Self-assembled small peptide C16FW and application thereof in preparation of medicine for treating cerebrovascular diseases

Abstract

The invention belongs to the field of biological medicines, and discloses a self-assembled small peptide C16FW and application thereof in preparation of drugs for treating cerebrovascular diseases. The invention provides an artificially synthesized self-assembled small peptide, and the molecular formula of the artificially synthesized self-assembled small peptide is C16-FFVLK-PEG4-WSPTKVH. The self-assembled small peptide provided by the invention can specifically wrap S100A9 protein and effectively reduce the level of S100A9, so that the S100A9 cannot be combined with any receptor, the structure is simple, the self-assembled small peptide can be mixed with various medicines for use, mutual immune response cannot be caused, and the effect of reducing intracranial inflammatory response and blood-brain barrier damage caused by cerebrovascular diseases is achieved.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biological medicine, and particularly relates to a self-assembled small peptide C16FW and application thereof in preparation of a drug for treating cerebrovascular diseases. BACKGROUND

[0002] Clinical research has found that the increase of the neutrophil count in the peripheral blood of an ischemic stroke patient is closely related to the aggravation of cerebral edema, the expansion of infarction volume and adverse prognosis. The infiltration of neutrophils can not only aggravate nerve damage, but also can lead to the occurrence of systemic inflammatory response, which is also a difficulty in current clinical treatment. Although in animal models, the reduction of neutrophil migration and infiltration can effectively reduce brain damage and improve nerve function. However, in related clinical research, this strategy does not show the expected effect. Because, excessive inhibition of the function of neutrophils can lead to inhibition of the immune system, thereby increasing the risk of secondary systemic infection.

[0003] The results of the previous basic research of the applicant show that S100A9 plays a key role in mediating the interaction between microglia and neutrophils after acute ischemic stroke: the applicant found that the S100A9 derived from neutrophils was specifically highly expressed in patients with severe cerebral edema by sequencing analysis of the plasma and peripheral blood neutrophils of patients with mild and severe cerebral edema after acute ischemic stroke. At the same time, it is found that: ① S100A9 inhibitor paquinimod and S100A9 knockout can reduce the damage of BBB after acute ischemic stroke and improve the prognosis of stroke. ② S100A9 can inhibit the phagocytosis of microglia to neutrophils and promote the aggregation of neutrophils in the intracranial. ③ S100A9 promotes microglial pyroptosis and aggravates intracranial inflammatory response. The above results verify that reducing the level of S100A9 is an effective therapeutic target for improving the prognosis of acute ischemic stroke. However, the current clinical dilemma is that there is no S100A9 inhibitor or antibody that has been successfully approved for clinical use. SUMMARY

[0004] The application aims to provide an artificially synthesized self-assembled small peptide. 16 -FFVLK-PEG4-WSPTKVH.

[0005] Another object of the application is to provide application of the artificially synthesized self-assembled small peptide in preparation of a drug for treating cerebrovascular diseases.

[0006] In order to achieve the above object, the application adopts the following technical scheme:

[0007] An artificially synthesized self-assembled small peptide C16FW has a molecular formula of C 16 -FFVLK-PEG4-WSPTKVH, and a structural formula of

[0008]

[0009] The scope of protection of the present application also includes:

[0010] The complex containing the self-assembling small peptide C16FW described above.

[0011] The application of the self-assembling small peptide C16FW described above or the complex described above in the preparation of a blocking drug of S100A9.

[0012] The application of the self-assembling small peptide C16FW described above or the complex described above in the preparation of a drug for treating inflammation caused by significant elevation of S100A9.

[0013] The application of the self-assembling small peptide C16FW described above or the complex described above in the preparation of a drug for treating cerebrovascular diseases and / or recovery period of cerebrovascular diseases.

[0014] The application described above, the cerebrovascular disease or the recovery period of cerebrovascular disease, the significant elevation of S100A9 in blood.

[0015] The application described above, the cerebrovascular disease includes but is not limited to: the disease caused by ischemic stroke, the disease caused by hemorrhagic stroke, the disease caused by traumatic brain injury, the disease caused by neutrophil-related intracranial inflammatory reaction.

[0016] In the application described above, the dosage form of the drug is all dosage forms acceptable in pharmacy, including but not limited to tablets, capsules, granules, injections, powders or drops, etc.

[0017] Compared with the prior art, the present application has the following advantages:

[0018] 1. The self-assembling small peptide (C16FW) provided by the present application can specifically wrap S100A9 protein, effectively reduce the level of S100A9, so that it cannot be combined with any receptor.

[0019] 2. The self-assembling small peptide (C16FW) provided by the present application has small molecular weight and can pass through the damaged BBB to reach the damaged brain tissue.

[0020] 3. The self-assembling small peptide (C16FW) provided by the present application can be administered intravenously: it is a safe, effective and easy-to-operate way.

[0021] 4. The self-assembling small peptide (C16FW) is a non-protein product with simple structure, which can be mixed with various drugs for use, and will not cause mutual immune reaction.

[0022] 5. The self-assembling small peptide (C16FW) provided by the present application uses materials and solvents approved by FDA for use in the human body.

[0023] 6. The self-assembling small peptide (C16FW) is easy to prepare and has low preparation cost, which can greatly reduce the treatment cost. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 Molecular structure diagram of the self-assembling small peptide (C16FW).

[0025] Figure 2 Electron microscope image of the self-assembling small peptide (C16FW);

[0026] Wherein: A is the electron microscope image of C16FW without co-incubation with recombinant S100A9 protein, and B is the electron microscope image of C16FW co-incubated with recombinant S100A9 protein.

[0027] Figure 3 Diagram showing that the self-assembling small peptide (C16FW) can significantly inhibit BBB of tMCAO model mice;

[0028] Wherein, A shows that immunofluorescence staining results show that the self-assembling small peptide (C16FW) can significantly reduce the leakage of fibrinogen after tMCAO; B is a statistical diagram of immunofluorescence staining results showing that the self-assembling small peptide (C16FW) can significantly reduce the leakage of fibrinogen after tMCAO.

[0029] Figure 4 Diagram showing that the self-assembling small peptide (C16FW) can significantly alleviate the neurological deficit of tMCAO model mice.

[0030] Figure 5 Diagram of the cytotoxicity experiment of the self-assembling small peptide (C16FW).

[0031] Figure 6 Diagram showing that the self-assembling small peptide (C16FW) can block the promotion of S100A9 to BBB model leakage in vitro;

[0032] Wherein, A: FITC-Dextran leakage experiment confirms that C16FW can block the promotion of S100A9 to BBB model leakage in vitro; B: TEER experiment confirms that C16FW can block the promotion of S100A9 to BBB model leakage in vitro.

[0033] Figure 7 Diagram showing that the self-assembling small peptide (C16FW) can gather in ischemic brain tissue through blood circulation. DETAILED DESCRIPTION

[0034] The present invention will be further described below with reference to specific embodiments. Unless otherwise specified, the technical solutions described in this invention are conventional solutions in the art. Unless otherwise specified, the reagents or materials used are all from commercial sources. The recombinant S100A9 protein used in this invention was purchased from Abcam.

[0035] Example 1:

[0036] Molecular structure of self-assembled small peptides (C16FW)

[0037] The self-assembled small peptide (C16FW) involved in this invention has the following structural formula and molecular formula: C16-FFVLK-PEG4-WSPTKVH, and is obtained through commercial synthesis.

[0038]

[0039] Example 2:

[0040] Some physicochemical properties of self-assembled small peptides (C16FW):

[0041] The C16FW group was treated with C16FW (10 μM) in PBS, while the C16FW+S100A9 group was treated with C16FW (10 μM) and recombinant S100A9 protein (1 mg / mL) in addition. After incubation for 24 hours, the morphological changes were observed under an electron microscope.

[0042] The results are as follows Figure 2 As shown, before the addition of S100A9, C16FW aggregates into a spherical structure. Figure 2 In C16FW, after the addition of S100A9, S100A9 can be captured by the S100A9 protein and form a network structure. Figure 2 (B)

[0043] Example 3:

[0044] Confocal analysis showed that the self-assembled small peptide (C16FW) significantly inhibited blood-brain barrier disruption in tMCAO model mice.

[0045] The tMCAO experimental group used 6-8 week old male C57BL / 6 mice to establish the tMCAO model:

[0046] First, the mice were anesthetized by inhaling isoflurane through a small animal anesthetic machine. Then, the skin of the mouse neck was disinfected, and the skin was incised along the median line of the neck to fully expose the left common carotid artery, internal carotid artery and external carotid artery. Next, a 6-0 silicon-coated nylon thread plug was inserted into the proximal end of the external carotid artery, and the thread plug was passed around the common carotid artery and slowly pushed to the middle cerebral artery starting part of the internal carotid artery to complete the middle cerebral artery blood flow blockage. After 1 hour of ischemia, the thread plug was removed to restore blood perfusion to the brain. Immediately after the blood flow of the mouse was restored, S100A9 inhibitor paquinimod (Paq group, 10 mg / kg) or C16FW (C16FW group, 200 μM, 100 μL per mouse) was injected through the tail vein, and the control group (veh group) was given the same amount of saline as the solvent control.

[0047] The sham group (sham group) mice received the same operation as the experimental group, but without middle cerebral artery embolization and drug injection.

[0048] At 24 hours after reperfusion, the mouse was perfused through the heart, and the brain tissue was removed and fixed in a 4% paraformaldehyde solution, then dehydrated with different concentrations of sucrose solution gradient, and finally sectioned using a freezing microtome. The sections were stained with Fibrin+Lectin at 4°C overnight, then DAPI staining solution was added and incubated at room temperature for 10 minutes, and the sections were photographed.

[0049] The results are shown in Figure 3 The results showed that C16FW could reduce the leakage of fibrinogen, indicating that C16FW could alleviate the damage of BBB in tMCAO mice, and the therapeutic effect was comparable to that of S100A9 inhibitor paquinimod.

[0050] Example 4:

[0051] Self-assembled small peptides (C16FW) can significantly alleviate the neurological deficits of tMCAO model mice

[0052] The neurological function of each group of mice in Example 3 was evaluated by the modified neurological severity score (mNSS) scale before and 24 hours after the tMCAO operation. The score of a normal mouse was 0, and the score of a mouse with maximum neurological dysfunction was 18. The mNSS scale covered multiple evaluation items, including the tail lifting experiment (0-3 points), the balance beam experiment (0-6 points), the loss of reflex and abnormal movement (0-4 points), the sensory experiment (0-2 points), and the straight line walking experiment (0-3 points). According to the performance of the mice in each experiment, the corresponding score was given, and the sum of each score was finally calculated to quantify the severity of the neurological dysfunction.

[0053] As shown in Figure 4 , the results showed that C16FW could reduce the mNSS score of tMCAO mice, indicating that C16FW could significantly alleviate the neurological dysfunction of tMCAO model mice, and its therapeutic effect was comparable to that of paquinimod.

[0054] Example 5:

[0055] Cytotoxicity experiment of self-assembled small peptide (C16FW)

[0056] The CCK8 method was used to detect the toxic effects of 5 μM, 10 μM, 20 μM, 40 μM, and 80 μM of C16FW on primary microglial cells.

[0057] As shown in Figure 5 , at a concentration of 80 μM, C16FW had an impact on the activity of primary microglial cells.

[0058] Example 6:

[0059] Self-assembled small peptide (C16FW) can block S100A9 to promote the leakage of in vitro BBB model

[0060] The permeability of the in vitro BBB model was measured by two methods: TEER (transendothelial electrical resistance) value and FITC-Dextran transendothelial permeability.

[0061] TEER (Transendothelial electrical resistance) value measurement: First, the endothelial cells were seeded in the upper chamber of the Transwell chamber membrane, and the microglial cells were seeded in the lower chamber. After OGD (oxygen-glucose deprivation) treatment for 6 hours, C16FW (20 mM) or paquinimod (10 mM) was added simultaneously with S100A9 protein (1 mg / mL) stimulation, and incubated for 24 hours. Then, the TEER value was measured by an endothelial voltmeter. The calculation formula of the TEER value was: TEER value (Ω x cm2) = [(cell insert resistance-blank insert resistance) x insert surface area]. The con group was treated by OGD without S100A9 protein treatment, and the veh group was treated by OGD and then treated by S100A9 protein.

[0062] FITC-Dextran transendothelial permeability measurement: First, 1 mg / mL of FITC-Dextran (molecular weight 70,000 Da) was added to the upper chamber of the Transwell chamber membrane, and incubated for 2 hours. Then, 50 mL of liquid was taken from the lower chamber and added to the enzyme-labeled plate. Subsequently, the absorbance of the lower chamber liquid was measured at a wavelength of 450 nm using a multifunctional enzyme-labeled instrument, and the transendothelial permeability of FITC-Dextran was calculated. Through these two tests, the changes in the barrier function of endothelial cells under different stimulation conditions can be evaluated. The con group was treated by OGD without S100A9 protein treatment, and the veh group was treated by OGD and then treated by S100A9 protein.

[0063] The results are shown in Figure 6 C16FW can reduce the leakage of FITC-Dextran induced by S100A9 and increase the TEER value, indicating that C16FW can significantly inhibit the in vitro BBB leakage induced by S100A9, and the effect of S100A9 is comparable to that of paquinimod.

[0064] Example 8:

[0065] Small animal live imaging confirmed that the self-assembled small peptide (C16FW) can be aggregated in ischemic brain tissue through blood circulation

[0066] First, the C16FW solution was co-incubated with Cy5 dye for 1 hour, and then 100 mL of C16FW-Cy5 solution (concentration 200 mM) was injected into the tMCAO mice prepared in Example 3 through the tail vein. After 24 hours of blood circulation, the mice were anesthetized and heart perfusion was performed. The brain, heart, liver and kidney tissues of the mice were removed, and the brain tissue was sliced and placed in a small animal imaging instrument (emission wavelength 660 nm, excitation wavelength 640 nm) for image acquisition.

[0067] The results are shown in Figure 7As shown, at 24 hours post-tMCAO, C16FW was successfully accumulated in the ischemic brain tissue region, while a large amount of C16FW was also metabolized by the liver.

Claims

1. A synthetic self-assembling small peptide C16FW having the formula: C 16 -FFVLK-PEG4-WSPTKVH.

2. A complex comprising the self-assembling small peptide C16FW of claim 1.

3. Use of the self-assembling small peptide C16FW of claim 1 or the complex of claim 2 for the manufacture of a blocking drug of S100A9.

4. Use of the self-assembling small peptide C16FW of claim 1 or the complex of claim 2 for the manufacture of a drug for the treatment of inflammation caused by a significant increase in S100A9.

5. Use of the self-assembling small peptide C16FW of claim 1 or the complex of claim 2 for the manufacture of a drug for the treatment of cerebrovascular diseases and / or recovery period of cerebrovascular diseases.

6. The use according to claim 5, wherein the cerebrovascular diseases or recovery period of cerebrovascular diseases are caused by a significant increase in S100A9 in blood due to ischemic stroke, due to hemorrhagic stroke, due to traumatic brain injury or due to neutrophil-related intracranial inflammatory reactions.

7. The use according to claim 6, wherein the cerebrovascular disease comprises:

8. The use according to claims 3, 4 or 5, wherein the drug is in a pharmaceutically acceptable dosage form.

9. The use according to claim 8, wherein the dosage form is a tablet, a capsule, a granule, an injection, a powder or a drop. ​

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