Hemostatic peptide capable of targeting to bleeding focus as well as preparation method and application thereof

Abstract

The invention discloses a hemostatic peptide capable of targeting to a bleeding focus as well as a preparation method and application of the hemostatic peptide, and the hemostatic peptide is characterized in that the amino acid sequence of the hemostatic peptide is RKLFFVRTA. According to the present invention, the key amino acid sequence combined with TF in the blood coagulation factor VII (FVII) is adopted as the basis for modification, the exposed TF (blood coagulation starting factor) after the blood vessel injury is specially identified, the specific expression of the TF at the bleeding site is adopted to achieve the precise targeting, the interference on the normal blood vessel is reduced, and the unnecessary thrombus risk is reduced from the source.

Description

TECHNICAL FIELD

[0001] The present application relates to a hemostatic peptide that can be targeted to a bleeding lesion, and a preparation method and application thereof, and relates to the field of polypeptides. BACKGROUND

[0002] The existing hemostatic peptide technology mainly includes the following categories:

[0003] 1. Complex carrier fixation technology

[0004] Graphene / chitosan composite sponge:

[0005] The thrombin receptor activating peptide (TRAP) is precisely fixed on the surface of the graphene / chitosan cross-linked sponge by thiol-ene photo-click chemistry to form the GCCS-TRAP material. This technology can not only quickly absorb blood (absorb droplets within 40 milliseconds), but also activate the coagulation cascade reaction through surface-fixed peptides, and avoid the risk of thrombosis caused by non-covalent binding. Advantages: water absorption capacity reaches 112 times of its own weight, good biocompatibility, suitable for traumatic massive hemorrhage scenarios.

[0006] 2. Light-responsive intelligent materials

[0007] Blue light triggered peptide hydrogel:

[0008] Composed of self-assembling peptide sequence PFV and methacrylated gelatin (GelMA), it forms a nanofiber network under the trigger of blue light endoscopy, and achieves ultra-fast hemostasis by wrapping blood cells and activating platelets. Experiments show that it can control non-varicose upper gastrointestinal bleeding within 60 seconds and significantly reduce the risk of rebleeding. Features: suitable for patients with coagulation dysfunction, with clinical transformation potential.

[0009] 3. Self-assembled nanofiber technology

[0010] Short peptide nanofiber biomaterials: self-assembling peptides form nanofiber hydrogels through ionic complementarity, which quickly stop bleeding through physical barrier action and do not interfere with the coagulation cascade reaction. For example AC5 and other products have been used in non-compressive torso bleeding, gastrointestinal mucosal covering and other scenarios, and have the functions of hemostasis and promoting wound healing. Advantages: high biocompatibility, can be combined with antibacterial or drug release functions to improve the wound microenvironment.

[0011] 4. Biomimetic platelet technology

[0012] Silk fibroin microspheres: silk fibroin microspheres that mimic the morphology of platelets, with surface modification to enhance interaction with blood cells, suitable for deep tissue hemostasis. Experiments show that it can effectively reduce bleeding by more than 60%, without the risk of high-temperature burns or distal thrombosis.

[0013] 5. Recombinant thrombin technology

[0014] As a thrombin drug, it directly promotes the conversion of fibrinogen into fibrin and accelerates the formation of blood clots. The domestic clinical application guidelines have been issued, emphasizing its advantages in surgical hemostasis.

[0015] However, the existing hemostatic peptide technology has significant limitations in targeting, mainly in two core problems of non-specific binding and poor lesion positioning ability. Taking polypeptide drug conjugate (PDCs) as an example, although it realizes directional delivery by combining the peptide chain with the target, clinical studies show that its targeting specificity is still limited by the structure characteristics of the polypeptide molecule. For example, linear polypeptides are easily degraded by plasma proteases, resulting in a short circulation half-life (usually less than 30 minutes), and unmodified polypeptides are easily combined with serum proteins in vivo, reducing the lesion enrichment efficiency. Although the graphene / chitosan composite sponge can quickly absorb liquid, the thrombin receptor activating peptide (TRAP) fixed on its surface may still activate platelets in non-bleeding areas, increasing the risk of thrombosis. In addition, although the biomimetic platelet technology simulates the shape of platelets, the surface modification of silk fibroin microspheres lacks the recognition ability of specific markers (such as von Willebrand factor vWF) at the site of vascular injury, resulting in insufficient targeting efficiency when bleeding in deep tissues. SUMMARY

[0016] In view of the above technical problems, the purpose of the present application is to provide a hemostatic peptide that can target bleeding lesions, its preparation method and application, to realize targeted binding at the bleeding site, reduce the amount of bleeding, and optimize the TF binding conformation of the original sequence and improve the lesion targeting of the hemostatic peptide.

[0017] In order to achieve the above purpose, the technical scheme of the present application is as follows: a hemostatic peptide that can target bleeding lesions, characterized in that the amino acid sequence is: RKLFFVRTA.

[0018] The hemostatic peptide of the present application takes tissue factor (TF) as the target, utilizes the characteristic that TF is exposed in the blood vessel after vascular injury, realizes targeted binding at the bleeding site, and reduces the amount of bleeding. TF is an important starting factor that plays a switching role in the coagulation process, and as a cell membrane surface receptor of coagulation factor VII (FVII), TF forms a complex with FVIIa (Activated Factor VII, an important coagulation factor) to start the extrinsic coagulation pathway. The hemostatic peptide of the present application is obtained by modifying the key amino acid sequence of FVII that binds to TF, which optimizes the TF binding conformation of the original sequence and improves the lesion targeting of the hemostatic peptide.

[0019] A preparation method of the hemostatic peptide that can target bleeding lesions, characterized in that it is prepared according to the following method:

[0020] First, the resin carrier is swelled and the protecting group is removed, and then reacted with the carboxyl group on the C-terminal of the first amino acid, after the first amino acid is connected to the resin carrier, the remaining amino acids are connected in sequence according to the amino acid sequence of the designed peptide, and after all the synthesis procedures are completed, the polypeptide on the resin carrier is cut off, the synthesized peptide is refined, separated and purified by high performance liquid chromatography, and finally freeze-dried to obtain.

[0021] In the above scheme, the resin is 4-methyl diphenyl methyl amine resin.

[0022] The application of a hemostatic peptide capable of targeting a bleeding lesion in the preparation of a hemostatic drug.

[0023] Compared with the existing hemostatic peptide technology, the hemostatic peptide sequence of the present application exhibits the following advantages by optimizing the tissue factor (TF) binding conformation and enhancing the lesion targeting: it can target the bleeding lesion and reduce the risk of non-specific binding. The existing technology lacks the ability to recognize specific markers of blood vessel damage sites, which easily activates the coagulation reaction in non-bleeding areas (such as TRAP peptide may cause non-targeted platelet activation) or non-specifically binds with serum proteins (such as linear polypeptide), increasing the risk of thrombosis or reducing the efficiency of lesion enrichment. The present application is based on the key amino acid sequence in coagulation factor VII (FVII) that binds to TF, which specifically recognizes TF (a coagulation initiator) exposed after blood vessel damage, uses the specific expression of TF at the bleeding site to achieve precise targeting, reduces interference with normal blood vessels, and reduces unnecessary thrombosis risk from the source. The present application enhances the structural stability of the peptide chain, prolongs the action time, and better fits the active site of TF, improves the binding affinity, and promotes the rapid enrichment of the hemostatic peptide in the bleeding lesion and accelerates the coagulation initiation. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 HPLC chart of rhodamine-labeled hemostatic peptide.

[0025] Figure 2 Mass spectrum chart of rhodamine-labeled hemostatic peptide.

[0026] Figure 3 Merge chart of brain tissue of the control group.

[0027] Figure 4 Merge chart of brain tissue of the hemostatic peptide group of the present application. DETAILED DESCRIPTION

[0028] The present application will be further described below by way of examples in conjunction with the accompanying drawings:

[0029] Example 1

[0030] The hemostatic peptide design sequence is: RKLFFVRTA

[0031] The manufacturing steps are as follows:

[0032] 1. Resin pretreatment

[0033] (1) Resin swelling

[0034] Weigh 0.5g Rink Amide MBHA Resin (benzyl amido methyl phenol resin, 0.7mmol / g, about 0.35mmol) into a synthesis reactor, add 10mL dichloromethane (DCM), shake at room temperature for 30 minutes to make the resin fully swell, remove the DCM by suction filtration, and repeat the swelling operation once.

[0035] (2) Initial Fmoc deprotection

[0036] Add Rink Amide MBHA Resin to 10mL 20% piperidine + 5% DBU / DMF solution, shake at room temperature for 10 minutes, suction filter, repeat the deprotection operation once, and wash the resin with DMF (3x10mL, 3 minutes each time) and DCM (3x10mL, 3 minutes each time) in turn, and dry by suction.

[0037] 2. Peptide chain extension

[0038] (1) Amino acid coupling

[0039] Prepare the amino acid activation solution: dissolve 4eq amino acid (1.4mmol), 4eq HATU (1.4mmol) and 4eq HOAt (1.4mmol) in 5mL DMF, add 8eq DIPEA (2.8mmol), and activate at room temperature for 2 minutes. Transfer the activation solution to the reactor, and react under microwave conditions (50°C, 400W, for hydrophobic amino acids L, F, V) or (30°C, 200W, for polar amino acids R, K, T, A) for 5 minutes. Suction filter, and wash the resin with DMF (3x10mL, 1 minute each time).

[0040] (2) Fmoc deprotection

[0041] After the first amino acid is coupled, add 10mL 20% piperidine + 5% DBU / DMF solution, and react under microwave conditions (30°C, 200W) for 5 minutes, suction filter, and wash the resin with DMF (3x10mL, 1 minute each time).

[0042] (3) Repeat step (1) amino acid coupling.

[0043] (4) For double coupling (for difficult coupling sites: F-F, V-R)

[0044] After the first F or V is coupled, do not deprotect, but directly repeat the coupling operation in step (1) once.

[0045] 3. Resin cleavage

[0046] (1) Pre-washing

[0047] Wash the resin with DCM (3 x 10 mL) and methanol (3 x 10 mL) sequentially, and dry.

[0048] (2) Cleavage reaction

[0049] Add 10 mL cleavage solution (TFA:TIPS:H2O = 92.5:2.5:5) and shake at room temperature for 2 hours in the dark.

[0050] (3) Product collection

[0051] Filter and retain the filtrate. Wash the resin with 2 mL TFA twice and combine the filtrates.

[0052] (4) Precipitation and washing

[0053] Slowly drop the filtrate into 100 mL pre-cooled anhydrous ether and stand at -20°C for 30 minutes. Centrifuge at 4000 rpm for 10 minutes and discard the supernatant. Wash the precipitate with cold ether (3 x 20 mL) and collect the solid by centrifugation.

[0054] 4. Purification and lyophilization

[0055] (1) Crude peptide dissolution

[0056] Dissolve the precipitate in 5-10 mL 50% acetonitrile / water (containing 0.1% TFA) solution and assist dissolution by ultrasonic.

[0057] (2) HPLC purification

[0058] Purify the crude peptide by reverse phase HPLC with a water-acetonitrile gradient containing 0.1% TFA as the mobile phase and collect the target peak fractions.

[0059] (3) Collection and lyophilization

[0060] Dissolve the purified peptide chain in HFIP, remove the HFIP under vacuum after freezing, redissolve with distilled water and lyophilize to obtain pure peptide powder.

[0061] As shown in Figure 1 and 2 , the theoretical molecular weight of the rhodamine-labeled hemostatic peptide is 1549.82 Da. In the ESI positive ion mode, if a three-charge ion is formed, the theoretical mass-to-charge ratio is (1549.82 + 3 x 1.0078) / 3 ≈ 517.6, which is completely consistent with the measured strongest peak 517.6 m / z, indicating that this peak is the characteristic ion peak of the target peptide.

[0062] Test Example

[0063] 1、The application of hemostatic peptide in the brain hemorrhage animal model, targeting the brain hemorrhage site

[0064] 12h after the collagenase brain hemorrhage modeling of rats, human blood thrombin (dose 10U / kg animal body weight) and the hemostatic peptide of the application (the final concentration of hemostatic peptide is 5mg / ml, the injection amount is 500μL / one, and one weighs about 250g) were injected into the tail vein of rats respectively, and each drug was labeled with rhodamine. 30min after administration, 400ml PBS was perfused in situ, the brain tissue was collected, quickly stored at-80℃, and a layer of OCT embedding glue was coated on the sample holder and quickly frozen for 10min. The tissue was placed on OCT, and a layer of OCT was coated on the tissue to completely cover the tissue, and frozen for 20min. The embedded tissue block was sliced to 10μm on a constant temperature freezing microtome.

[0065] Immunofluorescence observation:

[0066] After the frozen section was taken out and warmed at room temperature for 30min, it was immersed in PBS (0.01M) for 3 times, 3min each time; restain the nucleus: drop DAPI and incubate in the dark for 15min to stain the nucleus of the sample, and wash away the excess DAPI with PBS 5min*4 times; dry the liquid on the section with a water-absorbing paper, seal with a mounting medium containing an anti-fluorescence quencher, and then observe and collect images under a laser scanning confocal microscope, and the merge picture is as follows, the green light is the tissue autofluorescence, the red light is rhodamine (representing drug distribution), and the blue light is DAPI (representing the nucleus). The control group merge picture is mainly green and blue, and the red fluorescence is in the form of scattered points or weak flakes, without obvious regional enrichment. The hemostatic peptide group merge picture is in the form of large flakes with strong staining, and the overlapping area of the blue nucleus is concentrated in the hemorrhagic focus, and the green autofluorescence is covered by the red light. It shows that the hemostatic peptide of the application is specifically enriched in the hemorrhagic focus through the TF targeting mechanism, which is different from the diffuse distribution of the control group.

[0067] 2、Optimize the binding conformation, improve the targeting stability and efficiency

[0068] The linear sequence of the existing polypeptide technology (such as polypeptide drug conjugate PDCs) is easy to be degraded by plasma proteases, the circulation half-life is short (usually less than 30min), and the unstable binding conformation leads to low targeting efficiency.

[0069] The application improves the binding conformation with TF through sequence optimization, which may enhance the structural stability of the peptide chain, prolong the action time, and at the same time, better adapt to the active site of TF, improve the binding affinity, promote the rapid enrichment of the hemostatic peptide in the hemorrhagic focus, and accelerate the initiation of coagulation.

[0070] The strong fluorescence appeared 30 minutes after administration in the above case, which confirmed that TF was rapidly exposed after injury, and the hemostatic peptide of the application was not rapidly degraded by proteases.

[0071] 3. Adapt to the physiological mechanism of blood coagulation and enhance the specificity of hemostatic needles

[0072] The prior art (such as self-assembled nanofibers and light-responsive hydrogels) depends on physical barriers or non-specific activation of blood coagulation, and has insufficient targeting for patients with blood coagulation dysfunction or deep tissue bleeding.

[0073] The application directly targets the key initiator TF of the blood coagulation cascade, utilizes the exposure characteristics of TF after vascular injury, and accurately initiates the exogenous blood coagulation pathway (TF and FVIIa complex), is especially suitable for vascular injury type bleeding, and has stronger specificity for traumatic massive hemorrhage, surgical bleeding and the like, and more controllable hemostatic efficiency.

[0074] Rat liver hemorrhage model:

[0075] The experimental process is as follows:

[0076] Experimental animal grouping (5 rats in each group):

[0077] 1) Injury control group: only liver injury is made without intervention

[0078] 2) Human blood thrombin control group

[0079] 3) Hemostatic peptide intervention group of the application

[0080] Experimental process:

[0081] 3.1 Establishment of liver hemorrhage model

[0082] Anesthetize the rats, make a median incision (2-3 cm) under the xiphoid process, expose the left lateral lobe of the liver, and make a standardized incision on the edge of the left lobe of the liver with a scalpel: 5 mm long and 2 mm deep (penetrating the liver capsule to the parenchyma), immediately remove the bleeding caused by the blade. Observe the continuous bleeding of the liver wound and start timing.

[0083] 3.2 Intervention operation

[0084] Injury control group: only the wound is gently touched with sterile gauze (without pressing), and the natural hemostatic time (from the start of bleeding to the time when there is no visible bleeding on the wound, accurate to seconds) is recorded.

[0085] Human blood thrombin control group: human blood thrombin (dose 10 U / kg animal body weight) is injected through the tail vein 30 minutes later, and then the liver hemorrhage model is established, and the hemostatic time is recorded.

[0086] The hemostatic peptide intervention group of the application: 30 minutes after the tail vein injection of the hemostatic peptide of the application (the final concentration of the hemostatic peptide is 5 mg / ml, the injection amount is 500 μL per rat, and the weight of each rat is about 250 g), the liver hemorrhage model is established again, and the hemostasis time is recorded.

[0087] 3.3 Experimental results

[0088] The average hemostasis time of the liver of the damage control group of rats is 69 s, the average hemostasis time of the liver of the human blood thrombin control group of rats is 56 s, and the average hemostasis time of the liver of the hemostatic peptide intervention group of rats of the application is 43 s.

[0089] 4. Reduce system interference and improve biological safety

[0090] The prior art (such as recombinant thrombin) may cause systemic coagulation system activation and has a risk of distal thrombosis; unmodified polypeptides need a higher dose to take effect due to non-specific distribution, which may increase adverse reactions.

[0091] The application reduces systemic exposure through TF targeting, only enriches and plays a role at the bleeding site, reduces interference with the normal coagulation system, improves biological safety while ensuring hemostatic effect, and is especially suitable for clinical scenarios (such as patients with coagulation dysfunction) that require long-term or precise hemostasis.

[0092] The application is not limited to the above embodiments, and those skilled in the art can understand that various changes, modifications, replacements and variations can be made to these embodiments without departing from the principles and purposes of the application, and the scope of the application is defined by the claims and their equivalents.

Claims

1. A hemostatic peptide that can target bleeding lesions, characterized in that, Its amino acid sequence is: RKLFFVRTA.

2. A method for preparing the hemostatic peptide of claim 1, which can target bleeding lesions, characterized in that, Prepared according to the following method: First, the resin carrier is swollen and deprotected, and then reacted with the carboxyl group at the C-terminus of the first amino acid. After the first amino acid is attached to the resin carrier, the remaining amino acids are attached sequentially according to the designed peptide amino acid sequence. After all the synthesis procedures are completed, the peptide on the resin carrier is cut off. The synthesized peptide is purified, separated and purified by high performance liquid chromatography, and finally lyophilized to obtain the final product.

3. The method for preparing the hemostatic peptide that can target bleeding lesions according to claim 2, characterized in that: The resin is 4-methyldiphenylmethylamine resin.

4. Application of a hemostatic peptide that can target bleeding lesions in the preparation of hemostatic drugs.

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