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 invention belongs to the field of biomedicine, and specifically relates to a self-assembling small peptide C16FW and an application thereof in preparing a medicine for treating cerebrovascular diseases. Background Art

[0002] Clinical studies have found that an increase in peripheral blood neutrophil counts in patients with ischemic stroke is closely related to increased cerebral edema, increased infarct volume, and poor prognosis. Neutrophil infiltration can not only aggravate nerve damage, but also lead to the occurrence of systemic inflammatory response, which is also a difficulty in current clinical treatment. Although in animal models, reducing the migration and infiltration of neutrophils can effectively reduce brain damage and improve neurological function. However, in related clinical studies, this strategy has not shown the expected efficacy. Because excessive inhibition of neutrophil function may lead to suppression of the immune system, thereby increasing the risk of secondary systemic infection.

[0003] The applicant's previous basic research results show that S100A9 plays a key role in mediating the interaction between microglia and neutrophils after acute ischemic stroke: the applicant sequenced and analyzed the plasma and peripheral blood neutrophils of patients with mild and severe cerebral edema after acute ischemic stroke, and found that neutrophil-derived S100A9 was specifically highly expressed in patients with severe cerebral edema. At the same time, it was found that: ① The S100A9 inhibitor Paquimod and S100A9 knockout can reduce the destruction of BBB after acute ischemic stroke and improve stroke prognosis. ② S100A9 can inhibit the phagocytosis of neutrophils by microglia after stroke and promote the aggregation of neutrophils in the brain. ③ S100A9 promotes microglial pyroptosis and aggravates intracranial inflammatory response. The above results verify that lowering S100A9 levels is an effective therapeutic target for improving the prognosis of acute ischemic stroke. However, the current clinical dilemma is that there are no S100A9 inhibitors or antibodies that have been successfully approved for clinical use. Summary of the invention

[0004] The purpose of the present invention is to provide a synthetic self-assembling small peptide, the molecular formula of which is: 16 -FFVLK-PEG4-WSPTKVH.

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

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] A synthetic self-assembling peptide C16FW, whose molecular formula is: 16 -FFVLK-PEG4-WSPTKVH, the structural formula is:

[0008]

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

[0010] A complex containing the self-assembling small peptide C16FW.

[0011] Application of the artificially synthesized self-assembling small peptide C16FW or the complex in the preparation of S100A9 blocking drugs.

[0012] The use of the artificially synthesized self-assembling small peptide C16FW or the complex in preparing a drug for treating inflammation caused by a significant increase in S100A9.

[0013] Application of the artificially synthesized self-assembling small peptide C16FW or the complex in the preparation of drugs for treating cerebrovascular diseases and / or the prognosis recovery period of cerebrovascular diseases.

[0014] In the above application, the S100A9 in the blood of the patient with cerebrovascular disease or in the recovery period of cerebrovascular disease prognosis is significantly increased.

[0015] In the above-mentioned applications, the cerebrovascular diseases include but are not limited to: diseases caused by ischemic stroke, diseases caused by hemorrhagic stroke, diseases caused by traumatic brain injury, and diseases caused by neutrophil-related intracranial inflammatory response.

[0016] In the above-mentioned applications, the dosage form of the drug is all pharmaceutically acceptable dosage forms, including but not limited to tablets, capsules, granules, injections, powders or drops, etc.

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

[0018] 1. The self-assembling small peptide (C16FW) provided by the present invention can specifically encapsulate the S100A9 protein, effectively reducing the level of S100A9 and preventing it from binding to any receptor.

[0019] 2. The self-assembling small peptide (C16FW) provided by the present invention has a 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 invention can be administered intravenously: it is a safe, effective and easy-to-operate method.

[0021] 4. The self-assembling peptide (C16FW) is a non-protein product with a simple structure. It can be mixed with multiple types of drugs without causing mutual immune reactions.

[0022] 5. The materials and solvents used in the self-assembling peptide (C16FW) provided by the present invention are all materials approved by the 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 cost of treatment. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0025] Figure 2 This is the electron microscopy image of the self-assembled small peptide (C16FW);

[0026] Wherein: A is an electron micrograph of C16FW not incubated with recombinant S100A9 protein, and B is an electron micrograph of C16FW incubated with recombinant S100A9 protein.

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

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

[0029] Figure 4 Schematic diagram showing that the self-assembling small peptide (C16FW) can significantly alleviate the neurological deficits in tMCAO model mice.

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

[0031] Figure 6 Schematic diagram of the self-assembling small peptide (C16FW) that can block S100A9 from promoting leakage in the in vitro BBB model;

[0032] Among them, A: FITC-Dextran leakage experiment confirmed that C16FW can block the leakage effect of S100A9 in the in vitro BBB model; B: TEER experiment confirmed that C16FW can block the leakage effect of S100A9 in the in vitro BBB model.

[0033] Figure 7 The self-assembling small peptide (C16FW) can accumulate in ischemic brain tissue through blood circulation. DETAILED DESCRIPTION

[0034] The present invention is further described below in conjunction with specific examples. The technical solutions of the present invention are conventional solutions in the art unless otherwise specified. The reagents or materials are all from commercial channels unless otherwise specified. The recombinant S100A9 protein used in the present invention was purchased from Abcam.

[0035] Embodiment 1:

[0036] Molecular structure of self-assembling small peptide (C16FW)

[0037] The structural formula of the self-assembling peptide (C16FW) involved in the present invention is as follows. Its molecular formula is: C16-FFVLK-PEG4-WSPTKVH, obtained by commercial synthesis:

[0038]

[0039] Embodiment 2:

[0040] Some physical and chemical properties of self-assembling peptide (C16FW):

[0041] In the C16FW group, C16FW (10 μM) was added to PBS, and in the C16FW+S100A9 group, recombinant S100A9 protein (1 mg / mL) was added to C16FW (10 μM) and PBS. 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 aggregated into spherical structures ( Figure 2 Middle A), after adding S100A9, C16FW can capture S100A9 protein and form a network structure ( Figure 2 (middle B).

[0043] Embodiment 3:

[0044] Confocal microscopy of the self-assembled peptide (C16FW) can significantly inhibit the destruction of the blood-brain barrier 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 inhalation of isoflurane using a small animal anesthesia machine. Then, the skin of the mouse neck was disinfected, and the skin was incised along the midline 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 suture was inserted in the proximal direction of the external carotid artery, and the suture was bypassed around the common carotid artery and slowly advanced to the origin of the middle cerebral artery of the internal carotid artery to complete the blood flow blockade of the middle cerebral artery. After ischemia lasted for 1 hour, the suture was removed and brain blood perfusion was restored. After the blood flow of the mice was restored, the S100A9 inhibitor paquimod (Paq group, 10 mg / kg) or C16FW (C16FW group, 200 μM, 100 μL per mouse) was immediately injected through the tail vein, and the control group (veh group) was given an equal amount of normal saline as a solvent control.

[0047] The mice in the sham group received the same operation as the experimental group, but without occluding the middle cerebral artery and injecting drugs.

[0048] 24 hours after reperfusion, the mice were perfused through the heart, the brain tissue was removed, fixed in 4% paraformaldehyde solution, and then dehydrated with different concentrations of sucrose solution, and finally sliced ​​using a freezing slicer. The slices were stained with Fibrin+Lectin at 4°C overnight, then incubated with DAPI stain at room temperature for 10 minutes, and then spread and photographed.

[0049] The results are as follows Figure 3 As shown, laser confocal microscopy was used to detect BBB leakage, and ImageJ was used to calculate the ratio of fibrinogen leakage area to the entire image area. The results showed that C16FW could reduce fibrinogen leakage, indicating that C16FW could alleviate the destruction of BBB in tMCAO mice, and its therapeutic effect was comparable to that of the S100A9 inhibitor paquimod.

[0050] Embodiment 4:

[0051] Self-assembling small peptide (C16FW) can significantly alleviate neurological deficits in tMCAO model mice

[0052] By using the modified neurological severity score (mNSS) scale, the neurological function of each group of mice in Example 3 was evaluated before and 24 hours after tMCAO surgery. The score of normal mice was 0 points, and the score of mice with maximum neurological dysfunction was 18 points. The mNSS scale covers multiple evaluation items, including tail lifting test (0-3 points), balance beam test (0-6 points), loss of reflexes and abnormal movement (0-4 points), sensory test (0-2 points) and straight walking test (0-3 points). According to the performance of mice in each experiment, the corresponding score is assigned, and the sum of the scores is finally calculated to quantify the severity of its neurological dysfunction.

[0053] The results are as follows Figure 4 As shown, the results showed that C16FW could reduce the mNSS scores of tMCAO mice, indicating that C16FW could significantly alleviate the neurological deficits in tMCAO model mice, and its therapeutic effect was comparable to that of paquimod.

[0054] Embodiment 5:

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

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

[0057] The results are as follows Figure 5 As shown: C16FW affects the activity of primary microglia at a concentration of 80 μM.

[0058] Embodiment 6:

[0059] Self-assembling small peptide (C16FW) can block S100A9 from promoting leakage in an 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 determination: First, endothelial cells were planted in the upper chamber of the Transwell chamber membrane, and microglia were planted in the lower chamber. After OGD (oxygen glucose deprivation) treatment for 6 hours, C16FW (20 μM) or paquimod (10 μM) was added at the same time as S100A9 protein (1 mg / mL) stimulation. After incubation for 24 hours, the TEER value was measured using an endothelial volt-ohmmeter. The calculation formula of TEER value is: TEER value (Ω×cm2) = [(cell patch resistance-blank patch resistance)×patch surface area]. The con group was treated with OGD but not with S100A9 protein, and the veh group was treated with OGD and then with S100A9 protein.

[0062] FITC-Dextran transendothelial permeability assay: First, 1 mg / mL of FITC-Dextran (molecular weight 70,000 Da) was added to the upper chamber of the Transwell chamber membrane. After incubation for 2 hours, 50 μL of liquid was taken out from the lower chamber and added to the ELISA plate. Subsequently, the absorbance of the liquid in the lower chamber was measured at a wavelength of 450 nm using a multifunctional microplate reader to calculate the transendothelial permeability of FITC-Dextran. Through these two tests, changes in endothelial cell barrier function under different stimulation conditions can be evaluated. The con group was treated with OGD but not with S100A9 protein, and the veh group was treated with OGD and then with S100A9 protein.

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

[0064] Embodiment 8:

[0065] Small animal in vivo imaging confirmed that the self-assembling peptide (C16FW) can accumulate in ischemic brain tissue through blood circulation

[0066] The C16FW solution was first incubated with the Cy5 dye for 1 hour, and then 100 μL of the C16FW-Cy5 solution (concentration of 200 μM) 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 cardiac perfused, and the brain, heart, liver and kidney tissues of the mice were removed. After the brain tissue was sliced, it was placed in a small animal imager (emission wavelength 660 nm, excitation wavelength 640 nm) for image acquisition.

[0067] The results are as follows Figure 7As shown, 24 hours after tMCAO, C16FW could successfully accumulate in the ischemic brain tissue area, and at this time a large amount of C16FW also accumulated after liver metabolism.

Claims

1. An artificially synthesized self-assembling peptide C16FW, whose molecular formula is: C 16 -FFVLK-PEG4-WSPTKVH.

2. A complex containing the self-assembling small peptide C16FW according to claim 1.

3. Use of the self-assembling peptide C16FW according to claim 1 or the complex according to claim 2 in the preparation of S100A9 blocking drugs.

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

5. Use of the self-assembling peptide C16FW according to claim 1 or the complex according to claim 2 in the preparation of a drug for treating cerebrovascular diseases and / or the prognosis recovery period of cerebrovascular diseases.

6. The use according to claim 5, wherein the S100A9 in the blood of the patient with cerebrovascular disease or in the recovery period of cerebrovascular disease prognosis is significantly increased.

7. The use according to claim 6, wherein the cerebrovascular disease comprises: Disease due to ischemic stroke, disease due to hemorrhagic stroke, disease due to traumatic brain injury, or disease due to neutrophil-related intracranial inflammatory response.

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

9. The use according to claim 8, wherein the dosage form is tablets, capsules, granules, injections, powders or drops.

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