Neuroprotective agent capable of being combined with rt-PA and application of neuroprotective agent

Abstract

The invention relates to a neuroprotective agent capable of being combined with rt-PA and application of the neuroprotective agent, the neuroprotective agent is a synthetic small peptide, the amino acid sequence of the neuroprotective agent comprises at least one unmodified amino acid with neuroprotection, at least one of peptide bonds connected with the unmodified amino acid is a protease hydrolysis site, and at least one of the peptide bonds connected with the unmodified amino acid is a protease hydrolysis site. The amino acids can be hydrolyzed into free single amino acids and are used as pharmacologically active ingredients; at least one peptide end is connected with phenylalanine which can be transported by a transporter, so that the small peptide can penetrate through a cell membrane and / or a blood brain barrier; the phenylalanine is L-Phe (L-Phe) or D-Phe (D-Phe); experiments prove that the small peptide can effectively penetrate the blood brain barrier and release free single amino acid or short peptide after hydrolysis to play a role in neuroprotection, is a safe and effective neuroprotective agent, can be used for preparing a medicine for treating nervous system diseases, and particularly can be combined with rt-PA to prepare a neuroprotective medicine for treating cerebral apoplexy.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and more specifically to a neuroprotectant that can be used in combination with rt-PA and an application thereof. Background Art

[0002] According to the latest research, neurological diseases affect more than one-third of the world's population and have become one of the main causes of disability and health loss. Common neurological diseases include Alzheimer's disease, epilepsy, stroke, Parkinson's disease, etc. Among them, stroke is caused by the sudden rupture or blockage of blood vessels in the brain, which interrupts the blood flow to the brain tissue and causes brain tissue damage. The brain tissue has a very high demand for oxygen and nutrition, so when the blood flow is interrupted, brain cells, including neurons, will quickly be damaged or even die, and neuronal damage or death may lead to neurological dysfunction, such as loss of sensation, movement disorders or cognitive problems, etc., especially ischemic stroke will not only cause immediate neuronal damage, but also may trigger a series of secondary injuries.

[0003] Stroke includes ischemic stroke and hemorrhagic stroke. Ischemic stroke is mainly treated by intravenous thrombolysis, which performs intravascular thrombolysis to enhance vascular recanalization, such as intravenous injection of thrombolytic drug rt-PA (recombinant tissue plasminogen activator). Since ischemia and hypoxia cause nerve cell damage, neuroprotective therapy has become an important means of treating ischemic stroke. Neuroprotective agents mainly exert neuroprotective effects by targeting nerve cell damage and promoting cell survival targets. For example, NA-1 (Nerinetide) is used as a neuroprotective agent for the treatment of patients with acute ischemic stroke (AIS), but Mayor-Nunez D et al. found that the combination of NA-1 clinical trials with rt-PA would hydrolyze NA-1, making it difficult to exert neuroprotective effects (Plasmin-resistant PSD-95 inhibitors resolve effect-modifying drug-drug interactions between alteplase and nerinetide in acute stroke. Sci Transl Med. 2021; 13(588):eabb1498.).

[0004] Therefore, the development of more effective neuroprotective agents is of great significance for the treatment and prognosis of neurological diseases, especially neuroprotective agents that can be used in combination with rt-PA, which are more conducive to the treatment and prognosis of ischemic stroke. Summary of the invention

[0005] In view of the above defects or improvement needs of the prior art, the present invention provides a neuroprotectant that can be used in combination with rt-PA and its application, and its purpose is to provide a neuroprotectant that can be used in combination with rt-PA, which is a synthetic small peptide, and its amino acid sequence includes at least one unmodified amino acid with neuroprotective properties, and at least one peptide end is connected to a phenylalanine (L-Phe or D-Phe) that can be transported by a transporter, and is used as a transmembrane carrier to allow the small peptide to penetrate the blood-brain barrier and enter neurons. The small peptide can hydrolyze the unmodified amino acid with neuroprotective properties into free single amino acids in the brain to play its role in protecting nerves, can be used as a neuroprotectant, and can be used in combination with rt-PA, thereby solving the technical problem that the efficacy of existing neuroprotectants combined with rt-PA is significantly reduced, and it is difficult to achieve combination with rt-PA.

[0006] To achieve the above object, according to one aspect of the present invention, a neuroprotectant that can be used in combination with rt-PA is provided, which is a synthetic small peptide, and its amino acid sequence includes at least one unmodified amino acid with neuroprotective properties, wherein at least one of the peptide bonds connected to the unmodified amino acid is a protease hydrolysis site, so that it can be hydrolyzed into free single amino acids for use as a pharmacologically active ingredient;

[0007] At least one peptide end is connected to phenylalanine which can be transported by a transporter, so that the small peptide can penetrate the cell membrane and / or the blood-brain barrier; the phenylalanine is L-Phe or D-Phe; and the transporter includes LAT1.

[0008] Preferably, the neuroprotectant is a small peptide with a molecular weight of less than or equal to 600 Da, and its amino acid sequence includes the following:

[0009] X 1 -X 2 -X 3 -X 4 ;

[0010] If the C-terminus of the small peptide is phenylalanine F; X 3 is proline P, glycine G, cysteine ​​C, arginine R, alanine A, or lysine K; X 1 , X 2 is 0 or any amino acid;

[0011] If the N-terminus of the small peptide is phenylalanine F; X 2 is proline P, glycine G, cysteine ​​C, arginine R, alanine A, or lysine K; X 3 , X 4 is 0 or any amino acid.

[0012] Preferably, in the amino acid sequence of the small peptide of the neuroprotectant, except for phenylalanine F at one end, the remaining amino acids are amino acids with neuroprotective properties.

[0013] Preferably, the neuroprotectant is a dipeptide having an amino acid sequence as follows:

[0014] PF, GF, CF, FP, FG, FC, RF, FR, AF, FA, KF or FK;

[0015] The amino acid sequence of the tripeptide includes the following sequence:

[0016] GCF, PGF, GPF, CGF, FPG, FGP, FCG, RGF, FGR, AGF, FGA, KGF, FGK, RPF, FPR, APF, FPA, KPF or FPK, wherein F in GCF is D-Phe;

[0017] The amino acid sequence of the tetrapeptide is as follows:

[0018] PGPF, GPGF, CGPF, PGCF, RPGF, FGPR, APGF, FGPA, KPGF, FGPK, RGPF, FPGR, AGPF, FPGA, KGPF or FPGK, wherein P, G, C, A, K, or R is one of the pharmacologically active ingredients.

[0019] Preferably, the neuroprotectant is a tripeptide with an amino acid sequence of GCF, PGF, or GPF; and is a tetrapeptide with an amino acid sequence of PGPF.

[0020] According to another aspect of the present invention, there is provided a use of the neuroprotectant as described in the present invention in the preparation of drugs for treating nervous system diseases, wherein the nervous system diseases include neurodegenerative diseases and brain damage; wherein the neuroprotectant is used for preparing drugs for treating brain damage, and the neuroprotectant is administered intravenously, into the skull bone marrow, or into the nasal cavity.

[0021] Preferably, in the application, the neurodegenerative diseases include Alzheimer's disease and Parkinson's disease, and the brain injury includes stroke and traumatic brain injury.

[0022] Preferably, in the application, the neuroprotectant is used in combination with rt-PA to prepare a drug for treating ischemic stroke.

[0023] According to another aspect of the present invention, a composition is provided, which comprises one or more of the neuroprotective agents of the present invention and rt-PA.

[0024] Preferably, the composition comprises PF+rt-PA, GCF+rt-PA, or GF+rt-PA.

[0025] In general, the above technical solutions conceived by the present invention can achieve the following beneficial effects compared with the prior art:

[0026] The neuroprotectant provided by the present invention is a synthetic small peptide, and its amino acid sequence includes at least one unmodified amino acid with neuroprotection, wherein at least one of the peptide bonds connected to the unmodified amino acid is a protease hydrolysis site, so that it can be hydrolyzed into a free single amino acid for use as a pharmacologically active ingredient; and at least one peptide end is connected to phenylalanine that can be transported by a transporter, so that the small peptide can penetrate the cell membrane or penetrate the blood-brain barrier, and the phenylalanine is L-Phe or D-Phe. This type of small peptide can penetrate the blood-brain barrier under the carrier of phenylalanine, and hydrolyze into a single amino acid with neuroprotection, play a role in protecting the brain, and can be used as a neuroprotectant. Moreover, the neuroprotectant provided by the present invention can be used in combination with rt-PA for the treatment of ischemic stroke. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is to compare the effects of PF, AC-PF, and Phe on neuronal survival;

[0028] Figure 2 is to compare the effects of PF and AC-PF on the changes in Pro content in neurons;

[0029] Figure 3 is to compare the protective effects of PF, AC-PF, and Phe on brain injury;

[0030] Figure 4 It is a schematic diagram of co-staining to verify that PF can co-localize with nerves;

[0031] Figure 5 Yes Figure 4 The quantitative analysis results of

[0032] Figure 6 The protective effects of different neuroprotectants on brain damage in ischemia / reperfusion (I / R) model mice;

[0033] Figure 7 The protective effect of PF combined with rt-PA on brain injury in ischemia / reperfusion (I / R) model mice;

[0034] Figure 8 The protective effect of GCF combined with rt-PA on brain damage in MCAO model mice;

[0035] Fig. 9 The protective effect of GF combined with rt-PA on brain damage in MCAO model mice;

[0036] Fig.10 The effect of different administration methods on the effect of GCF in the treatment of ischemic stroke. DETAILED DESCRIPTION

[0037] In order to further explain the technical means and effects adopted by the present invention to achieve the predetermined invention purpose, the specific implementation mode, structure, characteristics and effects of the present invention are described in detail below in combination with the accompanying drawings and preferred embodiments.

[0038] The blood-brain-barrier (BBB) ​​is a dynamic structure, which refers to the barrier between plasma and cerebrospinal fluid and the barrier between plasma and brain cells. It is a barrier system formed by brain microvascular endothelial cells (BMEC) interacting with astrocytes, neurons and microglia. As a barrier structure for the exchange of substances between the central nervous system (CNS) and the blood, the BBB actively absorbs nutrients required by the CNS and expels or blocks the entry of substances that damage the CNS to ensure the physiological homeostasis of the CNS.

[0039] Generally, amino acids and small molecule drugs that can easily penetrate the blood-brain barrier need to have specific physical and chemical properties and structural characteristics, including appropriate lipophilicity, molecular size, electronegativity, etc., and the ability to be recognized and transported by specific transporters (transport proteins). However, transporters that can specifically transport a certain amino acid may not necessarily be able to transport polypeptides containing the amino acid. This may be because polypeptide molecules are larger and have more complex structures, which affect their transport capacity. In addition, the hydrolysis of polypeptides in the brain is also very complex. Whether it can be hydrolyzed to produce free single specific amino acids and then exert its neuroprotective effect is the key to whether the polypeptide can become a drug.

[0040] As is known to all, BBB has strict selective absorption of amino acids. For example, proline, alanine, glycine, cysteine, glutamic acid and aspartic acid are generally difficult to enter the brain. For example, in patent CN 106188236B (a synthetic peptide containing glycine, L-cysteine ​​and L-phenylalanine and its application), the hydrophobic amino acid L-phenylalanine is used as a carrier to penetrate the cell membrane, carrying the synthetic peptide molecule across the blood-brain barrier and into the brain, while glycine and cysteine ​​alone are difficult to penetrate the blood-brain barrier and enter the brain.

[0041] Existing studies have shown that arginine (Arg, R), lysine (Lysine, Lys, K) and alanine (Ala, A) all have neuroprotective effects. In addition, our previous research results also found that proline (Pro, P), glycine (Glycine, Gly, G) and cysteine ​​(Cysteine, Cys, C) also have significant neuroprotective effects, but they are difficult to penetrate the blood-brain barrier when used alone, which limits their clinical application. Further studies have found that not only L-phenylalanine (L-Phe) but also D-phenylalanine (D-Phe) can be used as transmembrane carriers, which can carry certain peptides through the blood-brain barrier and enter the brain.

[0042] Therefore, we propose a design scheme for a small peptide that can be used as a neuroprotective agent, using an unmodified amino acid with neuroprotective effect as the pharmacologically active ingredient of the small peptide, wherein at least one of the peptide bonds connected to the amino acid is a protease hydrolysis site, which can hydrolyze it into a free single amino acid; and at least one end of the small peptide is connected to a phenylalanine that can be transported by a transporter, which is used as a transmembrane carrier, so that the small peptide can penetrate the cell membrane or penetrate the blood-brain barrier to enter the brain tissue; the phenylalanine is L-Phe or D-Phe, and the transporter includes LAT1. The amino acids with neuroprotective effects include proline (Pro, P), glycine (Gly, G), cysteine ​​(Cys, C), arginine (Arg, R), lysine (Lys, K) and alanine (Ala, A).

[0043] Especially the synthesized small peptides with a molecular weight of less than or equal to 600Da. Experimental results show that this series of small peptides can penetrate the blood-brain barrier and enter the site of brain damage, and then release free single amino acids or short peptides after hydrolysis to exert neuroprotective effects, such as reducing the volume of cerebral infarction in ischemic stroke and alleviating brain damage, and can serve as neuroprotective agents.

[0044] In addition, considering that patients with ischemic stroke are usually given rt-PA (recombinant tissue plasminogen activator) when they are sent to the hospital in clinical practice, which is a drug used to treat acute ischemic stroke. In order to study whether rt-PA will affect the efficacy of this small peptide, we constructed an animal model (ischemia / reperfusion (I / R), MACO) to compare the therapeutic effects of PF, GCF, and GF on ischemic stroke after being combined with rt-PA. The results showed that the efficacy of free proline, glycine, and cysteine ​​generated by the hydrolysis of these peptides was not affected by rt-PA and could be combined with rt-PA to treat ischemic stroke.

[0045] Based on this, the present invention proposes a neuroprotectant that can be used in combination with rt-PA, wherein the neuroprotectant is a synthetic small peptide, and its amino acid sequence includes at least one unmodified amino acid with neuroprotective properties, wherein at least one of the peptide bonds connected to the unmodified amino acid is a protease hydrolysis site, so that it can be hydrolyzed into free single amino acids, and is used as a pharmacologically active ingredient to exert a neuroprotective effect;

[0046] At least one peptide end is connected to phenylalanine which can be transported by a transporter, so that the small peptide can penetrate the cell membrane and / or the blood-brain barrier; the phenylalanine is L-Phe or D-Phe; and the transporter includes LAT1.

[0047] In the present invention, it is found that the polypeptide synthesized by using acetylated proline has no neuroprotective effect. It is speculated that the polypeptide synthesized by using acetylated amino acids has good stability and is difficult to be hydrolyzed into free single amino acids, and thus cannot play the role of protecting the nerves.

[0048] Furthermore, the neuroprotectant is a small peptide with a molecular weight of less than or equal to 600 Da, and its amino acid sequence includes the following:

[0049] X 1 -X 2 -X 3 -X 4 ;

[0050] If the C-terminus of the small peptide is phenylalanine F; X 3 is proline P, glycine G, cysteine ​​C, arginine R, alanine A, or lysine K; X 1 , X 2 is 0 or any amino acid;

[0051] If the N-terminus of the small peptide is phenylalanine F; X 2 is proline P, glycine G, cysteine ​​C, arginine R, alanine A, or lysine K; X 3 , X 4 is 0 or any amino acid.

[0052] Such as X 4 is phenylalanine, F; X 3 is proline P, glycine G, cysteine ​​C, arginine R, alanine A, or lysine K; X 1 , X 2 is 0 or any amino acid, wherein "0" means no amino acid. 1 and / or X 2 It is an amino acid with neuroprotective properties.

[0053] More preferably, except for phenylalanine F at one end, the remaining amino acids in the amino acid sequence of the small peptide are amino acids with neuroprotective properties.

[0054] In some embodiments, the neuroprotectant is a dipeptide whose amino acid sequence comprises the following:

[0055] PF, GF, CF, FP, FG, FC, RF, FR, AF, FA, KF or FK. Phenylalanine (Phe, F) also includes D-Phe; PF, GF, CF are preferred, and CF is more preferred, as its solubility and efficacy are better. In this type of dipeptide, there is a protease hydrolysis site, i.e., the connection point between phenylalanine (Phe, F) at one end of the peptide and any amino acid, which can be hydrolyzed into free proline, glycine or cysteine ​​in the brain, thereby exerting its role in protecting nerves and reducing brain damage.

[0056] In some embodiments, the neuroprotectant is a synthetic tripeptide whose amino acid sequence contains an unmodified amino acid with neuroprotective properties, including the following sequence:

[0057] FGF, FCF, or FPF.

[0058] In some embodiments, the neuroprotectant is a tripeptide whose amino acid sequence contains two unmodified amino acids with neuroprotective properties, including the following sequence:

[0059] GCF, PGF, GPF, CGF, FPG, FGP, FCG, RGF, FGR, AGF, FGA, KGF, FGK, RPF, FPR, APF, FPA, KPF or FPK, wherein F in GCF is D-Phe.

[0060] GCF, PGF, GPF, CGF, FPG, FGP or FCG are preferred, wherein F in GCF is D-Phe; GCF, PGF and GPF are more preferred, which have significant brain protection effects. There are two protease hydrolysis sites in this type of tripeptide, one of which is the connection point between phenylalanine (Phe, F) at one end of the peptide and any amino acid, and the other is the connection point between glycine (Gly, G) and cysteine ​​(Cys, C) or the connection point between glycine (Gly, G) and proline (Pro, P); they can be hydrolyzed into free proline, glycine or cysteine ​​in the brain, thereby exerting their neuroprotective effects and reducing brain damage.

[0061] In some embodiments, the neuroprotectant is a tetrapeptide, the amino acid sequence of which comprises the following:

[0062] PGPF, GPGF, CGPF, PGCF, RPGF, FGPR, APGF, FGPA, KPGF, FGPK, RGPF, FPGR, AGPF, FPGA, KGPF or FPGK, among which P, G, C, A, K, R can be used as one of the pharmacologically active ingredients.

[0063] Preferred are PGPF, GPGF, CGPF or PGCF, wherein the proline in CGPF or PGCF is one of the pharmacologically active ingredients, and PGPF is more preferred. In this type of tetrapeptide, there are 2 to 3 protease hydrolysis sites, one of which is the connection point between the phenylalanine (Phe, F) at one end of the peptide and any amino acid, another is the connection point between glycine (Gly, G) and proline (Pro, P), or the third is the connection point between glycine (Gly, G) and cysteine ​​(Cys, C); they can be hydrolyzed into free proline, glycine or cysteine ​​in the brain, thereby exerting their role in protecting nerves and reducing brain damage.

[0064] In addition, the present invention also provides a use of the neuroprotective agent of the present invention in the preparation of drugs for treating nervous system diseases.

[0065] The nervous system diseases include neurodegenerative diseases and brain damage. When used to prepare a drug for treating brain damage, the neuroprotectant is administered by intravenous injection, skull bone marrow administration, or nasal administration; preferably, skull bone marrow administration.

[0066] The neurodegenerative diseases include Alzheimer's disease and Parkinson's disease, and the brain injuries include stroke, traumatic brain injury and other brain injury diseases.

[0067] rt-PA (recombinant tissue plasminogen activator) is a drug used to treat thrombosis-related diseases. Its main component is glycoprotein, which contains 526 amino acids. However, it has been found clinically that the neuroprotectant NA-1 (Nerinetide) used to treat acute ischemic stroke is used in combination with rt-PA, and rt-PA will hydrolyze NA-1, making it difficult to exert its efficacy. In order to study whether the neuroprotectant provided by the present invention will be affected by rt-PA, we constructed an animal model (ischemia / reperfusion (I / R), MACO), and compared it with the immediate administration of thrombus removal, and compared the therapeutic effects of PF, GCF, and GF on brain damage after being combined with rt-PA. The results showed that the efficacy of free proline, glycine, and cysteine ​​generated by the hydrolysis of these polypeptides is basically not affected by rt-PA, and can be used in combination with rt-PA, especially the efficacy of PF is not affected by rt-PA, and the two can be used in combination to treat ischemic stroke and reduce brain damage.

[0068] Furthermore, the neuroprotectant is used in combination with rt-PA to prepare a drug for treating ischemic stroke. rt-PA is used for intravascular thrombolysis to enhance vascular recanalization. The two are used in combination to treat ischemic stroke. It is recommended to give one of the components at an interval of more than 20 minutes before giving the other, such as giving rt-PA at an interval of 20 to 30 minutes before giving the neuroprotectant. It is generally not recommended to use the two at the same time.

[0069] In addition, the present invention also provides a composition, which comprises one or more of the neuroprotective agents described in the present invention and rt-PA.

[0070] In some embodiments, the composition comprises PF+rt-PA, GF+rt-PA, or GCF+rt-PA; preferably PF+rt-PA.

[0071] The following are examples

[0072] The following examples use adult male Sprague-Dawley (SD) rats and pregnant rats, male rats weighing 250g; pregnant rats with a gestation period of 17 to 20 days, all purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd. 2 to 3 rats are kept in each cage, the room temperature is 23 to 25 ° C, and there is sufficient water and bedding is changed regularly except for experimental requirements. The experimental rats were acclimatized in the animal room for one week before the experiment and randomly grouped during the experiment. Animal ethics number: QDU-AEC-2023423.

[0073] In the following examples, the polypeptide was synthesized by solid phase synthesis and purified and separated by HPLC, and its amino acid sequence is shown below:

[0074] PGPF, GCF, PGF, GPF, PF, GF, CF; F in GCF is D-Phe, and phenylalanine (Phe, F) in other polypeptides can be L-Phe or D-Phe. These polypeptides are purified and separated by HPLC, and the purity of each polypeptide product is measured to be above 98%.

[0075] Among them, phenylalanine (Phe, F): hydrophobic parameter 2.8, molecular weight 165.21; cysteine ​​(Cys, C): hydrophobic parameter 2.5, molecular weight 121.18; glycine (Gly, G): hydrophobic parameter -0.4, molecular weight 75.08; proline (Pro, P): hydrophobic parameter -1.6, molecular weight 115.15.

[0076] Example 1 Protective effect of unmodified PF on ischemia / reperfusion neurons and brain tissue

[0077] In this example, another dipeptide was synthesized, which was formed by linking acetylated proline and phenylalanine, and was abbreviated as AC-PF.

[0078] (1) Low-concentration PF treatment can enhance the activity of ischemic / reperfused neuronal cells

[0079] Construction of OGD / R model: Fetal rat primary neurons were extracted, and the primary neurons were cultured for about 12 days. The cell state was observed under a microscope, and the neuron culture medium with good neuron state and similar density was selected. The old neuron culture medium was aspirated and washed three times with PBS. The control group (Non-OGD group) was added with an appropriate volume of extracellular solution with sugar (ECS+) and placed in a 37°C cell culture incubator; the model group (OGD / R group) and the drug treatment group (OGD / R+drug group) were added with an appropriate amount of extracellular solution without sugar (ECS-) and placed in an anaerobic workstation (temperature: 37°C, gas parameters: 1% O 2 +94%N 2 +5% CO 2 ) for two hours to form a neuronal OGD model. Cell damage was 50%-60%, and re-glucose and re-oxygenation were performed to simulate reperfusion injury. The original culture medium of neurons in all groups was discarded and an appropriate amount of normal neuronal culture medium was added, which was OGD / R. After the model was successfully constructed, the specific drug treatments were as follows:

[0080] Drug group: After OGD, the neuron culture plate was taken out, the ECS-solution was replaced with neuron culture medium, and drug treatment was performed (PF: 200μmol / L; AC-PF: 200μmol / L; Phe: 200μmol / L), and placed in an incubator for reoxygenation for a specified time, with OGD / R as a blank control. At this time, the culture medium was also aspirated from the Non-OGD / R group and an equal volume of sterile PBS buffer was added.

[0081] After 24 hours, the CCK-8 method was used to detect the effects of supplementation of PF, AC-PF, and Phe on neuronal survival rate after OGD / R injury. Figure 1 HPLC was used to detect the changes in Pro content in PF and AC-PF neurons after OGD / R. The results are shown in Figure 2 shown.

[0082] Depend on Figure 1 and Figure 2 The results showed that among the supplementation of PF, AC-PF and Phe, only PF could improve the survival rate of neurons and significantly increase the Pro content in neurons, indicating that PF can be hydrolyzed into free natural proline and thus exert the neuroprotective effect of Pro, while acetylated proline has no neuroprotective effect.

[0083] (2) Low concentration PF treatment can reduce ischemia / reperfusion brain tissue damage

[0084] The MCAO model, or middle cerebral artery occlusion model, simulates the occurrence of ischemic stroke by blocking the middle cerebral artery (MCA). In this model, the MCA is blocked, causing ischemia in the area it supplies (usually the cerebral cortex and some subcutaneous structures). This model can be used to study the pathophysiology of cerebral ischemia, the mechanisms of neuronal damage, and to evaluate potential neuroprotective treatments. The MCAO model can be permanent or temporary. The latter, during the experiment, restores blood flow to simulate the reperfusion process in clinical treatment, forming an ischemia / reperfusion (I / R) model, i.e., an ischemia / reperfusion model.

[0085] The ischemia / reperfusion (I / R) model was constructed. After the preoperative work was prepared, the rats were fixed on the operating table in a supine position during the operation. The skin of the neck was prepared and disinfected, and the skin was neatly cut with tissue scissors. The subcutaneous tissue was separated with curved micro forceps to expose the superficial fascia and platysma. Under a low-power microscope, micro forceps were used to carefully and bluntly separate the neck muscles, blood vessels, nerves and other tissues, and the common carotid artery was exposed in the deep layer. The common carotid artery was separated upward to reveal the "Y"-shaped branches, which were the external carotid artery and the internal carotid artery.

[0086] Ligate the proximal end of the separated common carotid artery and tie a slipknot to reserve space for embolization. Tie a dead knot at the distal end of the external carotid artery and a slipknot that can be inserted at the proximal end. Tie a tight slipknot at the internal carotid artery or clamp it with a vascular clamp. After the ligation is completed, use ophthalmic scissors to make a "V"-shaped incision of appropriate size between the two ligature points of the external carotid artery, and insert the thread plug from the external carotid artery into the common carotid artery through the incision. After the thread plug enters the common carotid artery, tighten the slipknot at the proximal end of the external carotid artery.

[0087] Then, the blood vessel was cut at the distal end of the incision of the external carotid artery, and the internal carotid artery was entered from the common carotid artery. After the suture entered the internal carotid artery, the loose knot of the internal carotid artery was tied again, and the suture was inserted along the blood vessel to the distal end until the small black dot on the suture entered the incision of the external carotid artery. At this time, the suture entered the middle cerebral artery, which is the rat middle cerebral artery occlusion (MCAO) model.

[0088] 90 minutes after the embolization, the rats were anesthetized again with the same steps, the neck incision was opened, the embolism was slowly withdrawn, and the slipknot of the embolism at the proximal end of the external carotid artery was tied. The slipknot of the common carotid artery was opened, the blood supply was restored, and suture was performed. After the embolism was withdrawn, the blood supply of the middle cerebral artery was restored, and this was the rat ischemia / reperfusion (I / R) model.

[0089] After the successful modeling of ischemia / reperfusion (I / R), the drug treatment groups were immediately injected with 50 mg / kg PF, 50 mg / kg AC-PF, and 50 mg / kg Phe through the tail vein. TTC staining analysis of rat brain sections was performed 24 hours later. The results are shown in Figure 3 shown.

[0090] Figure 3 The results showed that compared with the ischemia / reperfusion (I / R) group, only the drug group injected with PF could reduce the volume of rat cerebral infarction and alleviate brain damage among the three drug treatment groups; this indicated that PF could penetrate the BBB into the ischemic side and hydrolyze into a single free proline in the brain tissue to play a neuroprotective role. It can be used as a safe and effective neuroprotective peptide for the preparation of potential drugs for the treatment of nervous system diseases, including neurodegenerative diseases, brain injury, cerebral palsy, and cerebrovascular diseases. Neurodegenerative diseases include Alzheimer's disease and Parkinson's disease, and brain damage includes brain damage caused by stroke, traumatic brain injury and other brain damage diseases, such as ischemic stroke, which can cause ischemic damage to brain tissue.

[0091] For intuitive visualization of the results, this example further uses NeuN and FITC-PF for co-staining to verify that PF can penetrate the BBB in a rat ischemia / reperfusion (I / R) model; specifically as follows:

[0092] After the ischemia / reperfusion (I / R) model was successfully established, the drug group was immediately injected with FITC-PF (0.13mmol / g) through the tail vein, and the injection of FITC (fluorescein isothiocyanate, which can emit green fluorescence under ultraviolet excitation, 0.13mmol / g) was used as a blank control; the sham group was also injected with equal amounts of FITC-PF and FITC. The co-localization of nerves and drugs in each group was detected, and the results are shown in Figure 4 As shown, FITC-PF (green), NeuN (red), scale bar = 50 μm; Figure 4 The quantitative analysis results are as follows Figure 5 shown.

[0093] The results showed that intravenous injection could enable FITC-PF to enter the ischemic brain tissue in large quantities and co-localize with nerves, indicating that PF could not only penetrate the blood-brain barrier, but also enter neurons and be hydrolyzed into single free proline in brain tissue, exerting its neuroprotective effect and reducing the volume of cerebral infarction in rats with ischemic stroke.

[0094] Example 2 Protective effect of neuroprotective agents on ischemia / reperfusion brain injury

[0095] A rat ischemia / reperfusion model was established, in which ischemia / reperfusion was performed by plugging the thrombus for 1.5 hours and then removing the thrombus and reperfusing it, simulating clinical thrombectomy and thrombolysis. After thrombus removal, the drug treatment group was immediately injected with GCF 150 mg / kg, PGF 150 mg / kg, PGPF 20 mg / kg, GPF 50 mg / kg, CF 50 mg / kg, PF 50 mg / kg, GF 150 mg / kg through the tail vein. Injection of NA-1 (7.5 mg / kg), (Edaravone, 3 mg / kg), NBP stock solution, and NBP Nacl injection was used as positive controls, and injection of normal saline (Normal Saline, NS) was used as blank control. Among them, NA-1 (Nerinetide) is a neuroprotective agent, mainly used to treat acute ischemic stroke (AIS); Edaravone is a brain protective agent, mainly used to treat acute cerebral infarction; NBP concentrate refers to butylphthalide (3-Butylphthalide), also known as Enbipu, which is a drug used to treat mild and moderate acute ischemic stroke.

[0096] After 24 hours, the rat brain TTC staining sections were analyzed and the cerebral infarction volume of each group was counted. Figure 6 As shown in the figure, the numbers marked on the columns such as "7, 8, 9" represent the number of samples, and the same applies below.

[0097] Depend on Figure 6 The results show that compared with the therapeutic effects of NBP stock solution, NBP Nacl injection and Edar, the volume of cerebral infarction in rats injected with the neuroprotectant provided by the present invention is significantly smaller, indicating that the neuroprotectant provided by the present invention has a better therapeutic effect on ischemic stroke than existing drugs and can be used as a potential drug for the treatment of ischemic stroke.

[0098] Example 3 Effect of rt-PA combination on the effect of neuroprotective agents in treating ischemic stroke

[0099] In clinical trials, the neuroprotectant NA-1 was used in combination with rt-PA, which hydrolyzed NA-1 and made it difficult for it to exert its efficacy. In order to study whether the therapeutic effect of the neuroprotectant provided by the present invention is affected by rt-PA, this example compares the therapeutic effects of PF, GCF, and GF after being used in combination with rt-PA, as follows:

[0100] (1) Combination of PF and rt-PA

[0101] Blank control group: rats were injected with 50 mg / kg normal saline immediately after thrombus removal after ischemia / reperfusion (I / R) as a control, and the brain was perfused and removed 24 hours later;

[0102] Drug treatment group 1: rats were injected with 50 mg / kg PF via tail vein immediately after ischemia / reperfusion (I / R), and the brains were harvested after perfusion 24 hours later;

[0103] Drug treatment group 2: rats were injected with 5 mg / kg rt-PA through the tail vein immediately after ischemia / reperfusion (I / R), and 50 mg / kg PF was injected through the tail vein again 30 minutes later. The brains were harvested after perfusion 24 hours later.

[0104] The statistical results of each group are as follows Figure 7 The results showed that compared with the control group, the whole brain infarction volume of rats in drug treatment group 1 (50 mg / kg PF group injected into the tail vein immediately after ischemia / reperfusion (I / R)) and drug treatment group 2 (5 mg / kg rt-PA was first given after ischemia / reperfusion (I / R), and 50 mg / kg PF was given 30 minutes later) was reduced, and there was no significant difference between the two, indicating that PF still has a neuroprotective effect after intravenous injection of rt-PA, and its efficacy is not affected by rt-PA. It can be seen that the neuroprotective agent provided by the present invention can be used in combination with rt-PA to treat ischemic stroke, but the simultaneous use of the two will affect the efficacy of the neuroprotective agent. Generally, rt-PA is given, and the neuroprotective agent is given after an interval of 20 to 30 minutes, or the neuroprotective agent is given first, and then rt-PA is given after an interval of 20 to 30 minutes.

[0105] (2) Combination of GCF and rt-PA

[0106] Blank control group: rats were injected with 150 mg / kg normal saline immediately after thrombus removal after MACO as a control, and the brain was perfused and removed 24 hours later;

[0107] Drug treatment group 1: rats were injected with 150 mg / kg GCF in the tail vein immediately after thrombus removal after MACO, and the brain was perfused and removed 24 hours later;

[0108] Drug treatment group 2: rats were given 1 mg / kg rt-PA 75 min after MACO embolization, and the brain was perfused and harvested 24 h later;

[0109] Drug treatment group 3: Rats were given 1 mg / kg rt-PA 75 minutes after MACO embolization. 150 mg / kg GCF was injected into the tail vein immediately after embolization. The brain was removed by perfusion 24 hours later.

[0110] The statistical results of each group are as follows Figure 8The results showed that compared with the control group, the whole brain infarction volume of rats in drug treatment group 1 (150 mg / kg GCF group injected into the tail vein immediately after MACO) and drug treatment group 3 (1 mg / kg rt-PA was given first after MACO, and then 150 mg / kg GCF) was reduced, and there was no significant difference between the two (P>0.05). Compared with drug treatment group 2 (injection of 1 mg / kgrt-PA), the brain infarction volume of rats in drug treatment group 3 was significantly reduced, indicating that GCF still has a neuroprotective effect after intravenous injection of rt-PA, that is, the neuroprotective effect of GCF is not affected by rt-PA, and the two can be used in combination to treat ischemic stroke.

[0111] (3) Combination of GF and rt-PA

[0112] Blank control group: rats were injected with 100 mg / kg normal saline immediately after thrombus removal after MACO as a control, and the brain was perfused and removed 24 hours later;

[0113] Drug treatment group 1: rats were injected with 100 mg / kg GF into the tail vein immediately after thrombus removal after MACO, and the brain was perfused and removed 24 hours later;

[0114] Drug treatment group 2: rats were given 5 mg / kg rt-PA 75 min after MACO embolization, and the brain was perfused and removed 24 hours later;

[0115] Drug treatment group 3: rats were given 5 mg / kg rt-PA 75 minutes after embolization, and 100 mg / kg GF was injected into the tail vein immediately after embolization. The brain was perfused and obtained 24 hours later.

[0116] The statistical results of each group are as follows Fig. 9 The results showed that compared with the control group, the whole brain infarct volume of rats in drug treatment group 1 (100 mg / kg GF injected into the tail vein after MACO) and drug treatment group 3 (5 mg / kg rt-PA and then 100 mg / kg GF after MACO) was reduced, and there was no significant difference between the two (P>0.05). Compared with drug treatment group 2 (injection of 5 mg / kg rt-PA), the brain infarct volume of rats in drug treatment group 3 was significantly smaller, indicating that the administration of GF after intravenous injection of rt-PA still has a neuroprotective effect, that is, the neuroprotective effect of GF is not affected by rt-PA, and the two can be used in combination to treat ischemic stroke.

[0117] Example 4 Effects of different administration methods on the effect of GCF in treating ischemic stroke

[0118] In this example, a rat MCAO model was constructed to simulate the ischemic nonperfusion process, and the effects of different administration methods on the effect of GCF in treating ischemic stroke were compared, as follows:

[0119] Drug treatment group 1: rats were injected with 150 mg / kg GCF intravenously (IV) immediately after MCAO, and the brain was perfused and removed 24 hours later;

[0120] Drug treatment group 2: rats were injected with 37.5 mg / kg GCF into the skull bone marrow (intravenous injection, IC) 1 hour after MACO embolism, and the brain was harvested by perfusion 24 hours later.

[0121] The statistical results of each group are as follows Fig.10 The results showed that the therapeutic effect of GCF in the treatment of ischemic stroke was significantly better than that of intravenous administration through skull bone marrow.

[0122] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not used to limit the present invention. Any technical personnel in this field can make some changes or modify the technical contents disclosed above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any brief modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A neuroprotectant that can be used in combination with rt-PA, characterized in that: It is a synthetic small peptide, the amino acid sequence of which includes at least one unmodified amino acid with neuroprotective properties, wherein at least one of the peptide bonds connected to the unmodified amino acid is a protease hydrolysis site, so that it can be hydrolyzed into free single amino acids, and used as a pharmacologically active ingredient; At least one peptide end is connected to phenylalanine which can be transported by a transporter, so that the small peptide can penetrate the cell membrane and / or the blood-brain barrier; the phenylalanine is L-Phe or D-Phe; and the transporter includes LAT1.

2. The neuroprotective agent according to claim 1, characterized in that It is a small peptide with a molecular weight of less than or equal to 600 Da, and its amino acid sequence includes the following: X1-X2-X3-X4; If the C-terminus of the small peptide is phenylalanine F; X3 is proline P, glycine G, cysteine ​​C, arginine R, alanine A, or lysine K; then X1 and X2 are 0 or any amino acid; If the N-terminus of the small peptide is phenylalanine F; X2 is proline P, glycine G, cysteine ​​C, arginine R, alanine A, or lysine K; then X3 and X4 are 0 or any amino acid.

3. The neuroprotective agent according to claim 2, characterized in that In the amino acid sequence of the small peptide, except for phenylalanine F at one end, the remaining amino acids are amino acids with neuroprotective properties.

4. The neuroprotective agent according to claim 3, characterized in that The amino acid sequence of the dipeptide is as follows: PF, GF, CF, FP, FG, FC, RF, FR, AF, FA, KF or FK; The amino acid sequence of the tripeptide includes the following sequence: GCF, PGF, GPF, CGF, FPG, FGP, FCG, RGF, FGR, AGF, FGA, KGF, FGK, RPF, FPR, APF, FPA, KPF or FPK, wherein F in GCF is D-Phe; The amino acid sequence of the tetrapeptide is as follows: PGPF, GPGF, CGPF, PGCF, RPGF, FGPR, APGF, FGPA, KPGF, FGPK, RGPF, FPGR, AGPF, FPGA, KGPF or FPGK, wherein P, G, C, A, K, or R is one of the pharmacologically active ingredients.

5. The neuroprotective agent according to claim 4, characterized in that The amino acid sequence of the tripeptide is GCF, PGF, or GPF; the amino acid sequence of the tetrapeptide is PGPF.

6. Use of the neuroprotective agent according to any one of claims 1 to 5 in the preparation of a drug for treating a nervous system disease, characterized in that: The nervous system diseases include neurodegenerative diseases and brain damage. When used in the preparation of drugs for treating brain damage, the neuroprotectant is administered by intravenous injection, skull bone marrow administration, or nasal administration.

7. The use according to claim 6, characterized in that The neurodegenerative diseases include Alzheimer's disease and Parkinson's disease, and the brain injuries include stroke and traumatic brain injury.

8. The use according to claim 7, characterized in that The neuroprotectant is used in combination with rt-PA to prepare a drug for treating ischemic stroke.

9. A composition, characterized in that The method comprises one or more of the neuroprotective agents according to any one of claims 1 to 5, and rt-PA.

10. The composition according to claim 9, characterized in that Including PF+rt-PA, GCF+rt-PA, or GF+rt-PA.

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