Composition and method for treating neonatal hypoxic-ischemic encephalopathy

Abstract

A use of an effective amount of an active peptide in the preparation of a drug for the prevention and / or treatment of neonatal hypoxic-ischemic encephalopathy, an amino acid sequence of the active peptide containing KKNRNKLRRQHSY or a functionally equivalent variant thereof. Optionally, the active peptide further comprises a signal peptide at the N-terminal or the C-terminal thereof.

Description

Compositions and methods for treating neonatal hypoxic-ischemic encephalopathy

[0001] This disclosure claims priority to Chinese patent application CN202311635832.6 filed on December 1, 2023, and the entire contents of the aforementioned patent application are incorporated into this disclosure by reference. Technical Field

[0002] The present disclosure relates to the field of research and development of small molecule polypeptide drugs, and in particular to the use of an effective amount of active peptides in the preparation of drugs for preventing and / or treating neonatal hypoxic-ischemic encephalopathy. Background Art

[0003] Hypoxic-ischemic encephalopathy (HIE) is a type of neonatal hypoxic-ischemic encephalopathy (HIE) that results from prenatal, perinatal, or postnatal asphyxia, resulting in brain hypoxia. Common contributing factors include various causes of fetal distress (approximately 50%), such as umbilical cord around the neck and amniotic fluid abnormalities. It is also commonly associated with asphyxia and hypoxia during delivery (approximately 40%) and congenital diseases (approximately 10%). The incidence of HIE in developed countries is 1-8 cases per 1,000 births, while in underdeveloped countries, the incidence is as high as 26 cases per 1,000 births. According to WHO data, globally, between 2000 and 2003, approximately 848,000 children under the age of five died annually from neonatal asphyxia, also known as HIE. 40%-60% of neonatal hypoxic-ischemic encephalopathy patients die before the age of 2 or suffer from sequelae such as mental retardation, epilepsy, and cerebral palsy.

[0004] Currently, there is no clinical drug that can prevent the death of nerve cells caused by neonatal hypoxic-ischemic encephalopathy. The only clear and effective treatment is mild hypothermia treatment for newborns. Generally, the child's head is targeted to be cooled to 34.5℃, or the whole body is cooled to 33.5℃. The hypothermia treatment is continued for 48-72 hours, and then the temperature is gradually raised back to 36.5℃ at a rate of 0.5℃ per hour. There are strict requirements for temperature control and treatment time of hypothermia treatment. When the temperature drops to 32℃, the neuroprotective effect is lost. When the temperature drops below 30℃, it will cause serious damage. In addition, hypothermia treatment is best performed within 6 hours of the occurrence of neonatal hypoxic-ischemic encephalopathy damage, otherwise it will lose its efficacy and the treatment time window is narrow. Therefore, even among children who received hypothermia treatment, less than half of the symptoms improved.

[0005] Although in recent years, some individual drugs have shown therapeutic effects in preventing nerve cell death in adult neurological injuries, the pathogenesis, pathological process, and treatment strategies of neonatal hypoxic-ischemic encephalopathy are different from those of adult neurological injuries such as ischemic stroke and traumatic brain injury. Therefore, these drugs have no clear therapeutic effect in neonatal hypoxic-ischemic encephalopathy.

[0006] The primary cause of traumatic brain injury is mechanical damage to neurons and nerve fibers caused by external forces. Current treatment strategies focus on controlling intracranial pressure and traumatic bleeding. However, the primary cause of neonatal hypoxic-ischemic encephalopathy (HIE) is energy depletion caused by oxygen and glucose deprivation, a completely different pathogenesis from TBI. Consequently, while mild hypothermia therapy has a proven therapeutic effect on neonatal HIE, its effectiveness in treating TBI in adults remains controversial.

[0007] Compared to ischemic stroke in adults, although both are caused by ischemia and hypoxia, the hypoxia-ischemia of adult stroke is primarily caused by localized vascular blockage caused by a thrombus, leading to focal brain tissue damage around the thrombus. Treatment includes thrombolysis, intravascular therapy, and antiplatelet drugs. In contrast, neonatal hypoxic-ischemic encephalopathy (HIE) primarily stems from asphyxia during delivery, causing widespread ischemia and hypoxia that affect the entire brain. Furthermore, compared to adults, neonatal brain tissue has a high metabolic rate and is more sensitive to hypoxia and ischemia. Brain glycogen is limited and dependent on the blood circulation, making hypoxia and ischemia more susceptible to hypoglycemia and impaired normal metabolic activity of the nervous system. Furthermore, the neonatal brain is characterized by fragile cerebral vessels and imperfect cerebrovascular autoregulation, making it less tolerant to hypoxia and ischemia. Therefore, neonatal HIE faces a more severe impact. More importantly, the neonatal brain is still developing. Compared to the adult brain, its neurons have not yet differentiated and matured. Significant differences exist in receptor expression, cell morphology / structure, synaptic density, axonal myelin coating, cell excitability, and susceptibility to cell death. Therefore, it is difficult to predict the effectiveness of treatments or drugs that are effective for ischemic stroke in adults for neonatal hypoxic-ischemic encephalopathy.

[0008] In summary, drug development for neonatal hypoxic-ischemic encephalopathy (HIE) needs to consider the specificity of the disease. Therefore, there is still an urgent need in the field for drugs that can effectively prevent neuronal cell death and protect neonatal brain tissue in HIE.

[0009] Overview

[0010] The present disclosure provides a use of an effective amount of an active peptide in preparing a medicament for preventing and / or treating neonatal hypoxic-ischemic encephalopathy.

[0011] In one embodiment, the present application provides a method for preventing and / or treating neonatal hypoxic-ischemic encephalopathy, comprising the steps of administering to a subject an effective amount of an active peptide of the present disclosure. In one embodiment, administering to a subject an effective amount of an active peptide of the present disclosure inhibits the death of at least one neuronal cell of the subject.

[0012] In one embodiment, the present application provides a method for preventing and / or inhibiting neural cell damage in neonatal hypoxic-ischemic encephalopathy, comprising exposing at least one neural cell of a neonatal subject to an effective amount of an active peptide, thereby inhibiting damage to at least one neural cell of the subject.

[0013] In one embodiment, the step of exposing at least one neural cell to an effective amount of an active peptide of the present invention or administering an effective amount of an active peptide of the present invention to a subject is performed within a short period of time after suffering from neonatal hypoxic-ischemic encephalopathy. In an exemplary embodiment, the active peptide can be administered within a period of time after neonatal hypoxic-ischemic encephalopathy, for example, within one month, two weeks, one week, 72 hours, 48 ​​hours, 24 hours, 18 hours, 12 hours, 8 hours, 6 hours, 4 hours, 2 hours, and 1 hour; preferably within two weeks, one week, 72 hours, more preferably 48 hours, still more preferably 24 hours or less, for example, within 18, 12, 8, 6, 4, 2, or 1 hour.

[0014] In one embodiment, the present disclosure provides the use of an effective amount of an active peptide in the preparation of a medicament for inhibiting nerve cell damage after neonatal hypoxic-ischemic encephalopathy.

[0015] In one embodiment, the present disclosure provides the use of an effective amount of an active peptide in the preparation of a medicament for inhibiting nerve cell damage after acute neonatal hypoxic-ischemic encephalopathy.

[0016] In another embodiment, the present disclosure provides the use of an effective amount of an active peptide in the preparation of a medicament for treating a subject after neonatal hypoxic-ischemic encephalopathy. The subject can be a mammal, such as a human.

[0017] In another embodiment, the present disclosure provides a composition for treating neural damage after neonatal hypoxic-ischemic encephalopathy, the composition comprising an active peptide of the present disclosure and a pharmaceutically acceptable carrier.

[0018] In another embodiment, the present disclosure provides the use of the active peptide of the present disclosure or a composition comprising the active peptide of the present disclosure for preventing and / or treating neonatal hypoxic-ischemic encephalopathy.

[0019] In another embodiment, the present disclosure provides the use of the active peptide of the present disclosure or a composition comprising the active peptide of the present disclosure for inhibiting nerve cell damage after neonatal hypoxic-ischemic encephalopathy.

[0020] In another embodiment, the present disclosure provides the use of the active peptide of the present disclosure or a composition comprising the active peptide of the present disclosure for inhibiting nerve cell damage after acute neonatal hypoxic-ischemic encephalopathy.

[0021] In another embodiment, the present disclosure provides an active peptide of the present disclosure or a method comprising the active peptide of the present disclosure for preventing and / or treating neonatal hypoxic-ischemic encephalopathy.

[0022] In another embodiment, the present disclosure provides an active peptide of the present disclosure or a method comprising the active peptide of the present disclosure for inhibiting nerve cell damage after neonatal hypoxic-ischemic encephalopathy.

[0023] In another embodiment, the present disclosure provides the active peptide of the present disclosure or a method comprising the active peptide of the present disclosure for inhibiting nerve cell damage after acute neonatal hypoxic-ischemic encephalopathy.

[0024] In another embodiment, the active peptide of the present disclosure is formulated into a dosage form for administration through the gastrointestinal tract or parenteral administration, wherein the dosage form is a pharmaceutically acceptable oral dosage form, injection dosage form or powder injection.

[0025] In another embodiment, the therapeutically effective amount of the active peptide of the present disclosure is in the range of 0.1-1000 mg / kg body weight, such as 1-100 mg / kg body weight, 10-50 mg / kg body weight.

[0026] In another embodiment, the peptide of the present disclosure may comprise SEQ ID NO: 1 (KKNRNKLRRQHSY) or a functionally equivalent variant thereof having 1, 2 or 3 conservative amino acid substitutions, additions or deletions. The peptide of the present disclosure may be selected from sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity to SEQ ID NO: 1.

[0027] In another embodiment, the peptide of the present disclosure may further comprise a signal peptide at its N-terminus or C-terminus. The signal peptide is selected from a TAT sequence, a MAP sequence, an MTS sequence, or an R9 sequence.

[0028] In another embodiment, the active peptides of the present disclosure may be chemically modified.

[0029] Without being bound by theory, surprisingly, the inventors of the present disclosure found that the active peptides of the present disclosure can inhibit neural damage after neonatal hypoxic-ischemic encephalopathy, significantly improve brain tissue survival after neonatal hypoxic-ischemic encephalopathy, and improve the overall health of newborns. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1. S1 pretreatment in vitro reduces neuronal damage induced by oxygen-glucose deprivation.

[0031] (1A) Experimental procedures for evaluating the neuroprotective effect of S1 pretreatment on excitotoxic injury after oxygen-glucose deprivation injury.

[0032] (1B) Lactate dehydrogenase (LDH) cytotoxicity assay quantitatively demonstrated the neuroprotective effect of S1 pretreatment.

[0033] (1C) MTT cell survival assay, quantitatively showing the neuroprotective effect of S1 pretreatment.

[0034] Figure 2. S1 treatment significantly improves the survival of neonatal rat brain tissue after neonatal hypoxic-ischemic encephalopathy injury.

[0035] (2A) Experimental procedures for evaluating the neuroprotective effect of S1 treatment on hypoxic-ischemic encephalopathy injury in neonatal rats.

[0036] (2B) TTC-stained brain tissue images of neonatal rats treated with S1 or scrambled peptide after neonatal hypoxic-ischemic encephalopathy injury.

[0037] (2C) Quantification of viable brain tissue.

[0038] Figure 3. S1 treatment significantly improved the weight gain of neonatal rats after neonatal hypoxic-ischemic encephalopathy injury.

[0039] Following hypoxic-ischemic encephalopathy, the rate of increase relative to pre-injury body weight was quantified in neonatal rats treated with S1 or scrambled peptide 24 and 48 hours after injury.

[0040] Figure 4. S1 treatment significantly improves the spatial learning and memory abilities of neonatal rats injured by hypoxic-ischemic encephalopathy in adulthood.

[0041] (4A) Neonatal rats treated with S1 or scrambled peptide after hypoxic-ischemic encephalopathy were tested for spatial learning in the Morris water maze four months after injury. The time it took the rats to reach the hidden platform in the water maze was measured on different days of training. (4B) Spatial memory was tested in the water maze. After the platform was removed from the water maze on day six, the time the rats spent swimming in the target quadrant where the platform had previously been located was measured. (4C) Swimming speed was measured in the water maze. DETAILED DESCRIPTION

[0042] For the purpose of promoting the understanding of the principle of the present disclosure, reference will now be made to the embodiments illustrated in the accompanying drawings and the embodiments will be described in detail. However, these descriptions are not intended to limit the scope of the present disclosure in any way.

[0043] The present disclosure provides various compositions, methods and uses for treating neural injury.In various embodiments of the present disclosure, the injury may be caused by neonatal hypoxic-ischemic encephalopathy injury.

[0044] As used herein, the term "mammal" refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sport, or pet animals, such as dogs, cats, cows, horses, sheep, pigs, goats, rabbits, and the like.

[0045] As used herein, the term "therapeutically effective amount" refers to an amount of a molecule, polypeptide, or other drug that is effective in "treating" neural damage or injury in a subject or mammal. In the case of neonatal hypoxic-ischemic encephalopathy, a therapeutically effective amount of a "drug" can reduce the level of neural cell death or damage; reduce the area of ​​neural tissue damage; and / or alleviate, to some extent, one or more symptoms associated with neonatal hypoxic-ischemic encephalopathy. In one embodiment, a therapeutically effective amount includes, but is not limited to, about 0.1-1000 mg / kg body weight, such as 1-100 mg / kg body weight, 10-50 mg / kg body weight.

[0046] Ways to prevent nerve damage:

[0047] In at least one embodiment of the present disclosure, nerve damage can be treated by introducing an inhibitor into the brain. For example, the disclosed method for inhibiting nerve cell damage may include the steps of exposing at least one nerve cell to an effective amount of an active peptide, thereby inhibiting damage to the at least one nerve cell. Additionally, the drug of the method may include a peptide, nucleotide, or molecule. Optionally, the drug may also include a pharmaceutically acceptable carrier.

[0048] According to the method for treating a subject with traumatic brain injury disclosed herein, the method comprises administering to the subject a therapeutically effective amount of an active peptide, whereby at least one nerve cell of the subject is inhibited. In at least one embodiment, the subject may be a mammal, such as a human. In addition, administration of a therapeutically effective amount of the peptide may be performed by the following routes: oral, intravenous, intramuscular, subcutaneous, retrograde intravenous, intraarterial, transdermal, inhalation, and surgical implantation. In an exemplary embodiment, the method for treating a subject with neonatal hypoxic-ischemic encephalopathy disclosed herein reduces the level of brain tissue damage in the subject and improves its spatial learning and memory abilities.

[0049] According to the method of the present disclosure, the step of exposing at least one neural cell to an effective amount of the peptide of the present disclosure or administering an effective amount of the peptide of the present disclosure to the subject is carried out within a short period of time after suffering from neonatal hypoxic-ischemic encephalopathy injury. In an exemplary embodiment, the active peptide of the present disclosure is administered within one week after neonatal hypoxic-ischemic encephalopathy injury, preferably 72 hours, more preferably 48 hours, still more preferably 24 hours or less, for example, within 18, 12, 8, 6, 4, 2 or 1 hour.

[0050] Peptides of the present disclosure:

[0051] In an exemplary embodiment, peptides suitable for the present disclosure include a peptide having SEQ ID NO: 1 (KKNRNKLRRQHSY) or a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or 100% identity thereto.

[0052] In an exemplary embodiment, peptides suitable for the present disclosure include functionally equivalent variants of a peptide having or consisting of SEQ ID NO: 1. For example, the variant has 1, 2, or 3 conservative amino acid substitutions, additions, or deletions and exhibits substantially similar in vivo or in vitro activity as the peptide examples of the present disclosure.

[0053] Variants of the peptides disclosed herein are also within the scope of the present disclosure. A peptide variant refers to an amino acid sequence in which one or more amino acids are altered. A variant can have "conservative" changes, wherein the substituted amino acids have similar structural or chemical properties, such as replacing leucine with isoleucine. Alternatively, a variant can have "non-conservative" changes, such as replacing glycine with tryptophan. Similar minor changes can also include amino acid deletions or insertions, or both. A specific form of a "variant" peptide is a "functionally equivalent" peptide, i.e., a peptide that exhibits substantially similar in vivo or in vitro activity to the peptide examples disclosed herein. Guidance for determining which amino acid residues can be substituted, inserted, or deleted without losing biological or immunological activity can be found using computer programs well known in the art, such as DNASTAR software (DNASTAR, Inc., Madison, WI). In addition, specific guidance is provided below, including those provided within the cited references, which are incorporated herein by reference.

[0054] In other embodiments, the specific position of the named residue can be varied slightly while still existing at a structurally and functionally analogous position in the peptide (see Chang, Y., et al., Biochemistry 37:3258-3271 (1998)).

[0055] In addition, the peptide of the present disclosure may further comprise a signal peptide at its N-terminus or C-terminus that can guide the peptide of the present disclosure across the blood-brain barrier and the neural cell membrane. Suitable signal peptides known in the art can be used in the present disclosure, as long as they can guide the peptide of the present disclosure across the blood-brain barrier and the neural cell membrane. Exemplary signal peptides can be selected from TAT sequence ("GRKKRRQRRR (SEQ ID NO: 3)", "YGRKKRRQRRR (SEQ ID NO: 4)", "YGRKKRRQRRRPPQ (SEQ ID NO: 5)" or "GRKKRRQRRRQ (SEQ ID NO: 6)"), model amphipathic peptide (modelamphipathic peptide, MAP) sequence ("KLALKLALKALKAALKLA (SEQ ID NO: 7)"), membrane translocation peptide (membrane translocating peptide, MTS) sequence ("AAVALLPAVLLALLAP (SEQ ID NO: 8)") or R9 ("RRRRRRRRR (SEQ ID NO: 9)") sequence. In one embodiment, the signal peptide of the present disclosure is a Tat sequence. The peptide of the present disclosure may be chemically modified to facilitate its passage across the BBB and nerve cell membranes, including but not limited to amidation, acetylation, or cyclization. In an exemplary embodiment, the peptide of the present disclosure comprises or consists of the sequence YGRKKRRQRRRKKNRNKLRRQHSY (SEQ ID NO: 2), which is designated as S1.

[0056] The scrambled peptide disclosed herein is Tat-NRRRNSKLQHKKY (SEQ ID NO: 10), the sequence of which is YGRKKRRQRRRNRRRNSKLQHKKY.

[0057] In an exemplary embodiment, the peptide of the present disclosure is a short-chain peptide with strong hydrophobicity, which enables it to easily cross the blood-brain barrier and nerve cell membrane with the help of a signal peptide.

[0058] Example

[0059] The following examples are only used to illustrate the present disclosure rather than to limit the present disclosure.

[0060] In the present disclosure, the inventors evaluated the neuroprotective effects of brain-penetrating peptides in inhibiting cellular excitotoxicity, enhancing neuronal survival, reducing brain tissue damage, and improving overall health status and behavioral outcomes after neonatal hypoxic-ischemic encephalopathy.

[0061] Example 1: In in vitro experiments, S1 can protect neurons from cellular excitotoxicity after oxygen and glucose deprivation through pretreatment.

[0062] This example was designed to examine the neuroprotective effect of S1 on excitotoxic injury in cells after oxygen and glucose deprivation, as well as the optimal dose in in vitro neural cells.

[0063] Cerebral cortical neuronal cell culture: Neonatal rat brains were dissected, the meninges removed, and the cerebral cortex isolated. Cortical tissue was digested with papain, and single cells were collected, plated on lysine-pretreated coverslips, and cultured in Neurobasal™ serum-free medium. To simulate hypoxic-ischemic encephalopathy injury in vitro, the Neurobasal medium of cells at 7 days old (DIV7) was replaced with DMEM medium and cultured in a 21% oxygen / 5% carbon dioxide atmosphere for 2.5 hours as the control group, or replaced with DMEM medium without glucose and cultured in a 0% oxygen / 5% carbon dioxide atmosphere for 2.5 hours as the oxygen-glucose deprivation treatment group. After treatment, both groups were returned to Neurobasal medium and cultured normally in a 21% oxygen / 5% carbon dioxide atmosphere for 24 hours.

[0064] S1 pretreatment: At the beginning of oxygen and glucose deprivation treatment, cells were simultaneously treated with S1 (final concentration of 0.01M, 0.1M, 1M or 10M), or treated with scrambled peptide Tat-NRRRNSKLQHKKY (SEQ ID NO: 10) or NaP (Davunetide) as a control.

[0065] Lactate dehydrogenase (LDH) kit cytotoxicity test: After resuming normal culture for 24 hours, the cell culture supernatant of each group was collected, and LDH detection working solution was added. The mixture was mixed and incubated at room temperature in the dark for 30 minutes. The absorbance was then measured at 490 nm, and the LDH release level, i.e., cytotoxicity, was calculated.

[0066] MTT cell survival assay: After resuming normal culture for 24 hours, MTT solution was added to each group and cultured for another 4 hours. After terminating the culture, the culture medium was aspirated and discarded, DMSO was added and shaken at low speed for 10 minutes, and then the absorbance was measured at 490 nm, and the MTT formation level, i.e., the cell survival rate, was calculated.

[0067] result

[0068] To evaluate the effects of S1 pretreatment on neuronal survival during hypoxia-glucose deprivation-induced injury, NaP (final concentration 2 mM), S1 (final concentrations 0.01 M, 0.1 M, 1 M, or 10 M), or scrambled peptide (final concentrations 0.01 M, 0.1 M, 1 M, or 10 M) were added to the cell culture medium at the beginning of hypoxia-glucose deprivation. LDH and MTT assays were performed 24 hours after injury to measure excitotoxicity and neuronal survival (Figure 1A). Following hypoxia-glucose deprivation, LDH release increased and MTT levels decreased in the untreated group, indicating that hypoxia-glucose deprivation caused neuronal cytotoxicity and reduced neuronal survival. NaP, a positive control for neuroprotective agents, significantly decreased LDH release (p < 0.05, Figure 1B) and increased MTT formation (p < 0.05, Figure 1C) compared to the untreated group, demonstrating a neuroprotective effect. S1 treatment, at concentrations of 0.1M and 1M, significantly reduced LDH release levels (p<0.05, Figure 1B) and significantly increased MTT formation levels (p<0.05, Figure 1B) compared to the oxygen-glucose deprivation (OGD) untreated group, indicating that S1 pretreatment has the effect of reducing excitotoxicity and improving neuronal cell survival. However, the scrambled peptide treatment group did not change LDH release levels or MTT formation levels compared to the untreated group at any concentration (Figures 1B and 1C). Therefore, S1 pretreatment at concentrations of 0.1-1M has a neuroprotective effect on cells in vitro.

[0069] Example 2: In an in vivo experiment, S1 protected brain tissue from neonatal hypoxic-ischemic encephalopathy, improved the overall health of animals after injury, and rescued the spatial learning and memory abilities of animals in adulthood.

[0070] This example was designed to test the neuroprotective effects of S1 after neonatal hypoxic-ischemic encephalopathy, as well as its improvement on the overall health of injured animals and their spatial learning and memory abilities in adulthood.

[0071] Neonatal hypoxic-ischemic encephalopathy animal model and S1 treatment (Figure 2A): 7-day-old newborn rats underwent unilateral common carotid artery clamping. After 2 hours of recovery, they were placed in an hypoxic environment containing 8% oxygen and 92% nitrogen for 2.5 hours. One hour after the end of the hypoxic treatment, S1 or scrambled peptide (10 mg / kg body weight) was injected into the tail vein.

[0072] TTC method for brain tissue damage detection: 48 hours after the end of hypoxia treatment, the animals were sacrificed and the brains were removed, sliced, and incubated in TTC solution at 37°C for 30 minutes. Then, photos were taken under an optical microscope, and the area of ​​brain tissue necrosis was measured and the area of ​​surviving brain tissue was calculated.

[0073] Body weight measurement was used to detect the overall health status of the animals: the body weight of the newborn rats was measured before surgery, 24 hours after neonatal hypoxic-ischemic encephalopathy injury, and 48 hours after neonatal hypoxic-ischemic encephalopathy injury, and the weight gain rate after surgery relative to that before surgery was calculated.

[0074] Morris water maze tests spatial learning and memory abilities: Four months after the hypoxia treatment, rats received five days of water maze training to learn to find a submerged platform. The time to reach the platform will be recorded to calculate the learning score. The day after the training task is completed, the rats will be tested for spatial memory using a transfer (probe) test. During the transfer (probe) test, the platform will be removed and the total distance the rats swim in the target quadrant will be recorded. The data will be plotted as a learning curve of the time to reach the platform during the five-day water maze test. The total time in the target quadrant during the 60-second transfer test will also be recorded, and the variables recorded will also include swimming speed. The cumulative delay time from the target quadrant to the area where the platform is located will also be recorded to calculate the memory score.

[0075] result:

[0076] To evaluate the effects of S1 or scrambled peptide treatment on brain tissue survival after neonatal hypoxic-ischemic encephalopathy injury, S1 (final concentration 10 mg / kg body weight) or scrambled peptide was administered via tail vein injection 1 hour after injury. Brain tissue was stained with TTC 48 hours after injury. Healthy brain tissue reduces TTC and stains red, while injured brain tissue is unable to reduce TTC and therefore does not stain red, appearing white (Figure 2B). In the untreated control group, neonatal hypoxic-ischemic injury caused significant brain necrosis, with large areas of brain tissue appearing white and visible damage to the brain surface. Only 54.9% of brain tissue survived (Figures 2B and 2C). In the S1-treated group, the area of ​​white brain tissue necrosis was significantly reduced compared with the untreated group, and the brain tissue surface damage was less obvious. The brain tissue survival rate increased to 75.4%, significantly higher than that in the untreated group (p < 0.05, Figures 2B and 2C). In the scrambled peptide-treated control group, the area of ​​white necrotic brain tissue was similar to that of the untreated injury group, and the brain tissue surface was significantly damaged. The brain tissue survival rate was only 52.5%, significantly lower than that of the S1-treated group (p < 0.05, Figures 2B and 2C). Therefore, S1 treatment significantly improved the brain tissue survival rate after neonatal hypoxic-ischemic encephalopathy injury and had a neuroprotective effect.

[0077] To evaluate the effects of S1 or scrambled peptide treatment on the overall health of animals after HIE injury, 7-day-old newborn rats were weighed and weight recorded before injury and then subjected to the aforementioned treatments. S1 (final concentration 10 mg / kg body weight) or scrambled peptide (final concentration 10 mg / kg body weight) was administered via tail vein injection 1 hour after injury, and body weights were measured and recorded 24 and 48 hours after injury. In normal 7-day-old newborn rats, weight continued to increase over the next two days, increasing by 13.47% on day 8 and 26.77% on day 9 (Figure 3). However, in rats subjected to HIE injury on day 7, weight gain decreased to only 5.42% 24 hours after injury and on day 8, and by only 13.4% on day 9, significantly lower than that in the sham-operated control group (p < 0.05, Figure 3). Therefore, HIE injury severely impacts the overall health of newborn rats. Neonatal rats in the S1-treated group gained 12.9% body weight 24 hours after surgery, or on the eighth day, and 23.32% 48 hours after injury, or on the ninth day. Both weight increases were significantly higher than those in the untreated group (p < 0.05, Figure 3). In contrast, neonatal rats in the scrambled peptide-treated group gained only 9.23% body weight 24 hours after injury, or on the eighth day, and 17.89% 48 hours after injury, or on the ninth day, significantly lower than those in the sham-operated control group (p < 0.05, Figure 3). There was no statistical difference between these groups and the untreated group. Therefore, S1 significantly improved the overall health of neonatal rats following neonatal hypoxic-ischemic encephalopathy injury.

[0078] To assess the effects of S1 or scrambled peptide treatment on spatial learning and memory in adulthood, 7-day-old neonatal rats, four months after HIE treatment, were tested in the Morris water maze for six days. On the first training day, there were no significant differences in escape times between the groups, ensuring that the animals began the test at similar behavioral levels. The escape time of the sham-operated group decreased daily over the first four days, reaching a peak on the fifth day (Figure 4A), indicating that they were learning the spatial location of the underwater platform. Although the escape time of untreated rats also gradually decreased during training, it remained significantly longer than that of the sham-operated group, indicating that although they were also learning the spatial location of the underwater platform, their learning ability was impaired (Figure 4A). The learning curves of the scrambled peptide-treated rats were similar to those of the untreated rats, with no significant differences, indicating that their learning impairment had not been ameliorated (Figure 4A). The escape times of rats in the S1-treated group remained consistently shorter than those of the untreated injury group during training. Although still higher than those of the sham-operated group, they were significantly lower than those of the untreated injury group on days 4 and 5 of learning (p < 0.05), indicating that their spatial learning ability had improved compared to the untreated injury group (Figure 4A). Therefore, S1 treatment significantly improved spatial learning ability in adult rats after neonatal hypoxic-ischemic encephalopathy injury. In the transfer (probe) test on day 6, rats in the sham-operated control group spent 51.16% of their time in the target quadrant, demonstrating their spatial memory of the underwater platform. However, rats in the untreated injury group spent only 29.49% of their time in the target quadrant, indicating impaired spatial memory (Figure 4B). The S1-treated rats spent approximately 47.86% of their time in the target quadrant, significantly higher than the untreated injury group (p < 0.05, Figure 4B), indicating a significant improvement in their spatial memory. In contrast, rats in the scrambled peptide-treated group spent only 29.12% of their time in the target quadrant, which was not significantly different from the untreated injury group. Therefore, S1 treatment significantly improved the spatial memory ability of adult rats after neonatal hypoxic-ischemic encephalopathy injury. However, there was no significant difference in swimming speed among rats in each group, eliminating the possibility that swimming speed affects behavior.

[0079] Although various embodiments of compositions and methods for treating neonatal hypoxic-ischemic encephalopathy injury have been described in great detail herein, such embodiments are provided merely as non-limiting examples of the disclosure described herein. Therefore, it will be understood by those skilled in the art that various changes and modifications may be made to the disclosure without departing from the scope of the disclosure. Indeed, the disclosure is not intended to be exhaustive or to limit the scope of the disclosure.

[0080] Furthermore, in the description of representative embodiments, the present disclosure has presented the methods and / or processes of the present disclosure in a specific order of steps. However, the methods or processes should not be limited to the specific order of steps described. Other order of steps are possible. Therefore, the specific order of steps disclosed herein should not be interpreted as limiting the present disclosure. In addition, the disclosure of the methods and / or processes should not be limited to performing their steps in the order described. Such an order can be varied and still be within the scope of the present disclosure.

Claims

1. Use of an effective amount of an active peptide in the preparation of a drug for preventing and / or treating neonatal hypoxic-ischemic encephalopathy, wherein the amino acid sequence of the active peptide comprises KKNRNKLRRQHSY (SEQ ID NO: 1) or a functionally equivalent variant thereof.

2. The use according to claim 1, wherein the amino acid sequence of the active peptide is KKNRNKLRRQHSY (SEQ ID NO: 1) or a functionally equivalent variant thereof.

3. The use according to claim 1 or 2, wherein the variant has 1, 2 or 3 conservative amino acid substitutions, additions or deletions.

4. The use according to any one of claims 1 to 3, wherein the active peptide has a sequence selected from KKNRNKLRRQHSY (SEQ ID NO: 1) that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identical.

5. The use according to any one of claims 1 to 4, wherein the active peptide is chemically modified.

6. The use according to any one of claims 1 to 5, wherein the active peptide further comprises a signal peptide at its N-terminus or C-terminus.

7. The use according to claim 6, wherein the signal peptide is selected from a TAT sequence, a MAP sequence, a MTS sequence or a R9 sequence.

8. The use according to claim 7, wherein the amino acid sequence of TAT is selected from GRKKRRQRRR (SEQ ID NO: 3), YGRKKRRQRRR (SEQ ID NO: 4), YGRKKRRQRRRPPQ (SEQ ID NO: 5) or GRKKRRQRRRQ (SEQ ID NO: 6).

9. The use according to claim 8, wherein the amino acid sequence of the active peptide is YGRKKRRQRRRKKNRNKLRRQHSY (SEQ ID NO: 2).

10. The use according to any one of claims 1 to 9, wherein the active peptide is prepared as a drug for administration within a period of time after the onset of neonatal hypoxic-ischemic encephalopathy, wherein the period of time is selected from one month, two weeks, one week, 72 hours, 48 ​​hours, 24 hours, 18 hours, 12 hours, 8 hours, 6 hours, 4 hours, 2 hours or 1 hour.

11. The use according to any one of claims 1 to 10, wherein the effective amount is in the range of 0.1-1000 mg / kg body weight.

12. The use according to any one of claims 1 to 11, wherein the active peptide is used to inhibit nerve cell damage in neonatal hypoxic-ischemic encephalopathy.

13. The use according to any one of claims 1 to 11, wherein the neonatal hypoxic-ischemic encephalopathy is acute neonatal hypoxic-ischemic brain damage.

14. The use according to any one of claims 1 to 13, wherein the active peptide is formulated into a dosage form for administration through the gastrointestinal tract or parenterally.

15. The use according to claim 14, wherein the dosage form is a pharmaceutically acceptable oral dosage form, injection dosage form or powder injection.

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