EA044400B1 - PEPTIDE COMPOSITION FOR THE TREATMENT OF DAMAGES ASSOCIATED WITH EXCITATIVE NEUROTOXICITY - Google Patents

Description

Field of technology

This application generally relates to the field of medicine. In particular, the present application provides compositions for the treatment of damage to the central nervous system and their use.

Prior Art

Strokes are common acute cerebrovascular diseases in middle-aged and elderly people, and tend to affect younger people. In the modern world, cerebrovascular diseases are among the three most dangerous diseases for people (cancer, cardio-cerebrovascular diseases and diabetes). It is estimated that about three million people die from cerebrovascular diseases every year in China. This number is 4 to 5 times higher than the US and European countries, 3.5 times higher than Japan, and even higher than some developing countries such as Thailand and India. The incidence rate of this disease is increasing by 8.7% per year. The relapse rate exceeds 30%, and the five-year relapse rate reaches 54%. Of those who survive a stroke, 75% lose their ability to work to a greater or lesser extent, and 40% become disabled.

Stroke can be broadly classified into two categories, namely, ischemic stroke and hemorrhagic stroke, and ischemic stroke patients account for 85% of the total stroke patients. Currently, therapeutic drugs for the treatment of ischemic strokes mainly include vasodilators (such as persantine), microcirculation and blood volume increasing drugs (such as low molecular weight dextran), thrombolytic drugs (such as urokinase), anticoagulant drugs, drugs anti-platelet aggregation agents (such as aspirin), Chinese medicine, neuroprotective agents, and so on. However, since most of these drugs have disadvantages, such as significant side effects, possible risks or lack of therapeutic efficacy, the study of the pathogenesis of stroke and the development of drugs aimed at eliminating the causes of pathogenesis are of great social importance for the prevention and treatment of the occurrence and development of cerebrovascular diseases. diseases.

Strokes are characterized by the death of nerve cells in areas of local ischemia, cerebral hemorrhage and/or trauma. Neuronal death or damage caused by cerebral ischemia occurs as a cascade process of damage, i.e. after the occurrence of cerebral ischemia, there is a decrease in tissue perfusion with blood, an increase in the level of excitatory neurotransmitters, which in turn activates K-methylIo-aspartate (NMDA) receptors and α-amino-3hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, causing the opening of ion channels and the influx of calcium ions further activates a large number of enzymes to initiate a signaling cascade, which leads to damage to nerve cells in numerous ways. Downstream in the signaling cascade, postsynaptic seal protein 95 (PSD-95) triggers a series of ischemic lesions through interactions with various proteins and is therefore a critical factor in cerebral ischemia injury and a promising target for drug therapy. Therefore, the development of PSD-95 inhibitors is of great medical importance from the point of view of nervous system damage caused by various types of excitatory neurotoxicity, including stroke.

In addition, research has shown that the excitatory neurotransmitter NMDA plays an important role in anxiety disorder, epilepsy and various neurodegenerative diseases such as Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Parkinson's disease or Huntington's disease. For example, studies have shown that overexcitation of the central glutamatergic system can cause anxiety disorders because the NMDA receptor (NMDAR) is the main element responsible for glutamic acid-induced neurotoxicity. An epileptic attack involves three distinct but long-term pathophysiological processes, including initiation, maintenance and development of seizures, as well as suppression of seizures. During this process, excitatory neurotransmitters such as glutamic acid and aspartic acid play an important role. In Alzheimer's disease, PSD-95 is involved in the mechanism of neurotoxicity of this disease through the GluR6-PSD-95-MLK3 (glutamate receptor 6-PSD-95-mixed lineage kinase 3) pathway. In addition, in Huntington's disease, PSD-95 is a mediator of neurotoxicity caused by NMDA receptors and mutant huntingtins. Therefore, the development of PSD-95 inhibitors is also important for the treatment, relief of symptoms and prevention of the above diseases.

Peptide drugs have limitations due to the storage conditions of the drugs and are characterized by a limited ability to withstand environmental influences. When peptides are stored at high temperatures and for long periods of time, changes in pH can occur, which can lead to degradation, decreased purity, significant changes in appearance and shortened shelf life, thereby affecting the effectiveness of the drug. In addition, there are high requirements for the transport of peptide drugs, which limits large-scale commercialization.

- 1 044400 use of peptide drugs. Thus, there is a need for technical improvements in peptide drugs.

Brief description of the invention

According to a first aspect, the present application provides a pharmaceutical composition comprising a peptide, a pH adjusting agent and an excipient, wherein the peptide contains the amino acid sequence YEKLLDTEI (SEQ ID NO: 1) or a functional variant thereof.

In some embodiments, the functional variant is a variant formed by one or more conservative substitutions in the LDTEI segment of SEQ ID NO: 1, preferably a conservative substitution selected from the group consisting of a substitution between D and E, a substitution among L, V, and I and substitutions between T and S.

In some embodiments, the functional variant is a variant formed by replacing the LDTEI segment in SEQ ID NO: 1 with a sequence selected from the group consisting of LDTEL, LDTEV, LDTDI, LDTDL, LDTDV, LDSEI, LDSEL, LDSEV, LDSDI, LDSDL, LDSDV , LETEI, LETEL, LETEV, LETDI, LETDL, LETDL, LETDL, LETDL, LETDL, LETDL, LETDL, IDTEI, IDTEL, IDTEV, IDTDI, IDTDL, IDTDV, IETEI, IETEL, IETEV, IETDI, IETDL, IETDL and IETDV.

In some embodiments, the peptide is a chimeric peptide comprising the amino acid sequence YEKLLDTEI (SEQ ID NO: 1) or a functional variant thereof, and an internalization peptide, wherein the internalization peptide facilitates uptake of the chimeric peptide into a cell.

In some embodiments, the internalization peptide comprises the amino acid sequence YGRKKRRQRRR (SEQ ID NO: 2).

In some embodiments, the chimeric peptide contains the amino acid sequence YGRKKRRQRRRYEKLTTLDTGGV (SEQ ID NO: 3).

In some embodiments, the pH adjusting agent is selected from the group consisting of histidine buffer, arginine buffer, sodium succinate buffer, potassium succinate buffer, sodium citrate buffer, gluconate buffer, acetate buffer, phosphate buffer, trisbuffer, and any combination thereof preferably, the pH adjusting agent is a citric acid/disodium phosphate buffer or a histidine/arginine buffer, and more preferably the pH adjusting agent is a histidine/arginine buffer.

In some embodiments, the pH of the composition is from about 5.5 to 8, preferably from about 6 to 7.5, more preferably from about 6 to 7, even more preferably from about 6.5 to 7, and most preferably is about 6.5.

In some embodiments, the amount of histidine/arginine in the histidine/arginine buffer is, by weight, from about 1 to 10%, preferably from about 3 to 10%.

In some embodiments, the excipient is selected from the group consisting of trehalose, mannitol, glucose, lactose, cyclodextrin, dextran-40, sorbitol, sucrose, glycine and any combination thereof, preferably the excipient is selected from the group consisting of trehalose, mannitol, glucose, lactose and any combination thereof, and more preferably the filler is trehalose.

In some embodiments, the weight ratio of peptide to trehalose is from about 1:0.05 to 1:10, preferably from about 1:0.5 to 1:5, more preferably from about 1:0.8 to 1:3, and most preferably is approximately 1:1.

In some embodiments, the excipient is trehalose and the pH adjusting agent is a histidine/arginine buffer.

In some embodiments, the mass ratio of peptide to trehalose is approximately 1:1.

In some embodiments, the pH of the composition is about 6.5 ± 0.5.

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

In some embodiments, the composition further comprises a deamidation inhibitor.

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

In some embodiments, the pharmaceutical composition is for use in the treatment, reduction of symptoms, or prevention of a disease selected from the group consisting of nervous system damage, disease or pain associated with nervous system damage, neurodegenerative disease, anxiety disorder, and epilepsy, in mammal or for use as a neuroprotective agent.

According to a second aspect, the present application provides a method of treating, alleviating symptoms of, or preventing a disease selected from the group consisting of nervous system injury, disease or pain associated with nervous system injury, neurodegenerative disease, anxiety disorder, and epilepsy in a mammal, comprising administering to a subject in need thereof a pharmaceutical composition according to the first aspect.

According to a third aspect, the present application provides the use of a pharmaceutical composition according to the first aspect in the preparation of a medicament for the treatment, alleviation of symptoms or prevention of a disease selected from the group consisting of nervous system damage, disease or pain associated with nervous system damage, neurodegenerative disease, anxiety disorder and epilepsy, in a mammal or in the preparation of a neuroprotective agent.

In some embodiments of any of the above aspects, the disease is a stroke or damage to the nervous system caused by a stroke.

In some embodiments of any of the above aspects, the stroke includes ischemic stroke, hemorrhagic stroke, and hemorrhagic stroke converted from ischemic stroke. Preferably, the stroke is an ischemic stroke.

In some embodiments of any of the above aspects, the nervous system damage is nervous system damage caused by excitatory neurotoxicity.

In some embodiments of any of the above aspects, the injury or pain is located in the peripheral nervous system or the central nervous system.

In some embodiments of any of the above aspects, damage to the nervous system caused by excitatory neurotoxicity includes damage selected from the group consisting of stroke, spinal cord injury, ischemic or traumatic injury to the brain or spinal cord, neuronal damage in the central nervous system (CNS), including acute CNS injury, ischemic stroke or spinal cord injury, hypoxia, ischemic or mechanical injury, and injury caused by neurodegenerative disease, anxiety disorder, epilepsy or stroke.

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

Brief description of graphic materials

In fig. Figure 1 shows the results of an affinity adsorption assay to detect the interaction between P5 and the PDZ1/2 domain. Lane M shows protein molecular weight markers; Lane 1 shows the His+PDZ1/2+P5 pattern; track 2 shows only one P5; lane 3 shows the His+P5 sample; Lane 4 shows the His+PDZ1/2 pattern. As shown in lane 1, the eluted fraction contained both P5 and PDZ1/2, confirming the ability of P5 to bind to the PDZ1/2 domain.

In fig. Figure 2 shows images of 2,3,5-triphenyltetrazolium chloride (TTC)-stained brain sections of rats in the middle cerebral artery occlusion (MCAO) model treated with P5 polypeptide: a) group of normal animals; b) simulation group; c) a group of model animals; d) group receiving positive control drug (Enbipu injection solution); e) NA-1 at a dose of 10 mg/kg body weight; f) YE-NA-1 at a dose of 10 mg/kg body weight; g) P5 at a dose of 10 mg/kg body weight; h) P5 at a dose of 3 mg/kg body weight; i) P5 at a dose of 1 mg/kg body weight; j) prophylactic administration of P5 at a dose of 10 mg/kg body weight; k) prophylactic administration of P5 at a dose of 3 mg/kg body weight; 1) prophylactic administration of P5 at a dose of 1 mg/kg body weight.

Fig. 3 is a bar graph showing statistics for cerebral infarct volume following therapeutic and prophylactic administration of various doses of P5 polypeptide to rats in the MCAO model. **p<0.01.

In fig. Figure 4 shows the distribution of P5 polypeptide in rat brain samples.

In fig. Figure 5 shows images of TTC-stained rat brain samples.

In fig. Figure 6 shows images of hematoxylin and eosin (HE)-stained paraffin sections of rat brain samples. A) group of normal animals; B) imitation group; C) a group of model animals; D) group receiving positive control drug; E) group receiving NA-1; F) group receiving YE-NA-1; G) group receiving P5; H) group receiving P5 for prophylactic purposes.

In fig. Figure 7 shows the effect of different excipients on the shape and stability of lyophilized compositions based on P5 (No. 0, No. 1 and No. 2) on day 0.

In fig. Figure 8 shows the effect of different excipients on the shape and stability of lyophilized compositions based on P5 (No. 3, No. 4 and No. 5) on day 0.

In fig. Figure 9 shows the effect of different excipients on the shape and stability of lyophilized P5-based formulations (No. 0, No. 1 and No. 2) after 1 week.

In fig. 10 shows the effect of different excipients on the shape and stability of lyophilized P5-based compositions (No. 3, No. 4 and No. 5) after 1 week.

In fig. 11 shows the results obtained after 1 week using histidine/arginine to adjust the pH of the composition (ie, pH range screening experiment II).

In fig. 12 shows the results obtained after 2 weeks using histidine/arginine to adjust the pH of the composition (ie, pH range screening experiment II).

- 3 044400

Detailed Description of the Invention

The present inventors have developed peptides for the treatment of damage to the central nervous system. To better utilize these peptides in industrial practice, the inventors, based on extensive research, have developed compositions containing the peptide, an excipient and a buffer for pH adjustment. Such compositions may have at least one of the following advantages.

1. The compositions are a white, loose, lyophilized mass with a pharmaceutically attractive appearance. The presence of the filler has a beneficial effect in terms of improving the decomposition temperature (white fluffy lyophilized mass), providing protection during lyophilization and increasing the stability of proteins during long-term storage.

2. The stability of the peptide is enhanced by providing an amorphous glassy matrix that binds to the protein via hydrogen bonds to replace the water molecules that must be removed during drying. This makes it easier to maintain peptide conformation, minimizes peptide degradation during the lyophilization cycle, and improves the long-term stability of the final product.

3. These compositions are resistant to environmental influences and do not decompose over a relatively long storage period. The appearance of the compositions, the purity of the compositions and the content of impurities therein may meet the requirements for clinical use.

Definitions

Unless otherwise specified, terms used in this application have the meaning as commonly understood by one of ordinary skill in the art.

The one-letter or three-letter abbreviations for amino acids used in this application are consistent with international regulations.

The term chimeric peptide means a peptide having two peptide components that are not naturally associated with each other. These two peptide components may form a fusion protein or may be joined via a chemical bond.

The term PDZ domain refers to a modular protein domain of approximately 90 amino acids characterized by high sequence identity (e.g., at least 60%) with the synaptic protein PSD-95, the Discs-Large (DLG) connexin protein from Drosophila, and the epithelial protein tight contacts Z01. The PDZ domain is also known as Discs-Large protein homologous repeats (DHRs) and GLGF repeats. The PDZ domain typically retains a core consensus sequence (Doyle, D. A., 1996, Cell, 85: 1067-76). Exemplary PDZ domain-containing proteins and PDZ domain sequences are described in US Application No. 10/714,537.

The term NMDA receptor or NMDAR refers to a membrane-associated protein that is known to interact with NMDA. These receptors can be in humans or in non-human animals (eg mice, rats, rabbits or monkeys).

The term specific binding refers to the binding of two molecules (eg, a ligand and a receptor), characterized by the fact that one molecule (eg, a ligand) is able to bind to another specific molecule (eg, a receptor) even in the presence of many other different molecules, i.e. the ability of one molecule to bind preferentially to another molecule in a mixture of heterogeneous molecules. Specific binding of a ligand to a receptor can also be confirmed if the binding of a detected labeled ligand to the receptor is attenuated in the presence of an excess of unlabeled ligands (ie, a competitive binding assay).

The term statistically significant means a p-value of <0.05, preferably <0.01, most preferably <0.001.

The term functional variant refers to a variant having the same or similar biological function and property as the parent variant. As a non-limiting example, a functional variant can be obtained by making one or more conservative substitutions in the original variant.

The term internalization peptide, also known as cell entry-facilitating peptide, is widely used in the field of protein therapeutics and is intended to facilitate the uptake and uptake into cells of an active peptide bound to the internalization peptide. As a non-limiting example, the peptide involved in internalization may be a Tat (transcription transactivator) peptide. One non-limiting example of Tat peptides is YGRKKRRQRRR (SEQ ID NO: 2).

According to a first aspect, the present application provides a pharmaceutical composition comprising a peptide, a pH adjusting agent and an excipient, wherein the peptide contains the amino acid sequence YEKLLDTEI (SEQ ID NO: 1) or a functional variant thereof. As used herein, a peptide is also referred to as an active peptide that acts as an active moiety in the chimeric peptides of the present invention for the treatment of central nervous system injuries or for use as a neuroprotective agent.

According to existing research, some active peptides that inhibit the interaction between NMDAR and PSD-95 are based on the NMDAR structure. For example, NMDAR2B

- 4 044400 (GenBank ID 4099612) has 20 amino acids FNGSSNGHVYEKLSSLESDV at its C-terminus and a PL (PDZ domain ligand binding) motif consisting of ESDV. Some known active peptides contain part of the amino acid sequence from NMDAR2B at the C-terminus, thereby competitively inhibiting the interaction of PSD-95 with NMDAR2B. Based on studies, it has been suggested that the ESDV or LESDV segment in the above-mentioned peptides plays an important role in inhibiting the interaction between NMDAR and PSD-95. Without being bound by any theory, the present inventors have unexpectedly discovered that the active peptide YEKLLDTEI (SEQ ID NO: 1) described herein (which does not contain two SS residues after KL compared to the C-terminal amino acid sequence of the described upstream of NMDAR2B and has the amino acid sequence YEKL extending from the N-terminus of the PL motif), enhances the interaction of the active peptide with the PDZ1/2 domain. At the same time, the LDTEI segment at the C-terminus of the peptide can be modified compared to the YEKL motif, and such modification is not expected to affect the activity of the active peptide or may even increase its activity. Accordingly, in some embodiments, a functional variant provided by this invention is a variant formed by one or more conservative substitutions in the LDTEI segment of SEQ ID NO: 1.

In some embodiments, the conservative substitution is selected from the group consisting of a substitution between D and E, a substitution among L, V, and I, and a substitution between T and S.

In certain specific embodiments, the functional variant is a variant formed by replacing the LDTEI segment in SEQ ID NO: 1 with a sequence selected from the group consisting of LDTEL, LDTEV, LDTDI, LDTDL, LDTDV, LDSEI, LDSEL, LDSEV, LDSDI, LDSDL, LDSDV, LETEI, LETEL, LETEV, LETDI, LETDL, LETDL, LETDL, LETDL, LETDL, LETDL, LETDL, IDTEI, IDTEL, IDTEV, IDTDI, IDTDL, IDTDV, IETEI, IETEL, IETEV, IETDI, IETDL, IETDL and IETDV.

In some embodiments, the functional variants described herein also contain an amino acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95% or even more identical to the peptides mentioned above. It is known in the art that the identity between two proteins can be determined by aligning the amino acid sequence of the first protein with the sequence of a second protein that contains conserved amino acid substitutions compared to the first protein. The level of identity between two proteins can be determined using computer algorithms and methods well known to those skilled in the art. The identity of two amino acid sequences is preferably determined using the BLASTP (Baseline Local Alignment Search Tool for Peptides) algorithm.

In some embodiments, the functional variants described herein include variants having substitutions, deletions, additions and/or insertions of amino acid residues at 1, 2, 3, 4, 5 or more positions compared to the peptides mentioned above, characterized in that most from the specific peptides described above.

As described above, a functional variant may differ from the particular peptide described above by one or more substitutions, deletions, additions and/or insertions. Such variants may be naturally occurring or may be synthesized. For example, one or more of the above-described peptide sequences disclosed herein may be modified and its biological activities may be assessed using any of a variety of methods well known in the art that are described herein.

In some embodiments, the peptide is a chimeric peptide comprising the amino acid sequence YEKLLDTEI (SEQ ID NO: 1) or a functional variant thereof, and an internalization peptide, wherein the internalization peptide is capable of facilitating uptake of the chimeric peptide into a cell.

Those skilled in the art will appreciate that the primary purpose of including an active peptide and an internalization peptide in a chimeric peptide is to improve delivery of the active peptide to its target. Therefore, the internalization peptides suitable for the present application are not limited to specific types as long as the goal of cellular penetration or internalization is achieved. It will also be appreciated by those skilled in the art that since the targets of action of the active peptide are located primarily within nerve cells, it is preferable that the internalizing peptide be particularly suitable for nerve cells. In some embodiments, the internalization peptide may be a Tat peptide. In some embodiments, the amino acid sequence of the Tat peptide is YGRKKRRQRRR (SEQ ID NO: 2). In some embodiments, the chimeric peptide contains the amino acid sequence YGRKKRRQRRRYEKLLDTEI (SEQ ID NO: 3).

It will be apparent that the internalized peptide can be linked to the active peptide via an amide bond to form a fusion peptide, but they can also be linked in another suitable manner, for example by forming a chemical bond. Re

- 5 044400 The act of combining two components can be carried out using a coupling agent or a conjugating agent. A large number of such reagents are commercially available and information about them can be found in S.S. Wong, Chemistry of Protein Conjugation and Cross-Linking, CRC Press (1991). Some examples of cross-linking reagents include N-succinimide-3-(2pyridine dithio)propionate (SPOP) or N,N'-(1,3-phenylene)bismaleumide; N,N'-ethylidene-bis-(iodoacetamide) or other similar reagents having 6 to 11 carbon methylene bridges (which are relatively specific for thiol groups) and 1,5-difluoro-2,4-dinitrobenzene (which forms irreversibly way of bonding, interacting with the amino group and with the tyrosine group). Other cross-linking reagents include P,P'-difluoro-m,m'-dinitrodiphenylsulfone (which forms an irreversible cross-link by reacting with the amino group and the phenolic group); dimethyl diethylamine hexanoate (specific to the amino group); phenol-1,4-disulfonyl chloride (which reacts mainly with the amino group); 1,6-hexamethylene diisocyanate or -diisothiocyanate or phenylazo-p-diisocyanate (which reacts mainly with the amino group); glutaraldehyde (which interacts with several different side chains) and bis-diazotized benzidine (which interacts mainly with tyrosine and histidine).

In addition, the peptides described above may optionally be modified (eg, acetylated, phosphorylated, and/or glycosylated) to increase their affinity for inhibitors, enhance the inhibitors' ability to cross cell membranes, or increase their stability.

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

In some embodiments, the pH adjusting agent is selected from the group consisting of histidine buffer, arginine buffer, sodium succinate buffer, potassium succinate buffer, sodium citrate buffer, gluconate buffer, acetate buffer, phosphate buffer, trisbuffer, and any combination thereof . In some embodiments, the pH adjusting agent is selected from the group consisting of a citric acid/disodium phosphate buffer and a histidine/arginine buffer. In some embodiments, the pH adjusting agent is selected from the group consisting of a histidine/arginine buffer.

In some embodiments, the pH of the composition is from about 5.5 to 8 (for example, about 5.5; 6; 6.5; 7; 7.5; 8). In some embodiments, the pH of the composition is from about 6 to 7.5. In some embodiments, the pH of the composition is from about 6 to 7. In some embodiments, the pH of the composition is from about 6.5 to 7. In some embodiments, the pH of the composition is about 6.5.

In some embodiments, the amount of histidine/arginine in the histidine/arginine buffer is from about 1 to 10% by weight (for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10%). In some embodiments, the amount of histidine/arginine in the histidine/arginine buffer is from about 3 to 10%.

In some embodiments, the excipient is selected from the group consisting of trehalose, mannitol, glucose, lactose, cyclodextrin, dextran-40, sorbitol, sucrose, glycine, and any combination thereof. In some embodiments, the excipient is selected from the group consisting of trehalose, mannitol, glucose, lactose, and any combination thereof. In some embodiments, the filler is trehalose.

In some embodiments, the mass ratio of peptide to trehalose is from about 1:0.05 to 1:10. In some embodiments, the mass ratio of peptide to trehalose is from about 1:0.5 to 1:5. In some embodiments, the mass ratio of peptide to trehalose is from about 1:0.8 to 1:3. In some embodiments, the mass ratio of peptide to trehalose is approximately 1:1.

In some embodiments, the excipient is trehalose and the pH adjusting agent is a histidine/arginine buffer.

In some embodiments, the mass ratio of peptide to trehalose is approximately 1:1.

In some embodiments, the pH of the composition is about 6.5 ± 0.5.

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

In some embodiments, the composition further comprises a deamidation inhibitor.

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

In some embodiments, administration of the composition may be parenteral, intravenous, subcutaneous, intraarterial, intracranial, intrathecal, intraperitoneal, topical, intranasal, or intramuscular. Intravenous administration is preferred.

In some embodiments, the pharmaceutical composition for parenteral administration is preferably sterile and substantially isotonic. In the case of injections, the composition containing the active peptide or chimeric peptide can be prepared in an aqueous solution, preferably in a physiologically compatible buffer such as Hank's solution, Ringer's solution or saline, or in an acetate buffer (to reduce discomfort at injection sites). The solution may contain composition-forming agents such as suspending, stabilizing and/or dispersing agents.

In addition to the compositions described above, the composition containing the active peptide or chimeric peptide can also be formulated as a reservoir type preparation. Such long-acting compositions can be administered by implantation (eg, subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compound may be formulated with a suitable polymeric or hydrophobic material (eg, emulsified in a suitable oil) or an ion exchange resin, or formulated as a sparingly soluble derivative, eg, a sparingly soluble salt.

In some embodiments, because the active peptides or chimeric peptides described herein may contain charged side chains or termini, they may be included in any of the above compositions as a free acid or base, or as a pharmaceutically acceptable salt. Pharmaceutically acceptable salts may be salts which substantially retain the biological activity of the free base and which are formed by reaction with inorganic acids. In general, pharmaceutical salts are more soluble in water and other protic solvents than the corresponding free base forms.

The active peptide or chimeric peptide is used in an amount effective to achieve the intended purpose (eg, to reduce the damaging effect resulting from stroke injuries and related conditions). A therapeutically effective amount means an amount of active peptide or chimeric peptide sufficient to significantly attenuate the damage caused by strokes in patients (or animal model populations) treated with the active peptide or chimeric peptide disclosed herein, compared to the damage to the central nervous system in a control population of patients (or animal models) not treated with the active peptide or chimeric peptide disclosed herein. If a treated patient achieves a better outcome than the average outcome (as measured by infarct volume or disability) in a matched control population of patients not treated with the methods described herein, then that amount is also considered therapeutically effective. A therapeutically effective amount is also considered to be an amount if the treated patient has a Rankin disability score of 2 or less and a Barthel disability score of 75 or more. If a population of treated patients exhibits a significantly improved distribution of disability scores (i.e., less disability) compared to comparable untreated populations, then that dose is also considered therapeutically effective, see Lees et al., N. Engl. J Med 2006, 354: 588-600. A therapeutically effective dosage regimen is the combination of a therapeutically effective dose and frequency of administration necessary to achieve the above purpose. Usually a single dose may be sufficient.

In some embodiments, the preferred dosage range contains from 0.001 to 20 μmol of active peptide or chimeric peptide per kg of patient's body weight in the 6 hours after the stroke attack, optionally from 0.03 to 3 μmol of active peptide or chimeric peptide per kg of patient's body weight. In some methods, 0.1-20 μmol of active peptide or chimeric peptide per kg of patient body weight is administered over a 6-hour period. In some methods, 0.1-10 μmol of active peptide or chimeric peptide per kg of patient's body weight is administered over a 6-hour interval, and more preferably, about 0.3 μmol of active peptide or chimeric peptide per kg of patient's body weight is administered over a 6-hour interval. In other cases, the dose range is from 0.005 to 0.5 μmol of active peptide or chimeric peptide per kg of patient body weight. The rat dose per kg body weight can be converted to humans by dividing by 6.2 to accommodate different surface area to mass ratios. A suitable dose of active peptide or chimeric peptide expressed in grams for use in humans may be from 0.01 to 100 mg/kg body weight of the patient, or more preferably from 0.01 to 30 mg/kg body weight of the patient, or from 0 .01 to 10 mg/kg, or from 0.01 to 1 mg/kg.

In some embodiments, the amount of active peptide or chimeric peptide administered depends on the subject being treated, the subject's weight, the severity of pain, the mode of administration, and adjustments prescribed by the attending physician. Treatment can be repeated when symptoms are detected or even undetected. Treatment may be provided as monotherapy or in combination with other drugs.

In some embodiments, a therapeutically effective dose of the active peptide or chimeric peptide described herein is capable of providing therapeutic benefit without causing significant toxicity. The toxicity of a chimeric peptide can be determined in cell cultures or experimental animals using standard pharmaceutical techniques, for example, by determining LD ₅₀ (the dose that causes the death of 50% of objects in the population) or LD1oo (the dose that causes the death of 100% of the population). The ratio of doses to produce a toxic effect and a therapeutic effect is the therapeutic index. Chimeric peptides or peptidomimetics exhibiting high therapeutic indices are preferred (see, for example, Fingl et al., 1975, in The Pharmacological Basis of Therapeutics, part 1, page 1).

In some embodiments, the pharmaceutical composition is used to treat, relieve symptoms of, or prevent damage to the nervous system or disease or pain caused by damage to the nervous system, or is used as a neuroprotective agent. In some embodiments, the damage to the nervous system is damage caused by excitatory neurotoxicity, and such damage is located in the peripheral nervous system or the central nervous system.

In some embodiments, nervous system injury caused by excitatory neurotoxicity includes injury selected from the group consisting of stroke, spinal cord injury, ischemic or traumatic brain or spinal cord injury, neuronal injury in the central nervous system (CNS), including acute CNS injury, ischemic stroke or spinal cord injury, hypoxia, ischemic or mechanical injury and injury caused by neurodegenerative disease, anxiety disorder, epilepsy or stroke. In some embodiments, the pharmaceutical composition is used to treat, relieve symptoms of, or prevent damage to the nervous system caused by ischemic stroke.

Stroke is a condition caused by disruption of blood flow to the central nervous system. Possible causes include embolism, hemorrhage, and thrombosis. Some nerve cells die immediately due to disruption of blood flow. These cells release their constituent molecules (including glutamic acid), which in turn activate the NMDA receptor, which increases intracellular calcium levels and intracellular enzyme levels, and this leads to the death of more nerve cells (cascading increases in excitatory neurotoxicity). The death of central nervous system tissue is called a heart attack. Infarct volume (ie, the volume of dead nerve cells in the brain caused by a stroke) can be used as an indicator of the extent of pathological damage caused by a stroke. Symptomatic effects depend on both the volume of the infarct and the location of the infarction in the brain. The disability index can be used as an indicator of symptomatic damage, for example, the Rankin stroke outcome index (Rankin, Scott. Med. J., 2: 200-15 (1957)) and the Barthel index.

The Rankin Scale is based on direct assessment of the patient's systemic status and is as follows:

0: no symptoms at all;

1: symptoms are present, but there is no significant loss of ability to work; the patient is able to perform all daily work and all usual activities;

2: minor disability; the patient is not able to perform all previous actions, but is able to care for himself without assistance;

3: moderate disability requiring some assistance, but the patient is able to walk without assistance;

4: moderately severe disability, the patient is unable to walk without assistance and is unable to satisfy his own bodily needs without assistance;

5: severe disability; the patient is bedridden, has urinary and fecal incontinence, and requires constant care and attention.

The Barthel Index is based on a series of questions about a patient's ability to perform 10 major activities of daily living, scored from 0 to 100, with lower scores indicating greater disability (Mahoney et al., Maryland State Medical Journal, 14: 56-61 (1965)).

Alternatively, stroke severity/outcome can be measured using the National Institutes of Health (NIH) Stroke Scale, which is available on the World Wide Web at ninds.nih.gov/doctors/NIH_Stroke_Scale_Booklet.pdf. This scale is based on the patient's ability to perform 11 sets of functions and includes assessment of the patient's levels of awareness, motor function, sensation, and speech function.

Ischemic stroke is more clearly defined as a type of stroke caused by blockage of blood flow to the brain. The possible pathology of such blockages is most often associated with the appearance of fatty deposits along the walls of blood vessels. This condition is called atherosclerosis. Ta

- 8 044400 Fat deposits can cause two types of obstruction. Cerebral thrombosis refers to the formation of a blood clot in a blocked part of a blood vessel. The term cerebral embolism usually means that various circulating emboli (such as mural thrombus in the heart, atherosclerotic plaque, fat, tumor cells, fibrocartilage tissue, or air) enter the cerebral artery along with the bloodstream, blocking the blood vessels. When there is insufficient collateral circulation to compensate, it causes ischemic necrosis of the brain tissue to which the artery supplies blood and focal neurological impairment. The second important cause of embolism is an irregular heartbeat called atrial fibrillation. It causes a condition in which a blood clot can form in the heart and then dislodge and travel to the brain. Other possible causes of ischemic stroke include hemorrhage, thrombosis, rupture of arteries or veins, cardiac arrest, shock from any cause (including hemorrhage), and iatrogenic causes such as damage to blood vessels in the brain or blood vessels leading to the brain in as a result of direct surgery, or heart surgery. Ischemic stroke accounts for approximately 83% of all stroke cases.

Several other neurological disorders can also cause neuronal death through NDMAR-mediated excitatory neurotoxicity. These disorders include neurodegenerative diseases, anxiety disorder, epilepsy, hypoxia, CNS damage unrelated to stroke, such as traumatic brain injury and spinal cord injury. Accordingly, in some embodiments, the pharmaceutical composition is used to treat, alleviate symptoms of, or prevent a neurodegenerative disease, anxiety disorder, or epilepsy, which neurodegenerative diseases may include Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Parkinson's disease, or Huntington's disease.

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

According to a second aspect, the present application provides a method of treating, alleviating symptoms of, or preventing nervous system damage and disease or pain associated with nervous system damage, a neurodegenerative disease, an anxiety disorder, or epilepsy, comprising administering to a subject in need thereof a pharmaceutical composition, as described in the first aspect.

In some embodiments, nervous system injury caused by excitatory neurotoxicity includes injury selected from the group consisting of stroke, spinal cord injury, ischemic or traumatic brain or spinal cord injury, neuronal injury in the central nervous system (CNS), including acute CNS injury, ischemic stroke or spinal cord injury, hypoxia, ischemic or mechanical injury and injury caused by neurodegenerative disease, anxiety disorder, epilepsy or stroke.

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

In some embodiments, the subject is a subject suffering from ischemic stroke. In some embodiments, administration of a pharmaceutical composition as described in the first aspect can reduce the volume of a cerebral infarct site caused by cerebral ischemia.

According to a third aspect, the present application provides the use of a pharmaceutical composition as described in the first aspect in the manufacture of a medicament for treating, alleviating symptoms of, or preventing damage to the nervous system and disease or pain associated with damage to the nervous system, neurodegenerative disease, anxiety disorder or epilepsy, or in the manufacture of a neuroprotective agent.

In some embodiments, nervous system injury caused by excitatory neurotoxicity includes injury selected from the group consisting of stroke, spinal cord injury, ischemic or traumatic brain or spinal cord injury, neuronal injury in the central nervous system (CNS), including acute CNS injury, ischemic stroke or spinal cord injury, hypoxia, ischemic or mechanical injury and injury caused by neurodegenerative disease, anxiety disorder, epilepsy or stroke.

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

In some embodiments, the drug is used to treat, relieve symptoms of, or prevent damage to the nervous system caused by ischemic stroke.

It should be understood that the foregoing detailed description is intended only to assist those skilled in the art to more clearly understand the present application, but is not intended to limit the present application in any way. Those skilled in the art can

- 9 044400 carry out various modifications and changes to the described embodiments.

Examples

The following examples are provided only to illustrate some embodiments of the present invention and are not intended to limit the invention in any way.

Example 1. Screening of active peptide molecules.

Based on the described study results, the Tat transmembrane peptide YGRKKRRQRRR (SEQ ID NO: 2) was selected and ligated with varying amounts of amino acids to produce a peptide library. Chimeric peptide molecules in the peptide library were tested for interaction with the PDZ1/2 domain expressed and purified in vitro, and the polypeptides were pre-screened for strength of interaction.

The molecule (ligand) attached to the stationary phase was the PDZ1/2 protein with a molecular weight of approximately 20 kDa at a concentration of 2 mg/ml. The mobile phase molecule (analyte) was a polypeptide intended for screening with a molecular weight of approximately 2 kDa at a concentration of 10 mg/ml. For fixation, a CM5 chip (carboxymethylated dextran chip) was used using a Biacore 3000 instrument. The electrophoresis buffer was PBS (phosphate buffered saline) with 0.005% Tween 20. Fixation was carried out using the amino coupling method. The ligand concentration was 10 μg/ml. The fixation buffer was a buffer based on 10 mM sodium acetate solution, pH 4.0. The amount that was recorded in flow cell 2 was 1400 RU. The flow rate used was 10 μL/mL and the ligand was loaded within 1 minute. For regeneration, a 10 mM Gly solution was used at pH 2.0-2.5. Regeneration was carried out at a flow rate of 30 μL/min. Loading time was 30 s.

Kinetic analysis was performed using the following conditions:

control channel: flow cell 1;

electrophoresis buffer: PBS;

mode: kinetic analysis Wizard;

concentration gradients: 6.25 nM, 12.5 nM, 25 nM, 50 nM, 100 nM, 200 nM, 400 nM;

loading time: 1 min;

dissociation time: 2 min; and flow rate: 30 µl/min.

Data approximation was carried out using Biaevaluation 4.1 approximation software. The fitting model was a 1:1 binding model. The value of the dissociation constant (KD) is inversely proportional to the strength of the interaction.

As a result of screening, a chimeric peptide was obtained that exhibits a strong ability to interact with the PDZ1/2 domain and was named P5. The sequence of the chimeric peptide is shown below:

To make a direct comparison with the same chimeric peptide from the above studies, a control chimeric peptide NA-1 was used with the following sequence:

NA-1: YGRKKRRQRRRKLSSIESDV (SEQ Ш NO: 4).

In addition, in order to compare P5 with NA-1 regarding their structural differences, the chimeric peptide YE-NA-1, having two YE residues added to the N-terminus of the active peptide in the chimeric peptide NA-1, and its sequence were additionally used shown below:

The chimeric peptides NA-1, YE-NA-1 and P5 were tested in parallel for interaction with the PDZ1/2 domain as mentioned above, and the results are shown in Table 1 below. 1.

Table 1

Detection of the strength of interaction of each of the three chimeric peptides with the PDZ1/2 domain

Chimeric peptides NA-1 YE-NA-1 P5 KD (M) 7.53E-08 5.44E-08 2.99E-08

As shown in table. 1, the chimeric peptides YE-NA-1 and P5 interacted with the PDZ1/2 domain more strongly than the control chimeric peptide NA-1, and the performance was better for P5. Thus, the inventors hypothesized that the two additional amino acid residues YE at the N terminus of the active peptide had a certain improving effect on the interaction of the polypeptide with the PDZ1/2 domain. In addition, P5 lacked two serine (SS) residues that are weakly hydrophobic compared to the carboxyl terminus of YE-NA-1. Based on this, the inventors hypothesized that this may further enhance the interaction of the polypeptide with the PDZ1/2 domain.

The chimeric P5 peptide was chosen for further testing in subsequent experiments, and NA-1 and YE-NA-1 were used as controls in some experiments.

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Example 2 Co-precipitation assay to test the interaction of P5 with the PDZ1/2 domain.

To confirm that P5 can interact with the PDZ1/2 domain, analysis was performed by a coprecipitation assay.

The column was equilibrated with 100 μL of His beads and 1 mL of metal chelate affinity chromatography (MCAC)-0 buffer for 5 min and shaken at 4°C. The mixture was centrifuged at 5000 g for 1 minute at 4°C and the supernatant was discarded. 1 mg of PDZ1/2 protein was added to the mixture and buffer was added until the volume reached 1 ml. The mixture was rotated for 1 hour at 4°C to effect binding. The mixture was centrifuged at 5000 g for 1 minute at 4°C and the supernatant was discarded. The mixture was washed three times, each time using 1 ml of MCAC-0 buffer, for 5 minutes (at 4°C, washing with shaking). 1 mg of P5 protein was added to the mixture and buffer was added until the volume reached 1 ml. The mixture was rotated for 2 hours at 4°C to effect binding. The mixture was centrifuged at 5000 g for 1 minute at 4°C and the supernatant was discarded. The mixture was washed three times, each time using 1 ml of lysis buffer, for 5 minutes (at 4°C, washing with shaking). After washing, 20 μl of MCAC-300 was added. After centrifugation, the eluate was collected for analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The results of the experiment are shown in Fig. 1.

As shown in FIG. 1, both P5 and the PDZ1/2 domain were contained in a single eluted fraction corresponding to the chimeric P5 peptide, thereby confirming the ability of the chimeric P5 peptide to bind to the PDZ1/2 domain.

Example 3: Therapeutic effect of chimeric peptide in a rat model of MCAO.

MCAO preparation method and assessment standard.

A model of focal cerebral ischemia-reperfusion was prepared according to the reversible middle cerebral artery occlusion (MCAO) ligation method proposed by Longhi with modifications taking into account the anatomical structure of the rat brain. Rats were anesthetized by intraperitoneal injection of 10% chloral hydrate at a dose of 0.3 ml/kg. After anesthesia, an incision was made along the midline of the cervical region to expose the common carotid artery (CCA), external carotid artery (ECA), and pterygopalatine artery. The working part (0.5 cm) of a monofilament nylon fishing line (0.26 mm) was covered with paraffin and a mark was made at a distance of 20 mm. In all rats, a fishing line was inserted through the right CCA incision, and the pterygopalatine artery was temporarily clamped to prevent misinsertion. The length of the fishing line used for occlusion was approximately 18-20 mm from the bifurcation site of the CCA, depending on the weight of the animal, thereby occluding the middle cerebral artery on the right side. The skin was then sutured and the very end of the line used for occlusion was partially fixed to the skin. At the end of the 2-hour ischemic period, the occlusion line was carefully removed for reperfusion. The steps of this procedure for the sham control group were the same as for the surgical group, except for the insertion of the nylon fishing line. Body temperature was maintained at 37 ± 0.5°C throughout the ischemia period and for 2 h after reperfusion. An indication of the success of this model is that the rats, upon awakening from anesthesia, exhibited paralyzed left limbs, unsteadiness when standing, and turning to one side when their tails were raised up.

Signs of neurological pathology were assessed using the 5-point method of Longhi and Bederson 24 hours after awakening the animals from anesthesia:

0: absence of any symptom of nerve damage;

1: inability to fully extend the opposite foreleg;

2: unfolding in the opposite direction;

3: tipping in the opposite direction;

4: inability to walk independently and loss of consciousness.

The higher the score, the more severe the animal's behavioral disorder.

Experimental animals and materials.

The animals used were adult male Sprague Dawley (SD; Vittalia) rats weighing 220-250 g with pathogen-free (SPF) status.

Instruments used included one straight scissors, two ophthalmic scissors, four curved clamps, #4, #5 surgical sutures, 6x17 triangular needles, occlusion line (0.26 mm diameter), and single needle holders. Agents used included Enbipu-containing sodium chloride injection (Shijiazhuang Group NBP Pharmaceutical Co., Ltd.), chloral hydrate, furosemide (20 mg/vial), gentamicin sulfate (80 mg/vial), cotton swabs, and medicinal pads. The tested peptides were synthesized at Kingsray Biotech Inc.

Division into experimental groups.

The experimental animals were divided into negative control group, sham group, model animal group, positive control drug group.

- 11 044400

Enbipu, group NA-1, group YE-NA-1 and group P5. Saline, positive drug Enbipu, NA-1 (10 mg/kg), YE-NA-1 (10 mg/kg) and P5 (10 mg/kg, 3 mg/kg and 1 mg/kg), respectively, was injected into each individual group of rats via the tail vein 1 h after ischemia.

The normal group and the sham group were not administered any drug.

Calculation of infarction volume.

After scoring, the rats were killed by decapitation. Brain tissues were quickly removed and refrigerated at -20°C. After 10 min, the tissues were placed at room temperature. Brain samples were placed in a mold to obtain rat brain slices. After removing the olfactory bulb, cerebellum, and lower brainstem, 5 coronal sections were made in the brain specimens, yielding six contiguous 2-mm-thick raw coronal sections in profile. Next, these brain sections were quickly placed in 5 ml of a solution containing 2% TTC and incubated at 37°C for 30 min in the dark, during which time the brain sections were turned over once every 5 min. When stained with TTC, tissue from normal animals will appear pinkish-red, but infarcted tissue will not stain and will remain white. Each group of brain slices was carefully arranged and photographed. Photographs were processed using system image analysis software and statistical analysis was performed. The infarct area for each brain section was calculated and multiplied by the thickness of each brain section (2 mm). The results obtained by multiplying the infarct area of an individual brain slice by the thickness were summed to obtain the cerebral infarct volume for each animal. Volumes were expressed as percentages based on each cerebral hemisphere to eliminate the effects of cerebral edema.

Experiment results.

The results of the experiment are shown in Fig. 2. Statistical histogram of cerebral infarct volume data shown in FIG. 3 were built on the basis of statistical analysis of the cerebral infarction volume data in FIG. 2, and specific statistics for the volume of cerebral infarction are given below in table. 2. These results showed that therapeutic administration and prophylactic administration of P5 peptide at the highest dose (10 mg/kg) could significantly reduce the volume of cerebral infarction in rats subjected to cerebral ischemia by approximately 50% (p < 0.01), in while the Enbipu positive group saw a decrease of only about 16% (p<0.01), the NA-1 group saw a decrease of about 16% (p<0.01) and the YE- group NA-1 showed a decrease of approximately 26% (p<0.01). Therapeutic administration and prophylactic administration of P5 peptide at a second high dose (3 mg/kg) also adequately reduced the volume of cerebral infarction. In addition, the data showed that the magnitude of infarct volume decreased with increasing dose of P5 peptide, indicating that the therapeutic effect was positively correlated with drug dose. The therapeutic effect of the YE-NA-1 polypeptide was significantly greater than that observed for NA-1. Based on this, we hypothesize that the addition of two YE amino acids may result in a greater therapeutic effect than that of NA-1 by improving the interaction of the polypeptide with the PDZ1/2 domain.

table 2

Therapeutic effect of P5 polypeptide on rats in the MCAO model

Groups Mean infarct volume percentage (%) Standard deviation Reduction in infarct volume as a percentage compared to the group of model animals t-test compared to group model animals tcriterion compared with P5 at a dose of 10 mg/kg Group of normal animals 0 0 Imitation group 0 0

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Group of model animals 45.96 3.35 Group receiving positive drug Enbipu 38.61 3.21 15.99 p<0.01 ΝΑ-l in dose 10 mg/kg 38.56 2.25 16.10 p<0.01 p<0.01 YE-NA-1 dose 10 mg/kg 33.96 2.40 26.11 p<0.01 p<0.01 P5 in dose 10 mg/kg 24.84 2.90 45.95 p<0.01 P5 in dose 3 mg/kg 36.54 2.35 20.50 p<0.01 P5 in dose 1 mg/kg 43.22 3.12 5.96 0.061 Preventative administration of P5 at a dose of 10 mg/kg 19.54 2.30 57.48 p<0.01 Preventative administration of P5 at a dose of 3 mg/kg 35.66 1.50 22.41 p<0.01 Preventative administration of P5 at a dose of 1 mg/kg 44.23 2.20 3.76 0.082

Example 4. Distribution of P5 in the brain of rats.

Normal control rats and rats in the MCAO model, respectively, were injected into the tail vein with a saline solution containing a fluorescently labeled polypeptide, FITC-P5, (10 mg/kg) 1 hour after model establishment. Rats were sacrificed 12 hours after administration. Brain tissue was quickly removed and placed in a small live animal imaging system for fluorescence detection. After fluorescence detection was completed, brain tissues were placed in TTC dye solution for staining to determine the correlation between ischemic area and drug distribution. As shown in FIG. 4 and 5, the brain of normal rats was completely stained with TTC, and no distribution of fluorescently labeled polypeptide was detected, while the ischemic area of the brain of ischemic rats was not stained with TTC, and fluorescently labeled polypeptide was distributed throughout the ischemia-affected area, with In this case, the main area damaged by ischemia was the middle artery area, which confirmed that the P5 polypeptide can target the area affected by ischemia and have a therapeutic effect, and its quantitative distribution was positively correlated with the degree of ischemia.

Example 5: HE staining to detect histological changes.

Rats in each group were decapitated 24 hours after ischemia, and the resulting brain specimen was incised coronally near the optic chiasm, producing sections approximately 4 mm thick. The sections were fixed using a 10% formalin solution and dehydrated with alcohol with a concentration gradient from 70 to 100%. Sections were permeabilized twice in xylene and embedded in paraffin. The paraffin block was carefully trimmed, fixed in a paraffin sectioning machine, and sections of 4 µm thickness were prepared. Paraffin sections were completely unwrapped, attached to a clean and dry glass plate, and stored in a refrigerator at 4°C. Traditional HE staining was performed and the staining results were observed using light microscopy.

The results of the experiment are shown in Fig. 6. Nerve cells from a normal animal's brain tissue showed a distinct nucleus, a spherical nuclear membrane, and an intact nuclear membrane. Tissues from the ischemic side of the brain of ischemic rat model animals showed severe necrosis of nerve cells, cell swelling, nuclear condensation, loose and lightly stained cytoplasm, and vacuolization. In the case of the therapeutic administration group and the prophylactic administration group of P5 peptide at a dose of 10 mg/kg, significant improvement in the above-mentioned pathological changes was observed, and the results were significantly better than those of the positive drug administration groups - Enbipu injection solution, NA-1 and YE-NA-1 (10 mg/kg).

Example 6 Acute toxicity assessment.

Acute toxicity testing was performed on rats. The results showed that P5 did not have any lethal effect or other obvious toxic side effects in rats at a dose of 200 mg/kg body weight.

Example 7. Preparation of lyophilized compositions based on P5.

A method for preparing lyophilized compositions using trehalose as a typical excipient and arginine solution as a typical pH adjusting agent is described below. Other lyophilized compositions were prepared in a similar manner.

The preparation of a lyophilized composition based on P5 included the following steps:

preparing a 5% arginine solution for use;

weighing the required amounts of trehalose and P5 peptide, respectively, and adding 80% of the total amount of water for injection, stirring until completely dissolved;

adding an arginine solution and adjusting the pH to 6.5±0.5;

adding water to the full volume with uniform stirring;

carrying out filtration using filters with pore diameters of 0.45 μm and 0.22 μm, respectively;

adding the filtrate to the bottle with partial capping;

carrying out vacuum lyophilization, including preliminary freezing at 30°C for 3 hours; sublimation at -20°C for 3 hours, -10°C for 5 hours and 5°C for 10 hours; and repeated drying at 30°C for 5 hours;

carrying out vacuum sealing; and application of a combined aluminum-plastic cover.

Example 8. Effect of different excipients on the shape and stability of lyophilized compositions based on P5.

Testing method.

To evaluate the effect of excipients on the shape and stability of lyophilized formulations based on P5, different excipients were selected. The samples were placed in a stability test chamber at 60°C for 2 weeks and a detection sample was taken at the end of the first and second weeks, respectively. Trehalose, cyclodextrin, mannitol, lactose and glucose were used as excipients, respectively, and the properties, clarity and color of the solution, pH, impurities and P5 content of various samples were evaluated.

Table 3

Fillers and quantities used

Test number 0 1 2 3 4 5 P5 0.20 g 0.20 g 0.20 g 0.20 g 0.20 g 0.20 g Filler / Trehalose 1.0 g Cyclodextrin 1.0 g Mannitol 1.0 g Lactose 1.0 g Glucose 1.0 g Water 20 ml 20 ml 20 ml 20 ml 20 ml 20 ml Volume/bottle 1 ml 1 ml 1 ml 1 ml 1 ml 1 ml

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Table 4

Information regarding reagents used in Example 8

Series Name Purity level Manufacturer Batch number 1 P5 Purity level "for injection" Synthesized and prepared in Hangzhou Zhongtai CQ-0400317 2 Trehalose Pharmaceutical grade Pfanstiehl 38540A 3 Hydroxypropylbetacyclodextrin Pharmaceutical grade Binzhou City Zhiyuan, Shandong Province 20160309- 1 4 Mannitol Pharmaceutical grade Nanning Chemical Pharmaceutical F431C 5 Lactose Pharmaceutical grade Zhenjiang fukang Biology 20160510 6 Glucose Pharmaceutical degree of purity Xiwang Pharmaceutical Industry 201605133 7 Acetonitrile Purity level "for chromatography" Mereda 8 Trifluoroacetic acid Purity level "for chromatography" Mereda

Evaluation method.

Appearance - Samples were visually examined and are expected to be white, friable, lyophilized masses or powders.

Transparency and color of the solution - the sample is dissolved using 1 ml of water.

The solution is expected to be clear and colorless. If the solution is turbid, it is compared to Turbidity Standard Solution No. 1 (General Rule 0902 Method 1) and should not be more turbid than the standard solution. If the solution is colored, it is compared with the yellow standard solution No. 1 (Chinese Pharmacopoeia IV, first method), and it should not be more colored than the standard solution.

pH measurement for a solution that meets the requirements for clarity and color is performed with reference to Method 0631 Determination of Solution Concentration of the Chinese Pharmacopoeia IV.

Impurities - Measurement is performed using high performance liquid chromatography under the following chromatographic conditions:

Column: Agilent C18 (4.6 mm x 150 mm, 5 µm);

mobile phase: A: 0.065% TFA - water; B: 0.05% TFA - acetonitrile;

elution: gradient elution, 0 to 30 min 5-65% B;

flow rate: 1.0 ml/min;

column temperature: 36°C;

detection wavelength: 220 nm;

injected volume: 10 µl.

A weighed amount of P5 peptide is dissolved in water, obtaining a solution containing approximately 2 mg of P5 peptide per 1 ml as a control solution.

The sample is dissolved in water and diluted to obtain a solution containing approximately 2 mg in 1 ml as the test sample. 10 µl of this solution is injected into the liquid chromatograph and the chromatogram is recorded. The content is calculated based on the peak area of the external standard for the instrument, and the amount of impurities is calculated by the area normalization method.

Finally, the clarity of the test sample is determined by comparison with the clarity measurement of a turbidity standard solution.

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Experiment results.

Table 5

The influence of various excipients on the shape and stability of lyophilized compositions based on P5 (on day 0)

Screening of fillers (on day 0) Number test Filler Appearance Transparency and color of the solution pH Content P5 (%) Impurities 0 / White friable lyophilizer. Transparent and colorless 5.88 109.27% Not defined weight 1 Trehalose White friable lyophilizer. weight Transparent and colorless 5.83 105.34% Not defined 2 Cyclodextrin White friable lyophilizer. weight Transparent and colorless 5.98 103.97% Not defined 3 Mannitol White friable lyophilizer. weight Transparent and colorless 5.90 111.40% Not defined 4 Lactose White friable lyophilizer. weight Transparent and colorless 5.70 107.60% Not defined 5 Glucose White friable lyophilizer. mass (packed) Transparent and colorless 5.73 100.88% Not defined

Table 6

The influence of different excipients on the shape and stability of lyophilized compositions based on P5 (after 1 week)

Screening of fillers (at 60°C for 1 week) Number test Fill tel Appearance Transparency and color of the solution pH value P5 content (%) Maksim. individual impurity content (%) All impurities (%) 0 / White friable lyophilizer. mass (slightly compacted) Transparent and colorless 6.07 107.91 0.53 1.04 1 Trehalose White friable lyophilizer. weight Transparent and colorless 5.76 105.35 0.21 0.30

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2 Cyclodextrin White friable lyophilizer. weight Transparent and colorless 5.89 102.42 0.89 1.36 3 Mannitol White friable lyophilizer. weight Transparent and colorless 6.09 103.05 1.34 3.91 4 Lactose White friable lyophilizer. weight Transparent and colorless 5.57 64.89 10.47 38.82 5 Glucose Yellow mass (considerably caked) Transparent, and the color is darker than yellow No. 10 4.62 57.58 75.28 75.51

Table 7

The influence of various excipients on the shape and stability of lyophilized ________________ compositions based on P5 (after 2 weeks)_____________________

Screening of fillers (at 60°C for 2 weeks) Number test Fill tel Appearance Transparency and color of the solution pH value P5 content (%) Maksim. individual impurity content (%) All impurities (%) 0 / White friable lyophilizer. mass (slightly compacted) Transparent and colorless 5.77 106.97 0.72 1.34 1 Trehalose White friable lyophilizer. weight Transparent and colorless 5.97 105.63 0.19 0.25 2 Cyclodextrin White friable lyophilizer. weight Transparent and colorless 6.30 101.04 1.01 1.72 3 Mannitol White friable lyophilizer. weight Transparent and colorless 6.29 102.05 1.61 4.66

On day 0, no impurities were detected in each of the samples (samples from No. 0 to No. 5). After 1 week, the maximum single impurity content of sample No. 1 (with trehalose) was 0.21%, and the total impurity content was 0.3%, which was significantly less than the control sample (No. 0), indicating that trehalose showed significant protective effects.

At the same time, regarding the content of all impurities in sample No. 2-5, it was found that the maximum content of a single impurity and the content of all impurities were higher than in sample No. 1 (with trehalose), indicating that cyclodextrin, mannitol, lactose and glucose were inferior to trehalose in terms of protective effect.

As for properties, all stored samples with trehalose, cyclodextrin, mannitol or lactose after 1 week were white, friable lyophilized masses, while the control sample was slightly caked, and sample No. 5 (with glucose) was strongly caked, having the appearance yellow mass. Samples containing trehalose, cyclodextrin or

- 17 044400 mannitol after 2 weeks were white, loose, lyophilized masses, while the control sample was slightly caked.

In terms of pH, no significant change was observed in the pH value for samples Nos. 0 and 1 after 1 and 2 weeks, while the pH values for samples Nos. 4 and 5 decreased after 1 week and the pH values for samples No. 2 and 3 increased after 2 weeks.

In terms of transparency and color, after 1 week, all samples Nos. 0-4 appeared transparent and colorless, while sample No. 5 appeared transparent but had a yellow color. After 2 weeks, all samples Nos. 0-3 looked transparent and colorless.

Taking into account the above evaluations in terms of appearance, clarity and color of the solution, pH value, impurity and drug content, trehalose was selected as the excipient for further testing.

Example 9. Effect of the amount of trehalose on the shape and stability of a lyophilized composition based on P5.

The form of lyophilized compositions based on P5 was evaluated using different amounts of trehalose. The methods described in example 8 were used as detection methods.

Table 8

Composition of samples from trehalose screening experiment I, in which the P5:trehalose ratios were 1:0, 1:0.25, 1:0.5, 1:0.75 and 1:1

Test number 0 1 2 3 4 P5 0.20 g 0.20 g 0.20 g 0.20 g 0.20 g Trehalose / 50 mg 0.10 g 0.15 g 0.20 g Water 2 ml 2 ml 2 ml 2 ml 2 ml Volume/bottle 100 µl 100 µl 100 µl 100 µl 100 µl

Table 9

Composition of samples from trehalose screening experiment II, in which the P5:trehalose ratio was 1:1.5, 1:2, 1:3, 1:4 and 1:5

Test number 5 6 7 8 9 P5 0.10 g 0.10 g 0.10 g 0.10 g 0.10 g Trehalose 0.15 g 0.20 g 0.30 g 0.40 g 0.50 g Water 2 ml 2 ml 2 ml 3 ml 4 ml Volume/bottle 100 µl 100 µl 100 µl 150 µl 200 µl

Each sample was placed in a stability test chamber at 60°C for 2 weeks and a detection sample was collected at the end of the first and second weeks, respectively. The properties, clarity and color of the solution, pH, impurity and drug content of different samples were evaluated for different ratios of P5:trehalose.

Table 10

Results of experiment I for screening the amount of trehalose (on day 0), with the applied amount being 10 mg/vial and using 7 ml vials

Experiment I for screening the amount of trehalose (on day 0) Number test P5: trehalose Appearance Transparency and color of the solution pH value P5 content (%) Maksim. individual impurity content (%) All impurities (%)

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0 / White friable lyophilizer. weight Transparent and colorless 5.92 91.19 0.10 0.10 1 1:0.25 White friable lyophilizer. weight Transparent and colorless 5.89 88.15 0.10 0.10 2 1:0.5 White friable lyophilizer. weight Transparent and colorless 5.73 89.68 0.10 oh and 3 1:0.75 White friable lyophilizer. weight Transparent and colorless 5.89 87.18 0.09 0.10 4 1:1 White friable lyophilizer. weight Transparent and colorless 5.93 88.84 0.08 0.10

Table 11

Results of Trehalose Amount Screening Experiment I (after 1 week)

Trehalose Amount Screening Experiment I (at 60°C for 1 week) Number test P5: trehalose Appearance Transparency and color of the solution pH value P5 content (%) Maksim. individual impurity content (%) All impurities (%) 0 / White friable lyophilizer. mass (packed) Transparent and colorless 6.10 87.68 0.65 1.78 1 1:0.25 White friable lyophilizer. mass (packed) Transparent and colorless 6.53 85.34 0.51 1.27 2 1:0.5 White friable lyophilizer. mass (packed) Transparent and colorless 6.40 89.56 0.23 0.48 3 1:0.75 White friable lyophilizer. mass (packed) Transparent and colorless 6.22 89.87 0.18 0.21 4 1:1 White friable lyophilizer. mass (packed) Transparent and colorless 6.16 87.61 0.17 0.17

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Table 12

Results of Trehalose Amount Screening Experiment I (after 2 weeks)

Screening Experiment I amount of trehalose (at 60°C for 2 weeks) Number test P5: trehalose Appearance Transparency and color of the solution pH value P5 content (%) Maksim. individual impurity content (%) All impurities (%) White loose 0 / lyophilizer mass (packed) White friable Transparent and colorless 6.88 87.91 0.88 2.74 1 1:0.25 lyophilizer mass (packed) White friable Transparent and colorless 6.97 87.48 0.51 1.15 2 1:0.5 lyophilizer mass (packed) White friable Transparent and colorless 6.89 92.77 0.37 0.88 3 1:0.75 lyophilizer mass (packed) Transparent and colorless 6.77 90.66 0.29 0.52 White loose 4 1:1 lyophilizer mass (packed) Transparent and colorless 6.81 91.10 0.26 0.47

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Table 13

Results of Experiment II for Trehalose Amount Screening Using 5 mg/vial applied using 7 ml vials

Experiment II to screen the amount of trehalose (on day 0) Nome P test Trehalose :P 5 Appearance Transparency and color of the solution pH value Containing P5 (%) Maksim. content of individual impurities (%) All impurities and (%) 5 1:1.5 White friable lyophilizer. weight Transparent and colorless 5.98 106.63 0.03 0.03 6 1:2 White friable lyophilizer. weight Transparent and colorless 5.89 97.19 0.05 0.05 7 1:3 White friable lyophilizer. weight Transparent and colorless 5.92 94.95 0.05 0.05 8 1:4 White friable lyophilizer. weight Transparent and colorless 5.83 99.88 0.03 0.03 9 1:5 White friable lyophilizer. weight Transparent and colorless 5.86 101.34 0.04 0.04

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Trehalose Amount Screening Experiment II (at 60°C for 1 week) Nome P test Trehalose :P 5 Appearance Transparency and color of the solution Meaning pH value Containing P5 (%) Maxim, content of individual impurities (%) All impurities and (%) 5 1:1.5 White friable lyophilizer. weight Transparent and colorless 6.18 98.13 0.23 0.30 6 1:2 White friable lyophilizer. mass (packed) Transparent and colorless 6.95 90.87 0.19 0.35 7 1:3 White friable lyophilizer. mass (packed) Transparent and colorless 6.64 87.27 0.17 0.30 8 1:4 White friable lyophilizer. mass (packed) Transparent and colorless 6.78 93.75 0.22 0.33 9 1:5 White friable lyophilizer. weight Transparent and colorless 6.33 95.45 0.23 0.31 Experiment II to screen the amount of trehalose (at 60°C for 2 weeks) Nome P test Trehalose: P 5 Appearance Transparency and color of the solution pH value Containing P5 (%) Maksim. content of one impurity (%) All impurities and (%) 5 1:1.5 White friable lyophilizer. mass (packed) Transparent and colorless 6.83 94.59 0.30 0.59

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6 1:2 White friable lyophilizer. mass (packed) Transparent and colorless 6.99 91.89 0.26 0.55 7 1:3 White friable lyophilizer. mass (packed) Transparent and colorless 7.20 86.25 0.27 0.52 8 1:4 White friable lyophilizer. mass (packed) Transparent and colorless 6.88 92.82 0.23 0.46 9 1:5 White friable lyophilizer. weight Transparent and colorless 6.95 94.42 0.23 0.47

Each sample was placed in a stability test chamber at 40°C for 3 months and a detection sample was taken at the end of the first and third month, respectively. The properties, clarity and color of the solution, pH, impurity and drug content of different samples were evaluated for different ratios of P5:trehalose.

Table 14

Results of Experiment I for trehalose amount screening (40°C) applying 0.1 ml (10 mg/vial) using 7 ml vials

Screening the amount of filler (at 40°C for 1 month) Number test P5: trehalose Appearance Transparency and color of the solution pH value P5 content (%) Maksim. individual impurity content (%) All impurities (%) 0 1:0 White friable lyophilizer. weight Transparent and colorless 7.05 91.52 0.98 1.09 1 1:0.25 White friable lyophilizer. weight Transparent and colorless 7.08 86.94 1.07 1.16 2 1:0.5 White friable lyophilizer. mass (packed) Transparent and colorless 7.07 91.43 0.85 0.92 3 1:0.75 White friable lyophilizer. mass (packed) Transparent and colorless 6.99 90.36 0.84 0.92 4 1:1 White friable lyophilizer. mass (packed) Transparent and colorless 6.94 90.36 0.80 0.88

Conclusions.

Regarding impurities, the impurity content (the maximum content of an individual impurity and

-23 044400 content of total impurities) in samples Nos. 1-9 after storage at 60°C for 1 or 2 weeks was significantly less than that in the control sample (No. 0), indicating that trehalose exhibited significant protective action.

As for experiment I (P5:trehalose is from 1:0.25 to 1:1), the impurity content gradually decreased as the amount of trehalose increased, indicating that the protective effect of trehalose was gradually enhanced.

Regarding experiment II (P5:trehalose is from 1:1.5 to 1:5), it was found that there is no significant difference in the impurity content in samples No. 5-9 and sample No. 4 (where P5:trehalose is 1:1) at the end of the first or second week, indicating that the protective effect of trehalose was no longer enhanced.

Thus, a 1:1 ratio of P5 to trehalose was found to be suitable.

Regarding pH, the change in pH value was observed for each sample and control sample, which were stored at 60°C for 2 weeks or at 40°C for one month, i.e. an increase from about 6.0 to about 7.0, indicating that it would be advisable to add a pH buffer to the composition to stabilize the pH value.

Example 10. The influence of different pH on the transparency of the solution and the content of impurities in lyophilized compositions.

The influence of different pH values on the transparency of the solution and the content of impurities in lyophilized compositions was assessed. The methods described in example 8 were used as detection methods.

Experiment I: pH adjustment of compositions using citric acid and disodium phosphate.

Table 15

Composition of samples from pH Range Screening Experiment I where the pH of the composition was adjusted to about 4, 5, 6, 7 and 8 using citric acid and disodium phosphate

Test number 0 1 2 3 4 5 pH value 5.43 4.10 5.00 6.04 6.92 7.84 P5 0.20 g 0.20 g 0.20 g 0.20 g 0.20 g 0.20 g Trehalose 1.0 g 1.0 g 1.0 g 1.0 g 1.0 g 1.0 g 0.1 M citric acid solution / 9.71 ml 11.3 ml 12.63 ml 16.47 ml 23.45 ml 0.2 M solution of dibasic sodium phosphate / 12.29 ml 9.7 ml 7.37 ml 3.53 ml 0.55 ml Water 20 ml / / / / / Application volume/bottle 1 ml 1 ml 1 ml 1 ml 1 ml 1 ml

Results and analysis of experiment I.

Table 17

Results of pH range screening experiment I (day 0)

pH range screening (on day 0) Number test pH Appearance Transparency and color of the solution pH value P5 content (%) Maksim. individual impurity content (%) All impurities (%) 0 Didn't let us down White friable lyophilizer. weight Transparent and colorless 5.73 102.46 0.10 0.11

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1 4.0 White friable lyophilizer. weight Transparency <No. 1 and colorless 4.32 102.31 0.13 0.13 2 5.0 White friable lyophilizer. weight Transparency = No. 2 and colorless 5.36 102.12 0.12 0.12 3 6.0 White friable lyophilizer. weight Transparency <No. 3 and colorless 6.52 99.22 0.09 0.09 4 7.0 White friable lyophilizer. weight Transparency <No. 3 and colorless 7.37 100.05 0.03 0.03 5 8.0 White friable lyophilizer. weight Transparency <No. 2 and colorless 8.20 98.39 0.05 0.05 Test number 0 Result! WITH pH Didn't let us down I experiment 'screening range. Appearance White friable lyophilizer. weight Screening pH zone I (at 6( Transparency and color of the solution Transparent and colorless /range )°C in those pH value 5.87 pH zone (read 1 not; P5 content (%) 101.28 Tab res 1 week) 1 ate) Maksim. contents of one impurities (%) 0.18 faces 18 All impurities (%) 0.23 1 4.0 White friable lyophilizer. mass (packed) Transparency <No. 1 and colorless 4.31 98.72 0.79 1.72

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2 5.0 White friable lyophilizer. mass (packed) Transparency <No. 1 and colorless 5.52 100.62 0.46 0.90 3 6.0 White friable lyophilizer. mass (packed) Transparency = No. 1 and colorless 6.65 97.39 0.28 0.36 4 7.0 White friable lyophilizer. mass (packed) Transparency <No. 1 and colorless 7.48 98.37 0.15 0.23 5 8.0 White friable lyophilizer. mass (packed) Transparency <No. 3 and colorless 8.40 96.66 0.15 0.15

Table 19

Results of pH Range Screening Experiment I (after 2 weeks)

pH range screening (at 60°C for 2 weeks) Number test pH Appearance Transparency and color of the solution pH value P5 content (%) Maksim. individual impurity content (%) All impurities (%) 0 Didn't let us down White friable lyophilizer. weight Transparent and colorless 5.76 102.81 0.22 0.28 1 4.0 White friable lyophilizer. mass (packed) Transparency <No. 1 and colorless 4.36 97.13 1.16 3.40

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2 5.0 White friable lyophilizer. mass (packed) Transparency <No. 1 and colorless 5.46 99.21 0.74 1.69 3 6.0 White friable lyophilizer. mass (packed) Transparency <No. 1 and colorless 6.46 97.87 0.39 0.58 4 7.0 White friable lyophilizer. mass (packed) Transparency <No. 1 and colorless 7.27 98.99 0.17 0.32 5 8.0 White friable lyophilizer. mass (packed) Transparency <No. 3 and colorless 8.20 95.76 0.24 0.33

In the case of screening experiment I, a citric acid-disodium phosphate buffer system was used to adjust the pH to a range of 4.0 to 8.0. The pH values of the control sample (No. 0) and samples No. 1 to No. 5 remained constant during storage at 60°C for 2 weeks. As can be seen from the transparency observations, the control sample (No. 0) had good clarity, and samples No. 1 to No. 4 showed little turbidity. After 1 week or after 2 weeks, the properties of the control sample (No. 0) did not change, while the samples with adjusted pH turned out to be significantly caked. In addition, it was also found that as the pH increased, the impurity content of the samples gradually decreased. Therefore, the inventors conducted Experiment II at a higher pH range using different pH adjusting agents.

Experiment II. Adjusting the pH of compositions using histidine/arginine.

Table 16

Composition of samples from pH Range Screening Experiment II where the pH of the composition was adjusted to about 6, 7, 8, 9 and 10 using histidine/arginine

Test number BUT H1 H2 nz H4 H5 pH value 5.5 7.1 7.0 7.9 8.9 9.5 P5 0.10 g 0.10 g 0.10 g 0.10 g 0.10 g 0.10 g Trehalose 0.20 g 0.20 g 0.20 g 0.20 g 0.20 g 0.20 g 3% histidine solution / 2.7 ml / / / / 10% arginine solution / / 80 µl 100 µl 250 µl 800 µl Water 2.7 ml / 2.7 ml 2.7 ml 2.7 ml 2.4 ml Application volume/bottle 150 µl 150 µl 150 µl 150 µl 150 µl 160 µl

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Results and analysis of experiment II.

Table 20

Results of pH range screening experiment II (day 0)

pH range screening (on day 0) Test number pH adjusting agent pH External view Transparency and color of the solution pH value P5 content (%) Maksim. individual impurity content (%) All impurities (%) BUT / / White friable lyophilizer. weight Transparent and colorless 5.90 101.13 0.22 0.26 H1 3% histidine 7.0 White friable lyophilizer. weight Transparency = No. 2 and colorless 7.14 102.94 0.06 0.06 H2 10% arginine 7.0 White loose lyophilizer weight Transparency <No. 1 and colorless 6.84 98.39 0.07 0.07 NZ 10% arginine 8.0 White loose lyophilizer weight Transparency <No. 1 and colorless 7.26 100.94 0.05 0.05 H4 10% arginine 9.0 White friable lyophilizer. weight Transparency <No. 2 and colorless 8.42 92.63 0.05 0.05 H5 10% arginine 10.0 White loose lyophilizer weight Transparency <No. 2 and colorless 9.08 95.46 0.07 0.07

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Table 21

Results of pH Range Screening Experiment II (after one week)

pH range screening (at 60°C for 1 week) Test number pH adjusting agent pH External view Transparency and color of the solution pH value P5 content (%) Maksim. contents of a separate impurities (%) All impurities (%) BUT / / White friable lyophilizer. weight Transparent and colorless 6.06 100.37 0.32 0.37 H1 3% histidine 7.0 White friable lyophilizer. weight Transparency = No. 2 and colorless 7.19 96.95 0.07 0.07 H2 10% arginine 7.0 White friable lyophilizer. weight Transparency <No. 1 and colorless 6.91 98.20 0.09 0.09 NZ 10% arginine 8.0 White loose lyophilizer weight Transparency <No. 1 and colorless 7.42 99.06 0.09 0.09 H4 10% arginine 9.0 White friable lyophilizer. weight Transparency <No. 3 and colorless 8.63 93.39 0.07 0.07 H5 10% arginine 10.0 White loose lyophilizer weight Transparency = No. 3 and colorless 9.15 94.33 0.24 0.30

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Table 22

Results of pH Range Screening Experiment II (after 2 weeks)

pH range screening (at 60°C for 2 weeks) Test number pH adjusting agent pH External view Transparency and color of the solution pH value P5 content (%) Maksim. contents of a separate impurities (%) All impurities (%) BUT / / White loose lyophilizer weight Transparent and colorless 6.47 100.89 0.31 0.38 H1 3% histidine 7.0 White friable lyophilizer. weight Transparency <No. 3 and colorless 7.22 103.64 0.09 0.09 H2 10% arginine 7.0 White friable lyophilizer. weight Transparency <No. 1 and colorless 7.10 101.57 oh and oh and NZ 10% arginine 8.0 White friable lyophilizer. weight Transparency <No. 2 and colorless 7.61 100.86 0.10 0.10 H4 10% arginine 9.0 White loose lyophilizer weight Transparency <No. 3 and colorless 8.64 91.40 0.21 0.29 H5 10% arginine 10.0 White loose lyophilizer weight Transparency <No. 4 and colorless 9.18 90.51 0.36 0.44

For Screening Experiment II, a histidine/arginine buffer system was selected to adjust the pH to a range of 7 to 10. After 2 weeks, the pH of the control sample (H0) changed (i.e., from 5.9 to 6 ,5), while the pH values for samples H1 to H5 remained constant. The impurity contents in the control sample (H0) increased significantly compared to those on day 0, while the impurity contents in samples H1, H2 and H3 did not increase. The clarity of the control sample (H0) was satisfactory, while samples H1 to H5 were turbid, with sample H2 having the highest turbidity. Thus, it is desirable to maintain the pH in the range of 6.5 ± 0.5.

In addition, in the absence of a pH adjusting agent, the pH changes of different samples were also different. For example, the pH value of sample No. 2 in the excipient screening experiment changed little when stored at 60°C for 2 weeks. The pH value of sample No. 0 in pH range screening experiment II changed little when stored at 60°C for 2 weeks. The pH value of sample H0 in pH range screening experiment II changed little when stored at 60°C for 2 weeks, with a range of change of 0.5. Know-

Claims (12)

The pH values of samples Nos. 1-5 in trehalose screening experiment II changed significantly when stored at 60°C for 2 weeks, with a range of 1.0. All publications and patent documents mentioned in this application are incorporated herein by reference as if each publication or patent were specifically and individually indicated by reference. Various changes and equivalent substitutions may be made to the various embodiments described herein without departing from the true spirit and scope of the invention. Any feature, step, or embodiment of the present invention may be used in combination with any other feature, step, or embodiment unless the context indicates otherwise. CLAIM 1. A pharmaceutical composition containing a therapeutically effective amount of a peptide, a pH-regulating agent and trehalose as an excipient, where the peptide contains the amino acid sequence YEKLLDTEI (SEQ ID NO: 1) or a functional variant thereof and wherein said functional variant is a variant having one or more than one conservative substitution in YEKLLDTEI (SEQ ID NO: 1), wherein said conservative substitution is selected from the group consisting of a substitution between D and E, a substitution among L, V, and I, and a substitution between T and S, and wherein the pH of the pharmaceutical composition is from about 6.5 to 7.5, where the mass ratio of peptide to trehalose is from about 1:0.5 to 1:1. 2. The pharmaceutical composition according to claim 1, wherein the functional variant is a variant formed by one or more than one conservative substitution in the LDTEI segment of SEQ ID NO: 1, and the conservative substitution is selected from the group consisting of a substitution between D and E, substitutions among L, V and I and substitutions between T and S. 3. The pharmaceutical composition according to claim 2, wherein the functional variant is a variant formed by replacing the LDTEI segment (SEQ ID NO: 6) in SEQ ID NO: 1 with a sequence selected from the group consisting of LDTEL (SEQ ID NO: 7 ), LDTEV (SEQ ID NO: 8), LDTDI (SEQ ID NO: 9), LDTDL (SEQ ID NO: 10), LDTDV (SEQ ID NO: 11), LDSEI (SEQ ID NO: 12), LDSEL (SEQ ID NO: 13), LDSEV (SEQ ID NO: 14), LDSDI (SEQ ID NO: 15), LDSDL (SEQ ID NO: 16), LDSDV (SEQ ID NO: 17), LETEI (SEQ ID NO: 18) , LETEL (SEQ ID NO: 19), LETEV (SEQ ID NO: 20), LETDI (SEQ ID NO: 21), LETDL (SEQ ID NO: 22), LETDV (SEQ ID NO: 23), VDTEI (SEQ ID NO: 24), VDTEL (SEQ ID NO: 25), VDTEV (SEQ ID NO: 26), VDTDI (SEQ ID NO: 27), VDTDL (SEQ ID NO: 28), VDTDV (SEQ ID NO: 29), IDTEI (SEQ ID NO: 30), IDTEL (SEQ ID NO: 31), IDTEV (SEQ ID NO: 32), IDTDI (SEQ ID NO: 33), IDTDL (SEQ ID NO: 34), IDTDV (SEQ ID NO : 35), IETEI (SEQ ID NO: 36), IETEL (SEQ ID NO: 37), IETEV (SEQ ID NO: 38), IETDI (SEQ ID NO: 39), IETDL (SEQ ID NO: 40) and IETDV (SEQ ID NO: 41). 4. The pharmaceutical composition according to claim 1, wherein the peptide is a chimeric peptide containing the amino acid sequence YEKLLDTEI (SEQ ID NO: 1) or a functional variant thereof and an internalization peptide, wherein the internalization peptide facilitates the uptake of said chimeric peptide into a cell. 5. The pharmaceutical composition according to claim 4, wherein the internalization peptide contains the amino acid sequence YGRKKRRQRRR (SEQ ID NO: 2), and preferably the chimeric peptide contains the amino acid sequence YGRKKRRQRRRYEKLLDTEI (SEQ ID NO: 3). 6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the pH adjusting agent is selected from the group consisting of histidine buffer, arginine buffer, sodium succinate buffer, potassium succinate buffer, sodium citrate buffer, gluconate buffer, acetate buffer, phosphate buffer, Tris buffer and any combination thereof, preferably the pH adjusting agent is a citric acid/disodium phosphate buffer or a histidine/arginine buffer, and more preferably the pH adjusting agent is a histidine/arginine buffer. 7. Pharmaceutical composition according to any one of claims 1 to 6, wherein the pH of the composition is approximately 6.5. 8. The pharmaceutical composition of claim 6, wherein the pH adjusting agent is a histidine/arginine buffer and the amount of histidine/arginine in the histidine/arginine buffer, by weight, is from about 1 to 10%, preferably from about 3 to 10%. 9. Pharmaceutical composition according to any one of claims 1 to 8, where the mass ratio of the peptide and trehalose is approximately 1:0.5. 10. The pharmaceutical composition according to claim 9, where the mass ratio of the peptide and trehalose is approximately 1:1. 11. The pharmaceutical composition according to any one of claims 1 to 10, which is in the form of a pre-lyophilized composition, or in the form of a lyophilized composition, or in the form of a reconstituted composition obtained by combining the lyophilized composition with an aqueous solution. 12. Pharmaceutical composition according to any one of claims 1-11 for treatment, reducing intensity -