Cyclic polypeptide CycloSD6 of targeted contact kinin system and application of cyclic polypeptide CycloSD6

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a cyclic polypeptide CycSD6 of a targeted contact kinin system and application of the cyclic polypeptide CycSD6. The cyclic polypeptide CycloSD6 provided by the invention comprises an amino acid sequence as shown in SEQ ID NO: 1; the head and the tail of the amino acid sequence as shown in SEQ ID NO: 1 form a ring through an amido bond. The head and tail amido bonds of the amino acid sequence as shown in SEQ ID NO: 1 are cyclized, the plasma stability of the straight-chain polypeptide as shown in SEQ ID NO: 1 can be improved, and compared with the straight-chain polypeptide, the cyclized cyclic polypeptide has the advantages that the inhibitory activity on plasma kallikrein and blood coagulation factor XIIa is further improved, the plasma recalcification time and the partial thromboplastin activating time can be prolonged, and the blood coagulation factor XIIa can be inhibited. The compound has good anti-thrombus and cerebral apoplexy activity effects, does not cause hemolysis and bleeding risks, and is low in cytotoxicity and high in safety.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to a cyclic polypeptide CycloSD6 targeting a contact kinin system and an application thereof. Background Art

[0002] According to statistics from the World Health Organization, 15 million people die from thromboembolic diseases every year. Thromboembolic diseases are characterized by high morbidity, high mortality and high disability rates. Common thromboembolic diseases include acute ischemic stroke, peripheral vascular disease, etc., which seriously endanger human health. Although there are many drugs such as heparin (including low molecular weight heparin LMWH such as enoxaparin), warfarin, platelet aggregation inhibitors (such as aspirin), hirudin and other drugs used in clinical practice, they also commonly have side effects such as thrombocytopenia and bleeding.

[0003] The contact kinin pathway plays a central role in the thrombotic pathology of ischemic stroke. When exposed to negatively charged surfaces or inorganic polyphosphates, coagulation factor XII (FXII) is partially activated and converted to active FXII (FXIIa), which subsequently initiates the contact kinin pathway. FXIIa also cleaves plasma prekallikrein (PK) to produce plasma kallikrein (PKa, KLKB1), which releases the inflammatory mediator bradykinin to promote inflammation. The activation between PKa and FXII produces a reciprocal cascade that further drives autoactivation within the contact kinin pathway and promotes thrombosis. Therefore, targeting key enzymes within the contact kinin system has great potential for the development of therapeutic agents aimed at treating and preventing stroke. However, there are currently few candidate drug molecules targeting the contact kinin pathway in the existing technology. Summary of the invention

[0004] The purpose of the present invention is to provide a cyclic polypeptide CycloSD6 targeting the contact kinin system and its application, which inhibits the activity of plasma kallikrein and coagulation factor XIIa, prolongs the plasma recalcification time and activated partial thromboplastin time, has good anti-thrombotic and anti-stroke activity, does not cause hemolysis, has low bleeding risk and cytotoxicity, and is highly safe.

[0005] In order to achieve the above object, the present invention provides a cyclic polypeptide CycloSD6, which comprises an amino acid sequence as shown in SEQ ID NO:1; the amino acid sequence as shown in SEQ ID NO:1 is ringed at the beginning and the end through an amide bond.

[0006] The present invention also provides the use of the cyclic polypeptide CycloSD6 described in the above technical solution in the preparation of a product for inhibiting the activity of plasma kallikrein and / or inhibiting the activity of coagulation factor XIIa.

[0007] The present invention also provides the use of the cyclic polypeptide CycloSD6 described in the above technical solution in the preparation of products for preventing and / or treating mental illness; the mental illness includes mental illness related to plasma kallikrein and / or coagulation factor XIIa.

[0008] The present invention also provides the use of the cyclic polypeptide CycloSD6 described in the above technical solution in the preparation of products for preventing and / or treating thromboembolic diseases.

[0009] Preferably, the thromboembolic disease includes one or more of arterial thrombotic disease, venous thrombotic disease and capillary thrombotic disease.

[0010] The present invention also provides the use of the cyclic polypeptide CycloSD6 described in the above technical solution in the preparation of anti-thrombotic products and / or anti-stroke products.

[0011] Preferably, the stroke includes ischemic stroke and / or hemorrhagic stroke; the ischemic stroke includes one or more of thrombotic cerebral infarction, embolic cerebral infarction, lacunar cerebral infarction, multiple cerebral infarction and transient ischemic attack.

[0012] Preferably, the product comprises a medicine and / or a reagent.

[0013] The present invention also provides an inhibitor of plasma kallikrein and / or coagulation factor XIIa, wherein the active ingredient of the inhibitor comprises the cyclic polypeptide CycloSD6 described in the above technical solution.

[0014] The present invention also provides an anti-thrombotic and / or anti-stroke drug, the active ingredient of which includes the cyclic polypeptide CycloSD6 described in the above technical solution.

[0015] Beneficial effects:

[0016] The present invention provides a cyclic polypeptide CycloSD6, which includes an amino acid sequence as shown in SEQ ID NO:1; the amino acid sequence shown in SEQ ID NO:1 is cyclically bonded at the head and tail. The present invention can improve the plasma stability of the linear polypeptide shown in SEQ ID NO:1 by cyclically bonding the head and tail amide bonds of the amino acid sequence shown in SEQ ID NO:1, and the cyclic polypeptide after cyclicization, compared with the linear polypeptide, has further improved inhibitory activity on plasma kallikrein and coagulation factor XIIa, can prolong plasma recalcification time and activated partial thromboplastin time, has good anti-thrombotic and anti-stroke activity, does not cause hemolysis and bleeding risks, has low cytotoxicity, and is highly safe. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0018] Figure 1 This is the structural diagram of the linear polypeptide SD6 of Example 1;

[0019] Figure 2 This is the structural diagram of the cyclic polypeptide cycloSD6 of Example 1;

[0020] Figure 3 This is a graph showing the plasma stability results of the linear peptide SD6 and the cyclic peptide cycloSD6 in Example 1;

[0021] Figure 4 This is the result of the chromogenic substrate method enzyme kinetic analysis of the effects of SD6 and cycloSD6 on the activity of PKa;

[0022] Figure 5 This is the result of the chromogenic substrate method enzyme kinetic analysis of the effects of SD6 and cycloSD6 on the activity of FXIIa;

[0023] Figure 6 The molecular docking result diagram of the sample to be tested (cycloSD6) and the protein to be tested (KLKB1 and FXII);

[0024] Figure 7 The results of the effect of cycloSD6 on PRT, APTT and PT in vitro are shown in Figure 2.

[0025] Figure 8 for the cytotoxic effect of cycloSD6;

[0026] Fig. 9 The effect of cycloSD6 on cell hemolysis;

[0027] Fig.10 The effect of cycloSD6 on the tail bleeding time of mice;

[0028] Fig.11 The effect of cycloSD6 on the levels of inflammatory factors in A549 cells and HUVEC cells after LPS induction;

[0029] Fig.12 The effect of cycloSD6 on carrageenan-induced tail thrombosis;

[0030] Fig.13 The effect of cycloSD6 on FeCl3-induced thrombosis in mice;

[0031] Fig.14The effect of cycloSD6 on the mouse cortical photothrombotic stroke model (CPS);

[0032] Fig.15 The effect of cycloSD6 on the Bederson score of cerebral ischemic injury in the tMCAO model;

[0033] Fig.16 The grip strength test of cycloSD6 on tMCAO model of cerebral ischemia injury;

[0034] Fig.17 TTC staining and statistical results of cycloSD6 on brain ischemic injury in tMCAO model;

[0035] Fig.18 Evans blue staining and statistical results of cycloSD6 on brain ischemic injury in tMCAO model;

[0036] In the figures, * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001. DETAILED DESCRIPTION

[0037] The present invention provides a cyclic polypeptide CycloSD6, which comprises an amino acid sequence as shown in SEQ ID NO: 1; the amino acid sequence as shown in SEQ ID NO: 1 is cyclically connected at the beginning and the end through an amide bond.

[0038] As an embodiment, the purity of the cyclic polypeptide CycloSD6 of the present invention is 98%. The present invention has no strict requirements on the preparation method of the cyclic polypeptide CycloSD6, and it can be obtained by conventional methods in the art, such as entrusting a company to perform artificial synthesis.

[0039] The present invention forms a loop at the beginning and the end of the amino acid sequence (SLGASD) shown in SEQ ID NO: 1, thereby increasing the stability in plasma, causing no risk of hemolysis or bleeding, having low cytotoxicity and high safety.

[0040] The present invention uses a chromogenic substrate method and enzyme kinetic analysis, and the results show that cycloSD6 inhibits the activity of plasma kallikrein (PKa) and the activity of coagulation factor XIIa (FXIIa) in a concentration-dependent manner, with the inhibition constant Ki value for PKa being 28.54 μM, and the inhibition constant Ki value for FXIIa being 41.27 μM. In view of this, the use of the cyclic polypeptide CycloSD6 described in the present invention in the preparation of products that inhibit the activity of plasma kallikrein and / or inhibit the activity of coagulation factor XIIa, as well as the use in the preparation of products for preventing and / or treating mental illness also fall within the scope of protection of the present invention. As an embodiment, the mental illness includes mental illness associated with plasma kallikrein and / or coagulation factor XIIa.

[0041] The present invention analyzes the effect of cycloSD6 on plasma recalcification time (PRT), activated partial thromboplastin time (APTT) and prothrombin time (PT) coagulation function in vitro. The results showed that cycloSD6 dose-dependently prolonged PRT and APTT time, and had no effect on PT, indicating that cycloSD6 specifically inhibits the endogenous coagulation pathway. In view of this, the use of the cyclic polypeptide CycloSD6 described in the present invention in the preparation of products for the prevention and / or treatment of thromboembolic diseases also falls within the scope of protection of the present invention. As an embodiment, the thromboembolic disease includes one or more of arterial thrombotic disease, venous thrombotic disease and capillary thrombotic disease.

[0042] The present invention also establishes three different mouse thrombosis models, and uses cycloSD6 to administer the drug. The results show that cycloSD6 dose-dependently inhibits the formation of mouse thrombosis. The present invention also establishes a 60-min cerebral ischemia-reperfusion stroke model (tMCAO), and uses cycloSD6 to administer the drug. The results show that cycloSD6 dose-dependently improves mouse muscle strength, alleviates nerve damage, and reduces the cerebral ischemic infarction injury area and blood-brain barrier damage in tMCAO mice. In view of this, the use of the cyclic polypeptide CycloSD6 described in the present invention in the preparation of anti-thrombotic products and / or anti-stroke products also belongs to the protection scope of the present invention. As an embodiment, the stroke includes ischemic stroke and / or hemorrhagic stroke. As an embodiment, the ischemic stroke includes one or more of thrombotic cerebral infarction, embolic cerebral infarction, lacunar cerebral infarction, multiple cerebral infarction and transient ischemic attack.

[0043] In one embodiment, the product comprises a drug and / or a reagent. In one embodiment, the reagent comprises an anticoagulant.

[0044] The present invention also provides an inhibitor of plasma kallikrein and / or coagulation factor XIIa, the active ingredient of the inhibitor comprising the cyclic polypeptide CycloSD6 described in the above technical solution. As one embodiment, the inhibitor is formulated as a single-use dosage form with 1 to 100 mg of cyclic polypeptide CycloSD6 / dose; as another embodiment, the inhibitor is formulated as a single-use dosage form with 4 to 80 mg of cyclic polypeptide CycloSD6 / dose; as another embodiment, the inhibitor is formulated as a single-use dosage form with 20 to 60 mg of cyclic polypeptide CycloSD6 / dose; as another embodiment, the inhibitor is formulated as a single-use dosage form with 40 to 50 mg of cyclic polypeptide CycloSD6 / dose.

[0045] The present invention also provides an anti-thrombotic and / or anti-stroke drug, the active ingredient of which includes the cyclic polypeptide CycloSD6 described in the above technical solution. As one embodiment, the drug is formulated as a single-use dosage form with 1 to 100 mg of cyclic polypeptide CycloSD6 per dose; as another embodiment, the drug is formulated as a single-use dosage form with 4 to 80 mg of cyclic polypeptide CycloSD6 per dose; as another embodiment, the drug is formulated as a single-use dosage form with 20 to 60 mg of cyclic polypeptide CycloSD6 per dose; as another embodiment, the drug is formulated as a single-use dosage form with 40 to 50 mg of cyclic polypeptide CycloSD6 per dose.

[0046] To further illustrate the present invention, a cyclic polypeptide CycloSD6 targeting the contact kinin system provided by the present invention and its application are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0047] Example 1

[0048] 1. The amino acid sequence of SEQ ID NO: 1 was synthesized by GLBiochem (China) and purified to obtain a linear polypeptide named SD6 (purity 98%). The structure of the linear polypeptide SD6 was shown in the figure using Chemdraw software. Figure 1 shown.

[0049] 2. GLBiochem (China) was commissioned to use the carboxyl group of the terminal aspartic acid (D) of the linear peptide SD6 in step 1 and the amino group of the first serine (S) to undergo a dehydration condensation reaction to form a ring from head to tail, and synthesize a 98% pure cyclic peptide named cycloSD6. The Chemdraw software was used to draw the structure of the cyclic peptide cycloSD6 as shown below: Figure 2 shown.

[0050] Example 2

[0051] Plasma stability analysis

[0052] The linear peptide (SD6) and cyclic peptide (cycloSD6) obtained in Example 1 were used as test samples respectively; 50 μL of 2 mg / mL of the test sample was mixed with 400 μL of fresh human plasma, and incubated at 37°C for different time periods (0, 15 min, 30 min, 45 min, 60 min, 90 min, 120 min, 150 min, 240 min, 300 min). The incubated samples were subjected to protein precipitation to remove macromolecular impurities such as proteins in the plasma, and then the supernatant was injected into the HPLC system. The retention time and peak area of ​​the peptide on the chromatographic column were used to quantitatively analyze the content of the peptide. The peak areas at different incubation time points were compared to calculate the remaining percentage of the peptide. The results showed that the half-life of SD6 in plasma was about 0.7 h, and the stable half-life of cycloSD6 in plasma was about 2.5 h, indicating that the stability of cycloSD6 after cyclization was higher than that of SD6 ( Figure 3 ).

[0053] Example 3

[0054] Protease activity detection - chromogenic substrate method enzyme kinetic analysis

[0055] PKa (40 nM, HPKa1303, Enzyme Research, USA) and FXIIa (30 nM, HFXIIA212a, Enzyme Research, USA) were used as the proteases to be detected; the linear polypeptide (SD6) and cyclic polypeptide (cycloSD6) obtained in Example 1 were used as the samples to be tested.

[0056] 10 μL of the test protease was mixed with 50 μL of 0.01 M phosphate buffered saline (PBS) containing different concentrations (0-100 μM) of the test sample and incubated at 37°C for 5 min. After incubation, 50 μL of the corresponding chromogenic substrate (0.2 mM S-2366 for FXIIa; 30 nM S-2302 for PKa, CHROMOGENIX, USA) was added. The OD was measured using an Epoch 109 BioTek, USA. 405The reaction kinetics were monitored at 400 nm, and the Dixon curve was generated by Dixon double reciprocal plotting to obtain the inhibition constant. The results showed that cycloSD6 inhibited the activity of PKa in a concentration-dependent manner. The inhibition constant of cycloSD6 on PKa was calculated by Dixon double reciprocal plotting. CycloSD6 competitively inhibited the activity of PKa with an inhibition constant Ki value of 28.54 μM, while the Ki value of SD6 was 36.34 μM, indicating that cycloSD6 increased the inhibitory activity on PKa ( Figure 4 ) ; cycloSD6 inhibited the activity of FXIIa in a concentration-dependent manner. The inhibition constant of cycloSD6 on FXIIa was calculated by the Dixon double reciprocal plot method. CycloSD6 competitively inhibited the activity of FXIIa with an inhibition constant Ki value of 41.27 μM, while SD6 had no effect on the activity of FXIIa ( Figure 5 ).

[0057] Example 4

[0058] Molecular docking assay

[0059] The protein domains of KLKB1 (PDB code: 6O1S) and FXII (AlphaFold prediction) were downloaded from the RCSB protein database and prepared using PyMOL software. The 3D model of the cyclic polypeptide (cycloSD6, amino acid sequence SLGASD) of Example 1 was generated using the PEP-FOLD server. AutoDockv4.2 was used to dock cycloSD6 with KLKB1 and FXII to evaluate their interactions. The results showed that cycloSD6 binds to KLKB1 and FXII at their structural sites ( Figure 6 ).

[0060] Example 5

[0061] Effects of plasma recalcification time (PRT), activated partial thromboplastin time (APTT) and prothrombin time (PT)

[0062] In the PRT experiment, the cyclic peptide (cycloSD6) obtained in Example 1 was used as the test sample. 20 μL of human plasma and 10 μL of cycloSD6 of different concentrations (0-100 μM) were placed in 60 μL of HEPES buffer and incubated at 37°C for 5 min. Subsequently, 60 μL of HEPES buffer solution containing 25 mM CaCl2 was added and the OD was immediately 650The mixture was continuously kinetically monitored at 100 nm. APTT (GMS10178.2, GenMed, USA) and PT (STY50101, Steelex, China) were analyzed using commercial kits according to the instructions provided. The results showed that 0-100 μM cycloSD6 prolonged PRT and APTT time in a dose-dependent manner and had no effect on PT, indicating that cycloSD6 specifically inhibits the intrinsic coagulation pathway ( Figure 7 ).

[0063] Example 6

[0064] Cytotoxic effects

[0065] Mouse peritoneal macrophages and spleen cells were cultured at 1×10 6 The cells were plated at a density of 10 cells / well on a 96-well plate (Thermo, 167008, China) and cultured at 37°C and 5% CO2 using RPMI1640 complete medium (10-040-CVR, Corning Co., Ltd., USA); after culturing for 12 hours, the cells were treated with different concentrations of cycloSD6 (0-200 μg / mL) for 12 hours. The effect of cycloSD6 on cell viability was determined using a cell counting kit-8 (CCK8) (GK10001, GLPBIO, USA). Cell viability was expressed as a percentage relative to the control group. The results showed that the cycloSD6 obtained in Example 1 did not inhibit the proliferation of mouse peritoneal macrophages and spleen cells in the concentration range of 0-200 μM, and had no toxic effect on the cells ( Figure 8 )

[0066] Example 7

[0067] Effect of cell hemolysis

[0068] Human blood was centrifuged at 3500 rpm and 25°C for 15 min. The supernatant was discarded and only the red blood cells were retained. These cells were incubated with TritonX-100 (0.1%) and different concentrations of cycloSD6 (0-200 μg / mL) in PBS (0.01 mol / L, pH 7.2) at 37°C for 30 min, and then centrifuged at 3500 rpm and 37°C for 5 min. The absorbance of the supernatant was detected at 540 nm using an ELISA reader. The results showed that cycloSD6 obtained in Example 1 did not cause hemolysis of red blood cells and was a potential safe anticoagulant ( Fig. 9 ).

[0069] Example 8

[0070] Effect of tail bleeding time in mice

[0071] Male C57BL / 6J mice (6 weeks old) were randomly divided into control group, 2500U / kg HS group, 4mg / kg C-SD6 group and 1mg / kg C-SD6 group, with 6 mice in each treatment group, and were treated as follows:

[0072] Control group: 0.9% saline was injected intravenously 10 min before tail amputation;

[0073] 2500U / kg HS group: heparin sodium (9041-08-1, BIOFROX, Germany) was injected at a dose of 2500U / kg 10min before censoring;

[0074] 4 mg / kg C-SD6 group: cycloSD6 obtained in Example 1 was injected at a dose of 4 mg / kg 10 min before tail censoring;

[0075] 1 mg / kg C-SD6 group: cycloSD6 obtained in Example 1 was injected at a dose of 1 mg / kg 10 min before tail censoring;

[0076] The tails of mice in each treatment group were amputated 2 mm from the tip and immediately immersed in sterile saline at 37°C, and the bleeding time was recorded. The results showed that the bleeding time of the tails of mice injected intravenously with cycloSD6 (1 and 4 mg / kg) was comparable to that observed in the saline-treated control group. In contrast, the bleeding time of mice treated with sodium heparin (2500 U / kg) was significantly prolonged, further confirming that cycloSD6 does not significantly increase the risk of bleeding in mice ( Fig.10 ).

[0077] Example 9

[0078] Effects of CycloSD6 on the levels of inflammatory factors in A549 cells and HUVEC cells induced by LPS

[0079] HUVEC and A549 cells were seeded into 12-well plates, respectively. After the cells adhered and reached 60% density, they were washed with 0.01M PBS and then incubated in complete medium containing 1% human plasma for 6 hours and randomly divided into Med group and LPS-induced group; LPS-induced group was then added with different concentrations of cycloSD6 (0-100μM) and LPS (1μg / mL), and Med group was added with different concentrations of cycloSD6 (0-100μM); after the treatment of each treatment group, the cells were cultured at 37℃ and 5% CO2 for 24 hours. The supernatant was collected and enzyme-linked immunosorbent assay (ELISA) of interleukin-6 (IL-6), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and interferon-α (IFN-α) was performed using detection kits. The results showed that the levels of pro-inflammatory cytokines (IL-6, IL-1β, TNF-α, and IFN-α) in the cell supernatant were significantly increased 24 h after LPS induction. Notably, cycloSD6 (4-100 μM) effectively inhibited the production of such cytokines, confirming its anti-inflammatory activity ( Fig.11 ).

[0080] Example 10

[0081] Effects of cycloSD6 on carrageenan-induced tail thrombosis

[0082] Male BALB / c mice (6 weeks old) were randomly divided into sham group, control group, 2500U / kg HS group, 4mg / kg C-SD6 group and 1mg / kg C-SD6 group, with 6 mice in each treatment group. Except for the sham group, the mice in other treatment groups were treated as follows:

[0083] Control group: intravenous injection of 0.9% saline, followed by intraperitoneal injection of 200 μL of 1% carrageenan (type I) (CC3171, Kulab, China) dissolved in 0.9% saline; the injection dose of carrageenan was 80 mg / kg;

[0084] 2500U / kg HS group: intravenous injection of 2500U / kg heparin sodium, followed by intraperitoneal injection of 200μL of 1% carrageenan (type I) (CC3171, Kulab, China) dissolved in 0.9% saline; the injection dose of carrageenan was 80mg / kg;

[0085] 4 mg / kg C-SD6 group: intravenous injection of 4 mg / kg cycloSD6 obtained in Example 1, followed by intraperitoneal injection of 200 μL of 1% carrageenan (type I) dissolved in 0.9% saline; wherein the injection dose of carrageenan was 80 mg / kg;

[0086] 1 mg / kg C-SD6 group: intravenous injection of 1 mg / kg cycloSD6 obtained in Example 1, followed by intraperitoneal injection of 200 μL of 1% carrageenan (type I) dissolved in 0.9% saline; wherein the injection dose of carrageenan was 80 mg / kg;

[0087] 24 hours after injection of carrageenan in each treatment group, the length of the tail thrombus was measured and imaged. The results showed that intravenous injection of cycloSD6 (1 and 4 mg / kg) significantly shortened the length of carrageenan-induced tail thrombus formation in mice. The antithrombotic effect of 4 mg / kg cycloSD6 was comparable to that of clinically used anticoagulant sodium heparin (2500 U / kg) ( Fig.12 ).

[0088] Embodiment 11

[0089] Effect of cycloSD6 on FeCl3-induced thrombosis in mice

[0090] Male C57BL / 6J mice (6 weeks old) were randomly divided into control group, 2500U / kg HS group, 4mg / kg C-SD6 group and 1mg / kg C-SD6 group, with 6 mice in each treatment group, and were treated as follows:

[0091] Control group: intravenous injection of 100 μL 0.9% saline;

[0092] 2500U / kg HS group: intravenous injection of 2500U / kg heparin sodium;

[0093] 4 mg / kg C-SD6 group: intravenous injection of 4 mg / kg cycloSD6 obtained in Example 1;

[0094] 1 mg / kg C-SD6 group: intravenous injection of 1 mg / kg cycloSD6 obtained in Example 1;

[0095] Each treatment group was anesthetized 10 min after treatment, the right carotid artery was isolated, and a circular pad with a radius of 2 mm was placed under the carotid artery to minimize background blood flow. A circular filter paper soaked in 10% ferric chloride (w / v) solution was then applied to the artery for 30 seconds and then removed. Thrombosis in the region of interest (ROI) was monitored using a real-time laser speckle perfusion (LSP) imaging system (RWD, China) with SIMBFI software (SIM BFI HRPro, Simonto, China). Thrombotic occlusion was identified by a color change of the vessel from red to blue. Data recording lasted for 30 min, and mice exposed to FeCl3 for more than 5 min or experiencing severe surgical bleeding were excluded from further analysis. The results showed that at 5 min, the blood flow of the control group injected with normal saline decreased by 38.17% ± 4.81%, while the blood flow of mice treated with 1 mg / kg and 4 mg / kg cycloSD6 decreased by 32.33% ± 4.72% and 23.67% ± 2.64%. At 10 minutes, the blood flow of the control group mice injected with normal saline was completely blocked by the formed thrombus, while the blood flow of the vascular blood vessels of the cycloSD6-treated group was not completely blocked. CycloSD6 has a significant inhibitory effect on FeCl3-induced thrombosis in the common carotid artery of mice ( Fig.13 ).

[0096] Example 12

[0097] Effects of cycloSD6 on the mouse cortical photothrombotic stroke model (CPS)

[0098] Male C57BL / 6J mice (6 weeks old) were intraperitoneally injected with 50 mg / kg Rose Bengal 1 h before surgery, and then randomly divided into sham group, control group, 2500 U / kg HS group, 4 mg / kg C-SD6 group and 1 mg / kg C-SD6 group 10 min before surgery, with 10 mice in each treatment group. Except for the sham group, the mice in other treatment groups were treated as follows:

[0099] Control group: intravenous injection of 100 μL 0.9% saline;

[0100] 2500U / kg HS group: intravenous injection of 2500U / kg heparin sodium;

[0101] 4 mg / kg C-SD6 group: intravenous injection of 4 mg / kg cycloSD6 obtained in Example 1;

[0102] 1 mg / kg C-SD6 group: intravenous injection of 1 mg / kg cycloSD6 obtained in Example 1;

[0103] Except for the sham group, mice in other treatment groups were anesthetized after 10 min of treatment, and the skin was incised along the midline from the eyes to the neck to expose the skull. The right brain area was irradiated with a 15mW 562nm yellow-green laser (R-LG561-100-A5, RWD, China) for 7 min. One day after CPS surgery, mice were imaged using the LSP imaging system, and the survival rate was monitored every day for 15 days. The results showed that mice treated with 4mg / kg cycloSD6 showed cerebral blood flow comparable to that of the sham group, significantly higher than that of mice treated with 2500U / kg heparin sodium, and mice treated with cycloSD6 showed improvement. By day 15, the survival rate of mice in the control group was 40%, while mice treated with 1 and 4mg / kg cycloSD6 showed higher survival rates of 50% and 60%, respectively, highlighting the potential of cycloSD6 to improve survival in thrombotic stroke ( Fig.14 ).

[0104] Embodiment 13

[0105] CycloSD6 reduces cerebral ischemic injury in transient middle cerebral artery occlusion (tMCAO) model

[0106] Male C57BL / 6J mice (6 weeks old) were randomly divided into sham group, control group, 4mg / kg DX-88 group, 4mg / kg C-SD6 group and 1mg / kg C-SD6 group, with 6 mice in each treatment group. Except for the sham group, mice in other groups were anesthetized and a thread plug (1623-50A4, China) was inserted into the internal carotid artery (ICA) through the external carotid artery (ECA). The mice were treated as follows 10 minutes before the thread plug was removed:

[0107] Control group: intravenous injection of 0.9% saline;

[0108] 4 mg / kg DX-88 group: intravenous injection of 4 mg / kg DX-88;

[0109] 4 mg / kg C-SD6 group: intravenous injection of 4 mg / kg cycloSD6 obtained in Example 1;

[0110] 1 mg / kg C-SD6 group: intravenous injection of 1 mg / kg cycloSD6 obtained in Example 1;

[0111] 60 minutes after the suture plug was inserted into the internal carotid artery, the suture plug was removed to restore perfusion. Mice that died within 24 hours after tMCAO surgery or whose surgery time exceeded 15 minutes were excluded from endpoint analysis. At 24 hours after tMCAO surgery, neurological function and the extent of cerebral infarction were assessed using Bederson and grip strength scores. The mice were then killed for 2% 2,3,5-triphenyltetrazolium chloride (TTC) staining. In order to evaluate the blood-brain barrier (BBB) ​​function in the brain, a portion of the mice were intravenously injected with 200 μL of 0.5% Evans blue staining solution (R20616, Yuanye Biotech, China) immediately after tMCAO. Coronal brain sections (2 mm thick) were imaged 24 hours later, and the absorbance of the supernatant of the homogenized brain sections was measured at OD640nm to quantify Evans blue extravasation; among them,

[0112] The Bederson neurological function scoring system was as follows: 0 points, no behavioral disorder; 1 point, inability to extend the right forelimb; 2 points, rotation to the right; 3 points, tilt to the right; 4 points, no spontaneous activity with impaired consciousness; 5 points, death.

[0113] Grip strength test: The grip strength was tested using a grip dynamometer (DS2-50N, Sansbio, China) according to the manufacturer's instructions.

[0114] TTC (T-8877, Sigma-Aldrich, USA) staining: The infarct volume of mice was evaluated. After euthanasia of mice, the brain was removed and immediately precooled at -20°C for 3 min. The brain was then cut into 2 mm thick coronal slices using a mouse brain slice mold (Harvard Instruments, Holston, Massachusetts, USA). The slices were stained with 2% TTC at 37°C for 20 min and then imaged. The infarct volume was measured using ImageJ software according to the following equation: V = Sd / T2, where S represents the infarct area, d represents the thickness, and T is the linear magnification.

[0115] Evans blue staining: To evaluate the protective effect of cycloSD6 on the blood-brain barrier, mice were injected with 200 μL of 0.5% Evans blue staining solution (R20616, Yuanye Biotech, China) immediately after tMCAO surgery and sacrificed 24 h later. Brain slices were homogenized after imaging, and the supernatant was quantitatively analyzed at 650 nm using a microplate reader.

[0116] The results showed that cycloSD6 treatment resulted in a decrease in Bederson score compared with the control group ( Fig.15 ) and improved grip strength ( Fig.16), indicating that neurological function was significantly improved after tMCAO. In addition, TTC staining showed red-stained living cells and white-stained ischemic injury areas, and quantitative analysis showed that cycloSD6 significantly reduced brain tissue damage after ischemia-reperfusion ( Fig.17 ). These findings demonstrate the protective effect of cycloSD6 in tMCAO mice by alleviating thrombosis and highlight its potential as a therapeutic candidate for ischemic stroke. Blood-brain barrier disruption in tMCAO mice was exacerbated by cerebral thrombosis. However, intravenous injection of 1 and 4 mg / kg of cycloSD6 reduced the OD value after Evans blue staining ( Fig.18 ), indicating that the integrity of the blood-brain barrier was improved. 1-4 mg / kg cycloSD6 dose-dependently increased the muscle strength of mice, alleviated nerve damage, and reduced the lesion area of ​​cerebral ischemia and infarction and blood-brain barrier damage in tMCAO mice.

[0117] According to the above content, it can be seen that the cycloSD6 provided by the present invention can target and inhibit the activity of plasma kallikrein and coagulation factor XIIa (FXIIa), inhibit inflammation, thrombosis and ischemic stroke injury, and has the potential to become an anti-inflammatory, thrombotic and stroke therapeutic drug.

[0118] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A cyclic polypeptide CycloSD6, characterized in that: The cyclic polypeptide CycloSD6 includes the amino acid sequence shown in SEQ ID NO: 1; the amino acid sequence shown in SEQ ID NO: 1 is formed into a ring through amide bonds at the beginning and the end.

2. Use of the cyclic polypeptide CycloSD6 according to claim 1 in the preparation of a product for inhibiting the activity of plasma kallikrein and / or inhibiting the activity of coagulation factor XIIa.

3. Use of the cyclic polypeptide CycloSD6 according to claim 1 in the preparation of products for preventing and / or treating mental illness; the mental illness includes mental illness related to plasma kallikrein and / or coagulation factor XIIa.

4. Use of the cyclic polypeptide CycloSD6 according to claim 1 in the preparation of a product for preventing and / or treating thromboembolic diseases.

5. The use according to claim 4, characterized in that: The thromboembolic disease includes one or more of arterial thrombotic disease, venous thrombotic disease and capillary thrombotic disease.

6. Use of the cyclic polypeptide CycloSD6 according to claim 1 in the preparation of anti-thrombotic products and / or anti-stroke products.

7. The use according to claim 6, characterized in that: The stroke includes ischemic stroke and / or hemorrhagic stroke; the ischemic stroke includes one or more of thrombotic cerebral infarction, embolic cerebral infarction, lacunar cerebral infarction, multiple cerebral infarction and transient ischemic attack.

8. The use according to any one of claims 2 to 7, characterized in that: The products include drugs and / or reagents.

9. An inhibitor of plasma kallikrein and / or coagulation factor XIIa, characterized in that The active ingredient of the inhibitor includes the cyclic polypeptide CycloSD6 described in claim 1.

10. An anti-thrombotic and / or anti-stroke drug, characterized in that: The active ingredient of the drug includes the cyclic polypeptide CycloSD6 described in claim 1.

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