[0216] Hemolysis refers to the destruction of the cell membrane of red blood cells, resulting in increased transparency and a deep red color. Certain drug components and excipients contain hemolytic agents, which can cause hemolytic reactions in the human body, leading to adverse reactions such as local swelling and impaired blood circulation. Based on the principle that hemoglobin released from ruptured red blood cells absorbs light in the visible light spectrum, a solution of the test compound was added to a suspension of rabbit red blood cells, incubated, and the degree of hemolysis was measured using an enzyme-linked immunosorbent assay (ELISA) reader.

[0217] 3.3.1 Experimental Objective

[0218] This test case examines whether polypeptide compounds induce hemolysis in rabbit whole blood.

[0219] 3.3.2 Experimental Methods

[0220] Preparation of red blood cell suspension: Take 100 μL of fresh rabbit whole blood, then add 900 μL of 1×PBS solution, place on a plate shaker, shake at 30 rpm for 5 min, then centrifuge at 1000 g for 5 min, and discard the supernatant; repeat the above washing steps until the supernatant no longer appears red, for testing purposes.

[0221] Preparation of test peptide solutions: Add an appropriate amount of 1×PBS solution to the peptide powder to dissolve and obtain a standard stock solution. Then dilute with 1×PBS to obtain test solutions with concentrations of 1, 3, 10, 30, 100, and 300 μg / mL, with two replicates for each concentration. Simultaneously, blank 1×PBS was used as a negative control, and a 1×PBS solution containing 0.1% Triton X-100 was used as a positive control.

[0222] Incubation process: Add 500 μL of test solution to the red blood cell suspension, then shake on a plate shaker at 30 rpm for 5 min to mix thoroughly. Then incubate the suspension at 37°C for 1 hour, and then centrifuge at 1000g for 5 min.

[0223] 3.3.3 Sample Analysis and Data Processing

[0224] Transfer 100 μL of the supernatant to a single well of an ELISA plate and measure the absorbance at 540 nm using a SpectraMax M5 microplate reader. Analyze the data using a GraphPad Prism 9. The hemolysis rate (%) is calculated as (test sample absorbance - negative control absorbance) / (positive control absorbance - negative control absorbance) × 100%. A result less than 5% indicates no hemolysis; a result greater than 5% indicates hemolysis.

[0225] 3.3.4 Risk assessment of hemolysis in rabbit whole blood by polypeptide compounds

[0226] The experimental results are shown in Figure 1. The results indicate that in rabbit whole blood, the compound of formula (I) and the positive control drugs NA-1 and NoNO42 all meet the requirement of hemolysis rate being less than 5% in the concentration range of 1 to 50 μM, and there is no risk of hemolysis.

[0227] 3.4 In vitro stability study of peptide compounds in rat and human plasma

[0228] Plasma contains a variety of hydrolytic enzymes that can break down and metabolize drug molecules, resulting in a rapid decrease in the concentration of drug molecules in the plasma, which fails to reach an effective concentration. This leads to a high clearance rate and a short half-life, resulting in poor pharmacokinetic and pharmacodynamic properties. Therefore, plasma stability is an important indicator affecting drug efficacy.

[0229] 3.4.1 Experimental Objective

[0230] This test case examines the in vitro stability of peptide compounds in rat and human plasma, and is divided into two parts: 1) the stability of the peptide compound alone in rat and human plasma; 2) the stability of the peptide compound in rat and human plasma when it is co-administered with alteplase (rt-PA).

[0231] 3.4.2 Experimental Methods

[0232] Solution preparation: Weigh an appropriate amount of the test peptide compound and dissolve it in an appropriate amount of 1×PBS to obtain a 1mM test solution. NA-1 is the positive control, bromhexine is used as the human plasma test control, and lovastatin is used as the rat plasma test control.

[0233] Plasma stability test: Add 12.5 μL of 1 mM test solution to 495 μL of pre-incubated plasma sample. Aliquot the prepared plasma sample into 50 μL portions into centrifuge tubes for different incubation times (0 min, 10 min, 20 min, 30 min, 60 min, 120 min), and incubate at 37°C with shaking at 60 rpm. Perform duplicate incubation for each time. Add 200 μL of stop solution to stop incubation and vortex for 5 min. Then centrifuge at 10000 rpm at 4°C for 10 min to remove protein. Transfer 70 μL of supernatant to a new 96-well plate (add 70 μL of water to each well before mixing).

[0234] Plasma stability test with added alteplase: Weigh an appropriate amount of alteplase solid, add an appropriate volume of water, vortex to dissolve, and obtain an alteplase 1 mg / mL solution. Take 250 μL of the 1 mg / mL alteplase solution and add it to 4750 μL of plasma to obtain plasma containing 50 μg / mL alteplase. Other procedures are the same as in the previous "Plasma Stability Test", except that plasma containing 50 μg / mL alteplase is used instead of regular plasma.

[0235] 3.4.3 Sample Analysis and Data Processing:

[0236] The content of the remaining compounds at each time point was detected by LC-MS / MS, and the relative content of the remaining compounds at other time points was calculated with 0 min as the standard (100%).

[0237] Sample pretreatment method: Take 30 μL of plasma sample, add 120 μL of 5% formic acid methanol solution containing 1 ng / mL internal standard (verapamil), vortex for 5 min, centrifuge at 10000 rpm for 10 min at low temperature, take 70 μL of supernatant, add 70 μL of 0.1% formic acid aqueous solution, mix well, and analyze by LC-MS instrument.

[0238] The LC-MS analysis method was as follows: (1) Chromatographic conditions: Mobile phase A was 0.1% formic acid aqueous solution, mobile phase B was 0.1% formic acid acetonitrile solution; flow rate was 0.5 mL / min; injection volume was 10 μL; chromatographic column was Nanomicro Unisil C18aq (4.6 mm × 150 mm, 5 μm); column temperature was 40℃. (2) Mass spectrometry conditions: Mass spectrometry was performed using electrospray ionization (ESI), positive ion analysis mode, and multiple reaction monitoring (MRM) scanning mode. The experimental results are shown in Table 9, Figure 2A and Figure 2B.

[0239] Table 9. Plasma stability of polypeptide compounds

[0240] 3.4.4 Experimental Conclusions

[0241] The results showed that NA-1 underwent significant degradation in rat plasma within 2 hours; the degradation rate of NA-1 accelerated after co-incubation with alteplase. These results are consistent with the phase III clinical trial outcomes of NA-1, indicating that the addition of alteplase rapidly disrupts the molecular structure of NA-1, rendering it biologically inactive. The plasma resistance of peptide compound 14 to alteplase makes it a promising molecule to compensate for the clinical disadvantages of NA-1.

[0242] Compound (I) is stable in human and rat plasma. After the addition of alteplase, compound (I) remains stable in human plasma, but its degradation rate in rat plasma is significantly slower than that of NA-1.

[0243] The resistance of compound (I) to alteplase in plasma allows it to compensate for the clinical disadvantages of NA-1.

[0244] 3.5 Pharmacokinetic Study of Peptide Compounds in Rats

[0245] 3.5.1 Experimental Objective

[0246] Using male SD rats as test animals, the pharmacokinetic behavior of a single intravenous bolus injection of a polypeptide compound in rat plasma was studied.

[0247] 3.5.2 Experimental Methods

[0248] Male SD rats weighing 170-200 grams and aged 4-6 weeks were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. After preparing a polypeptide compound solution using 1×PBS, the drug was administered intravenously at a dose of 3 nmol / g over 10 minutes, with 3 animals per group. Blood samples of 0.2 mL were collected at 2, 8, 15, 30, 45, 60, and 120 minutes after infusion. Immediately after collection, enzyme inhibitors were added, and the blood samples were transferred to centrifuge tubes containing EDTA-K2 anticoagulant. Whole blood samples were centrifuged at 4000g for 5 minutes at 4°C to obtain plasma, which was then stored at -80°C.

[0249] 3.5.3 Sample Analysis and Data Processing

[0250] The plasma concentrations of each analyte were determined using the method described in Test Example 3.4.3. Plasma concentration-time curves were plotted, and pharmacokinetic parameters were calculated using PKSolver software. The experimental results are shown in Table 10.

[0251] 3.5.4 Experimental Conclusions

[0252] The results in Table 10 show that the addition of enzyme inhibitors reduced the degradation of NA-1 after blood collection, resulting in improved AUC and Cmax values. Compared to NA-1, the half-life of compound (I) was significantly prolonged by 29 times, the area under the curve (AUC) increased by 19.4 times, and the maximum plasma concentration (Cmax) increased by 2.3 times.

[0253] Table 10. Pharmacokinetic parameters of rats after intravenous bolus injection (n=3)

[0254] 3.6 Pharmacokinetic Study of Peptide Compounds in Beagle Dogs

[0255] 3.6.1 Experimental Objective

[0256] Using ordinary beagle dogs as test animals, this study investigated the pharmacokinetic behavior of a single intravenous infusion of a polypeptide compound in beagle dogs (plasma).

[0257] 3.6.2 Experimental Methods

[0258] Compound (I) was administered at two dose groups of 1.2 nmol / g and 0.4 nmol / g, and NA-1 was administered at a dose group of 1.2 nmol / g. Each group consisted of four animals, half male and half female. The polypeptide compound solution was prepared by dissolving it in 1×PBS and administered via intravenous infusion over 10 minutes. Blood was collected at the following time points: before infusion (0 h), 5 min after the start of the bolus, and 0 min, 2 min, 5 min, 10 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, and 24 h after the end of the bolus. 0.5 mL of blood was collected at each time point and transferred to centrifuge tubes containing EDTA-K2 anticoagulant. 50 μL of protease inhibitor was added simultaneously, and the mixture was centrifuged at 2000g for 10 min (4 °C). Plasma was separated within 1 h, aliquoted into cryovials, and stored at -60 to -90 °C. The entire process from blood collection to centrifugation was performed under ice bath conditions.

[0259] 3.6.3 Sample Analysis and Data Processing

[0260] The plasma concentrations of each analyte were determined using the method described in Test Example 3.4.3. Plasma concentration-time curves were plotted, and pharmacokinetic parameters were calculated using PKSolver software. The experimental results are shown in Table 11 and Figure 3.

[0261] 3.6.4 Experimental Conclusions

[0262] The results showed that the pharmacokinetic properties of the tested compound (I) were significantly better than those of NA-1. At the same dose, the average T in dogs... 1 / 2 The average AUC for NA-1 was 2.7 min, while that for compound (I) was 5.1 h; the average AUC for compound (I) was 21.3 times that of NA-1; the average C... max The concentration of compound (I) was 1.3 times that of NA-1. The drug exposure of compound (I) was positively correlated with the administered dose.

[0263] Table 11. Pharmacokinetic parameters of intravenous infusion in beagle dogs

[0264] 3.7 Test of the ability of peptide compounds to induce histamine release

[0265] 3.7.1 Experimental Objective

[0266] Using beagle dogs as test animals, this study investigated the ability of a single intravenous infusion of a polypeptide compound to induce histamine release in canine plasma.

[0267] 3.7.2 Experimental Methods

[0268] The experiment was conducted concurrently with test case 3.6, with 4 animals in each group. Blood collection points were set as follows: before drug administration (0h), 2 min, 5 min, and 15 min after the intravenous infusion.

[0269] 3.7.3 Sample Analysis and Data Processing

[0270] The contents of histamine and 3-methylhistamine at each time point were detected by LC-MS / MS. The LC-MS method for detecting the compounds was as follows: (1) Chromatographic conditions: Mobile phase A was acetonitrile / water / 100mM ammonium acetate, 50 / 45 / 5 (v / v / v), 2% FA; Mobile phase B was acetonitrile / 100mM ammonium acetate, 95 / 5 (v / v), 2% FA; Flow rate was 0.4 mL / min; Column was Waters BEH HILIC 1.7 μm, 2.1 × 150 mm; Column temperature was 50℃. Injection volume was 20 μL; (2) Mass spectrometry conditions: Mass spectrometry was performed using electrospray ionization (ESI), positive ion analysis mode, and multiple reaction monitoring (MRM) scanning mode.

[0271] Sample pretreatment method: Take 30 μL of plasma sample and add it to 200 μL of acetonitrile solution containing tetradeuterated histamine (D4-Histamine, 100 ng / mL). Vortex for 1 min, then centrifuge at 5800 rpm for 10 min. Take 100 μL of supernatant into the sample tray for instrument analysis. Table 12 and Figure 4 show the summation results of the detected histamine and 3-methylhistamine.

[0272] Table 12. Histamine levels at different time points

[0273] 3.7.4 Experimental Conclusions

[0274] Compared with the pre-administration values, no significant changes in histamine levels were observed at either of the two doses of compound (I), indicating good safety.

[0275] 3.8 Pharmacodynamic effects of peptide compounds in rat tMCAO model

[0276] 3.8.1 Experimental Objective

[0277] This study aimed to investigate the pharmacodynamic effects of peptide compounds on a rat tMCAO (transient midbrain artery occlusion) model. High-dose NA-1 (3 nmol / g) was used as a positive control, and three doses of compound (I) were set: high (3 nmol / g), medium (1 nmol / g), and low (0.3 nmol / g). By comparing the infarct area and neurological function scores of rats, and combining with previous tests, a safe and effective dosage reference was provided for the in-depth evaluation of compound (I).

[0278] 3.8.2 Experimental Methods

[0279] Modeling and Drug Administration: Male SD rats weighing 240-260 grams and aged 6-8 weeks were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. A rat MCAO model was established using the suture occlusion method. Drug administration was administered 60 minutes after suture insertion, and the suture was removed 90 minutes later for reperfusion. Inclusion criteria included a baseline cerebral blood flow before suture insertion, a 50% or greater reduction in cerebral blood flow from baseline after insertion, and immediate recovery of cerebral blood flow to at least 50% of pre-reperfusion levels after suture removal. Animals not meeting these criteria were excluded. The polypeptide compound was dissolved in physiological saline to the required concentration. One hour after MCAO suture insertion, the polypeptide compound was slowly injected via a single tail vein injection over 4-5 minutes. The model control group received intravenous injection of physiological saline for 4-5 minutes. The endpoint was 24 hours after drug administration. The overall condition of the test animals during the drug administration period was observed: spontaneous activity, fasting and water intake status, mortality, and other abnormal manifestations.

[0280] Infarct area measurement: 24 hours after MCAO, surviving rats in each group were dissected, their hearts were perfused with pre-cooled PBS, and their heads were severed to obtain the whole brain. Brain sections were stained in 2% red tetrazolium (TTC) solution to calculate the infarct area and the percentage of infarct area. Percentage of cerebral infarct area = Infarct area / Total brain area × 100%.

[0281] Neurological function impairment severity assessment: Before modeling (excluding animals with abnormal pre-modeling behavioral scores) and 24 hours after drug administration, the degree of neurological function impairment was assessed using a blinded method according to the scoring criteria in the attached table. The total score is 16 points. A higher score indicates a more severe degree of impairment. Detailed scoring criteria are shown in Table 13.

[0282] Table 13. Scoring criteria for the degree of neurological function impairment in MCAO rats

[0283] 3.8.3 Sample Analysis and Data Processing

[0284] IBM SPSS Statistics 25.0 statistical software was used for data analysis. The experimental data were presented as follows: The results indicate that the LSD test was used to compare data with homogeneous variance, and the Dunnett's T3 test was used to compare data with unequal variance. A p-value < 0.05 was considered statistically significant. Specific experimental results are shown in Table 14, Figures 5A and 5B.

[0285] Table 14. Summary of pharmacodynamic results in the rat tMCAO model

[0286] 3.8.4 Experimental Conclusions

[0287] The results showed that a single dose of compound (I) improved neurological function and cerebral infarction area in tMCAO rats in a dose-dependent manner. The medium dose (1 nmol / g) of compound (I) was comparable in efficacy to the high dose (3 nmol / g) of the positive control drug NA-1.

Claims

1. A pharmaceutical composition comprising an active ingredient, a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein the compound of formula (I) or a pharmaceutically acceptable salt thereof is selected from the amino acid sequence rKKRrQRRrG-Aib-Dab-T-Tle-Tle-TDV (SEQ ID NO. 01) (I); in, The pH of the pharmaceutical composition is 5.0-9.0; Preferably, the pH of the pharmaceutical composition is 6.0-8.0; More preferably, the pH of the pharmaceutical composition is 6.5-7.

5.

2. The pharmaceutical composition according to claim 1, wherein the concentration of the active ingredient or its pharmaceutically acceptable salt is 10 mg / mL to 500 mg / mL (as a free base); Preferably, the concentration of the active ingredient or its pharmaceutically acceptable salt is 20 mg / mL to 90 mg / mL (as a free base). More preferably, the concentration of the active ingredient or its pharmaceutically acceptable salt is 55 mg / mL to 65 mg / mL (as a free base).

3. The pharmaceutical composition according to claim 1 or 2, wherein the pharmaceutically acceptable salt of the active ingredient is selected from chloride salts, acetates, or bicarbonates; Preferably, the pharmaceutically acceptable salt of the active ingredient is a chloride salt.

4. The pharmaceutical composition according to any one of claims 1-3, wherein the pharmaceutical composition further comprises a buffer; Preferably, the buffer is selected from one or more of acetate buffer, histidine buffer, glycine buffer, phosphate buffer, succinate buffer, citrate buffer, and tris(hydroxymethyl)aminomethane buffer; More preferably, the buffer is selected from one or more of acetate buffers, histidine buffers, phosphate buffers, succinate buffers, and citrate buffers; Most preferably, the buffer is selected from histidine buffers.

5. The pharmaceutical composition according to claim 4, wherein the concentration of the buffer is from 5.0 mM to 50.0 mM; Preferably, the concentration of the buffer is from 10.0 mM to 50.0 mM; More preferably, the concentration of the buffer is 10.0 mM to 20.0 mM.

6. The pharmaceutical composition according to any one of claims 1-5, wherein the pharmaceutical composition further comprises a filler.

7. The pharmaceutical composition according to claim 6, wherein the filler is selected from one or more amino acids and sugars.

8. The pharmaceutical composition according to claim 7, wherein the sugar is selected from one or more of mannitol, sorbitol, xylitol, trehalose, glucose, sucrose, maltose, and dextran; Preferably, the sugar is selected from one or more of mannitol, trehalose, glucose, sucrose, and maltose.

9. The pharmaceutical composition according to claim 8, wherein the sugar is selected from mannitol and has a concentration of 40 mM-400 mM; Preferably, the concentration of mannitol is 200 mM-400 mM; More preferably, the concentration of mannitol is 240mM-360mM.

10. The pharmaceutical composition according to claim 8, wherein the sugar is selected from trehalose and has a concentration of 20 mM-200 mM; Preferably, the concentration of trehalose is 60mM-120mM.

11. The pharmaceutical composition according to claim 7, wherein the amino acid is selected from one or more of glycine, alanine, arginine, aspartic acid, asparagine, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, methyllysine, ornithine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

12. The pharmaceutical composition according to claim 11, wherein the amino acid is selected from glycine and has a concentration of 100 mM-400 mM; Preferably, the concentration of glycine is 120mM-360mM.

13. The pharmaceutical composition according to any one of claims 1-12, wherein the pharmaceutical composition further comprises a pH adjuster; Preferably, the pH adjuster comprises sodium hydroxide and / or hydrochloric acid.

14. The pharmaceutical composition according to any one of claims 1-13, wherein the pH of the pharmaceutical composition is 6.5 to 7.5, wherein: (1) The pharmaceutical composition comprises: 55 mg / mL to 65 mg / mL (as free base) of the compound represented by formula (I) or its pharmaceutically acceptable salt; 10.0 mM to 20.0 mM histidine buffer, and 240mM to 360mM mannitol; (2) The pharmaceutical composition comprises: 55 mg / mL to 65 mg / mL (as free base) of the compound represented by formula (I) or its pharmaceutically acceptable salt; 10.0 mM to 20.0 mM citrate buffer, and 240mM to 360mM mannitol; (3) The pharmaceutical composition comprises: 55 mg / mL to 65 mg / mL (as free base) of the compound represented by formula (I) or its pharmaceutically acceptable salt; 10.0 mM to 20.0 mM phosphate buffer, and 240mM to 360mM mannitol; (4) The pharmaceutical composition comprises: 55 mg / mL to 65 mg / mL (as free base) of the compound represented by formula (I) or its pharmaceutically acceptable salt; 10.0 mM to 20.0 mM histidine buffer, and 60mM to 120mM trehalose; (5) The pharmaceutical composition comprises: 55 mg / mL to 65 mg / mL (as free base) of the compound represented by formula (I) or its pharmaceutically acceptable salt; 10.0 mM to 20.0 mM citrate buffer, and 60mM to 120mM trehalose; (6) The pharmaceutical composition comprises: 55 mg / mL to 65 mg / mL (as free base) of the compound represented by formula (I) or its pharmaceutically acceptable salt; 10.0 mM to 20.0 mM phosphate buffer, and 60mM to 120mM trehalose; (7) The pharmaceutical composition comprises: 55 mg / mL to 65 mg / mL (as free base) of the compound represented by formula (I) or its pharmaceutically acceptable salt; 10.0 mM to 20.0 mM histidine buffer, and 120mM to 360mM glycine; (8) The pharmaceutical composition comprises: 55 mg / mL to 65 mg / mL (as free base) of the compound represented by formula (I) or its pharmaceutically acceptable salt; 10.0 mM to 20.0 mM citrate buffer, and 120mM to 360mM glycine; or (9) The pharmaceutical composition comprises: 55 mg / mL to 65 mg / mL (as free base) of the compound represented by formula (I) or its pharmaceutically acceptable salt; 10.0 mM to 20.0 mM phosphate buffer, and 120mM to 360mM glycine; The pharmaceutical composition optionally includes a pH adjuster, which comprises sodium hydroxide and / or hydrochloric acid.

15. The pharmaceutical composition of claim 14, wherein the pH of the pharmaceutical composition is 6.5 to 7.5, wherein the pharmaceutical composition comprises: 55 mg / mL to 65 mg / mL (as free base) of the compound represented by formula (I) or its pharmaceutically acceptable salt; 10.0 mM to 20.0 mM histidine buffer, and 240mM to 360mM mannitol; The pharmaceutical composition optionally includes a pH adjuster, which comprises sodium hydroxide and / or hydrochloric acid.

16. The pharmaceutical composition according to any one of claims 1-15, wherein the composition is a liquid formulation obtained by reconstitution of a lyophilized formulation or a liquid formulation prior to lyophilization.

17. A method for preparing a pharmaceutical composition according to any one of claims 1-16, comprising the step of dissolving a compound of formula (I) or a pharmaceutically acceptable salt thereof.

18. A lyophilized formulation, wherein the lyophilized formulation, upon reconstitution, can form a pharmaceutical composition according to any one of claims 1-16, or the lyophilized formulation is obtained by freeze-drying a pharmaceutical composition according to any one of claims 1-16.

19. A method for preparing the lyophilized formulation of claim 18, comprising the step of lyophilizing the pharmaceutical composition of any one of claims 1-16.

20. A reconstituted solution, wherein the reconstituted solution is prepared by reconstituted lyophilized formulation of claim 18.

21. An article comprising a container containing a pharmaceutical composition as claimed in any one of claims 1 to 16, a lyophilized formulation as claimed in claim 18, or a reconstituted solution as claimed in claim 20.

22. Use of the pharmaceutical composition according to any one of claims 1-16, the lyophilized formulation according to claim 18, the reconstituted solution according to claim 20, or the article according to claim 21 in the preparation of a pharmaceutical product; The drug is used to treat and / or prevent stroke, cerebral ischemia, traumatic injury to the central nervous system, reperfusion injury, subarachnoid hemorrhage, concussion, pain, anxiety, epilepsy, neurodegenerative diseases and / or diseases with the above risks.

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