Supermolecular anticoagulant peptide and application thereof

Abstract

The invention discloses a supramolecular anticoagulant peptide and application thereof, and belongs to the technical field of medicines. The supramolecular anticoagulant peptide is constructed by adopting a peptide thrombin inhibitor, a self-assembly motif and a response motif of thrombosis related enzyme. The supramolecular anticoagulant peptide can form a semi-solid reservoir hydrogel in a physiological environment, and the supramolecular anticoagulant hydrogel not only has a long-acting anticoagulant characteristic, but also can realize accurate drug release according to the in-vivo thrombus formation level, and can significantly reduce the bleeding risk while ensuring the anticoagulant curative effect, thereby greatly improving the clinical medication safety, and having good clinical application prospects. And a new thought is developed for treatment of thromboembolic diseases.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular relates to a class of supramolecular anticoagulant peptides and applications thereof. Background Art

[0002] Thrombosis, a pathological process triggered by an imbalance in the coagulation and fibrinolytic systems, is a common causative factor in a variety of fatal cardiovascular diseases and plays a key role in the pathogenesis of conditions such as pulmonary embolism, acute coronary syndrome, and ischemic stroke. These cardiovascular diseases have become a major global public health challenge, with high morbidity and mortality. Anticoagulation has become a core strategy for the clinical prevention and treatment of thrombotic diseases. Hirudin, a peptide extracted from the salivary glands of leeches, is a direct thrombin inhibitor. In 2000, the FDA approved the recombinant hirudin derivative, bivalirudin (trade name Angiomax™), which has demonstrated excellent anticoagulant efficacy in a variety of conditions, including percutaneous coronary intervention, interventional treatment for acute coronary syndromes, and heparin-induced thrombocytopenia. However, bivalirudin has a half-life of 20-30 minutes in vivo, requiring continuous intravenous infusion to maintain efficacy. Although the risk of bleeding is lower than that of traditional heparin preparations, a key challenge in developing long-acting sustained-release preparations is the potential increased risk of unexpected bleeding. Therefore, developing a more convenient and safe anticoagulant drug delivery system is of great value in improving patient medication compliance, drug treatment safety, and clinical prognosis.

[0003] Micro-nanotechnology has become a very promising drug delivery platform, aiming to improve the treatment of various diseases including thrombotic diseases and reduce adverse reactions. For example, She et al. constructed a nanocarrier that can self-assemble into micelles by coupling DSPE-PEG (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide (polyethylene glycol)-2000]) with bivalirudin. This micellar formulation significantly extended the half-life of bivalirudin from 20 minutes to 210 minutes and improved the safety of the drug by reducing systemic exposure. In addition, the researchers developed a thin film engineering technology that can be used for anti-thrombotic coating by modifying bivalirudin (Biva) on the surface of a polymer-based stent. However, although a large number of studies have been devoted to improving the therapeutic effect and patient compliance of anticoagulant drugs, there is still an urgent need for the clinical development of a new anticoagulant preparation that is simple to prepare, has long-lasting anticoagulation and low bleeding risk. Summary of the Invention

[0004] One of the objectives of the present invention is to provide a class of supramolecular anticoagulant peptides that can self-assemble in situ to form hydrogels under physiological conditions and have prodrug properties, which can restore biological activity on demand and prolong the in vivo half-life of peptide thrombin inhibitors.

[0005] Specifically, the supramolecular anticoagulant peptide of the present invention is composed of three parts: a peptide thrombin inhibitor, a self-assembly motif, and a response motif of a thrombosis-related enzyme.

[0006] The peptide thrombin inhibitor can be any peptide thrombin inhibitor reported in the prior art, such as lepirudin, desirudin, bivalirudin, tsetse fly salivary gland extract (MKFFTVLFFLLSIIYLIVAAPGEPGAPIDYDEYGDSSEEVGGTPLHEIPGIRL), Rhodniin (EGGEPCACPHALHRVCGSDGETYSNPCTLNCAKFNGKPELVKVHDGPCEPDEDEDVCQE CDGDEYKPVCGSDDITYDNNCRLECASISSSPGVELKHEGPCRT), Ornithodorin (LNVLCNNPHTADCNNDAQVDRYFREGTTCLMSPACTSEGYASQHECQQACFVGGEDHS SEMHSSCLGDPPTSCAEGTDITYYDSDSKTCKVLAASCPSGENTFESEVECQVACGAPIEG), Variegin(SDQGDVAEPKMHKTAPPFDFEAIPEEYLDDES), Heamadin(IRFGMGKVPCPDGEVGYTCDCGEKICLYGQSCNDGQCSGDPKPSSEFEEFEIDEEEK), etc.

[0007] The self-assembly motif is a self-assembly peptide selected from the following sequences:

[0008] (RADA)n, n=4-20;

[0009] (EAK)n, n=4-20;

[0010] AEAEAKAKAEAEAKAK;

[0011] [(ME)n(MK)m]k,n=1-8, m=1-8, k=1-10;

[0012] VKVKVKVKV D PPTKVKVKVKV;

[0013] K(SL)6KGPRKLYDY;

[0014] KLDLPVGLIGKLDL;

[0015] VEVSVSVEV D PPTEVSVEVEVGGGGRGDV;

[0016] CGELENEVAQLEREVRSLEDEAAELEQKVSRLKNEIEDLKAE;

[0017] KSLSLSLRGSLSLSLLKGKLTWQELYQLKYKGI;GKYGFYTHVFRLKKWIQKVIDQFGE.

[0018] The thrombosis-related enzyme is coagulation factor VIIa (FVIIa), coagulation factor IXa (FIXa), coagulation factor Xa (FXa), coagulation factor XIa (FXIa), coagulation factor XIIa (FXIIa), coagulation factor XIIIa (FXIIIa) or thrombin.

[0019] Furthermore, the supramolecular anticoagulant peptide is composed of a self-assembly motif, a response motif of a thrombosis-related enzyme, and a peptide thrombin inhibitor connected in sequence from the N-terminus to the C-terminus.

[0020] In a specific embodiment of the present invention, the self-assembly motif is L-type and D-type (RADA)n peptide, which is a self-assembly building block composed of positively charged arginine (R), negatively charged aspartic acid (D) and hydrophobic alanine (A), and can self-assemble into β-pleated hydrogels.

[0021] A second object of the present invention is to provide the use of the supramolecular anticoagulant peptide in the preparation of anticoagulant products.

[0022] In order to improve the sustained-release effect of peptide thrombin inhibitors in vivo and prolong their half-life, the present invention is designed to construct them into hydrogel reservoirs. Specifically, the present invention uses three parts: peptide thrombin inhibitors, self-assembly motifs, and response motifs of thrombosis-related enzymes to construct supramolecular anticoagulant peptides. In preliminary experiments conducted in the early stage, the inventors found that introducing additional modifications to the N-terminus of the peptide thrombin inhibitor would lead to the loss of its biological activity. Therefore, it was designed to connect the self-assembly motif to the N-terminus of the peptide thrombin inhibitor; at the same time, the response motif of the thrombosis-related enzyme was introduced between the peptide thrombin inhibitor and the self-assembly motif.

[0023] In one embodiment of the present invention, the self-assembly motif is an L-type or D-type 32-peptide of 8 repeating units of RADA ( L -RADA)8 and ( D -RADA)8, the response motif of thrombosis-related enzymes is the response motif IEGR of coagulation factor FXa, and the peptide thrombin inhibitor is bivalirudin (Bivalirudin, Biva), the three are connected to each other to form a supramolecular anticoagulant peptide ( L -RADA)8-B and (D -RADA)8-B. Figure 1 As shown, after subcutaneous injection of the supramolecular anticoagulant peptide, (RADA)8-B hydrogel (including ( L -RADA)8-B hydrogel and ( D -RADA)8-B hydrogel), can form a semi-solid reservoir under physiological conditions. ( D -RADA)8-B hydrogel exhibits a longer in vivo retention time and can maintain longer-lasting anticoagulant activity. As the hydrogel slowly and continuously releases (RADA)8-B subcutaneously and diffuses into the blood. N-terminal modification can completely block the biological activity of Biva; inert (RADA)8-B circulates in the blood, and under the specific action of relevant enzymes at the lesion site, the response motif is recognized and cleaved by FXa, releasing Biva to exert anticoagulant activity. This supramolecular anticoagulant hydrogel can not only maintain long-term anticoagulant properties, but also achieve precise drug release according to the level of thrombosis in the body, significantly reducing the risk of bleeding while ensuring the anticoagulant effect, thereby greatly improving the safety of clinical medication and opening up new ideas for the treatment of thromboembolic diseases.

[0024] The present invention innovatively develops a new class of anticoagulants, whose molecular structure and mechanism of action are significantly different from traditional anticoagulant drugs (such as heparin, warfarin and hirudin, etc.). While maintaining long-term anticoagulant activity, it has high safety and provides a new solution for the treatment of thrombotic diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Schematic diagram of the supramolecular anticoagulant peptide hydrogel construction and drug release principle in Example 1.

[0026] Figure 2 To study the in vitro anticoagulant activity of Biva, Met-biva and Biva-met.

[0027] Figure 3 for( L -RADA)8-B solution and ( D -RADA)8-B solution after incubation with FXa.

[0028] Figure 4 for( L -RADA)8-B hydrogel and ( D Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) images of the 8-B hydrogel.

[0029] Figure 5 for( L -RADA)8-B hydrogel and ( D-RADA)8-B hydrogel rheological measurements in dynamic time sweep mode.

[0030] Figure 6 for( L -RADA)8-B hydrogel and ( D -RADA)8-B hydrogel incubation experiment with whole blood.

[0031] Figure 7 Rho-( L -RADA)8-B hydrogel and Rho-( D -RADA) Images of the in vitro degradation of 8-B hydrogels.

[0032] Figure 8 Cy5.5-( L -RADA)8-B hydrogel and Cy5.5-( D -RADA)8-B hydrogel residence time image in vivo.

[0033] Figure 9 Representative fluorescence images of ferric chloride-induced carotid artery embolism in mice 30 minutes and 1 day after administration to different treatment groups.

[0034] Figure 10 Representative fluorescence images of carotid artery embolism in mice induced by ferric chloride 2 hours after administration of different treatment groups

[0035] Figure 11 for( L -RADA)8-B and ( D Representative fluorescence images of carotid artery embolization in mice treated with (RADA)8-B hydrogel.

[0036] Figure 12 for( D Bleeding time of mice in the RADA)8-B hydrogel-treated group was measured. DETAILED DESCRIPTION

[0037] The preferred embodiments of the present invention will be described in detail below with reference to the examples. It should be understood that the following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0038] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0039] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0040] In the following examples, the synthetic peptide sequences are shown in Table 1.

[0041] Table 1 Synthetic peptide sequences

[0042]

[0043] Bivalirudin D F stands for D-phenylalanine; B stands for Bivalirudin, D Represents D-amino acids.

[0044] Example 1

[0045] This example designs a class of supramolecular anticoagulant peptides based on bivalirudin

[0046] 1. L-type and D-type prodrug peptides ( L -RADA)8-B and ( D Synthesis of -RADA)8-B

[0047] Modification of the N-terminus of hirudin can disrupt its interaction with the thrombin active pocket, thereby significantly affecting the activity of hirudin. Currently, there are few reports on the effects of Biva terminal modification. The present invention introduces a methionine (met) at the N-terminus and C-terminus of Biva, respectively, to obtain Met-biva and Biva-met, respectively, to explore the effect of terminal extension on their biological activity. The anticoagulant activity of different Biva-derived peptides (Met-biva and Biva-met) was evaluated using a chromogenic substrate method. Specifically: 40 μL of Met-biva or Biva-met was mixed with 40 μL of thrombin (2 U / mL) and incubated at 37°C for 5 minutes; then 40 μL of chromogenic substrate S2238 (1 mM) was added and incubated for another 10 minutes; the absorbance of the reaction solution at 405 nm was detected by a microplate reader Infinite Pro M200 (Tecan, Switzerland).

[0048] like Figure 2 As shown, free Biva exhibits strong anticoagulant activity. Introducing a single methionine at the C-terminus of Biva has no effect on its biological activity, while introducing a methionine at the N-terminus results in a loss of biological activity, indicating that Biva's anticoagulant activity requires exposure of the N-terminus. This finding lays a solid foundation for the design of Biva-derived anticoagulant prodrugs.

[0049] The supramolecular anticoagulant peptide of this embodiment ( L -RADA)8-B and ( D -RADA)8-B includes Biva and L-type or D-type self-assembly motifs ( L -RADA)8 and ( D-RADA)8, in which Biva and the self-assembly motif are connected by a FXa enzyme responsive motif containing a 4-peptide IEGR. At the same time, four glycine (GGGG) as the linker ( D -RADA)8-GB, as ( D -RADA)8-B non-responsive control group. The structure of the supramolecular anticoagulant peptide prepared by solid phase peptide synthesis is shown below:

[0050]

[0051] 2.( L -RADA)8-B solution and ( D Study on the anticoagulant activity of -RADA)8-B solution

[0052] The in vitro anticoagulant activity of the supramolecular anticoagulant peptide was determined by thrombin titration. Specifically: 200 μL of 0.5% fibrinogen and 10 μL of supramolecular anticoagulant peptide were added to a 96-well plate in sequence and gently mixed. After 5 minutes, 5 μL of thrombin standard solution (100 NIH / mL) was added to each well each time and gently mixed. The formation of a clot within 1 minute was considered to be the termination of the reaction. The number of additions of the thrombin standard solution was recorded until the reaction was terminated. In order to evaluate the FXa-responsive bioactivity recovery of the fusion protein, the sample was pretreated with FXa before the addition of the fibrinogen solution. After incubation for a predetermined time interval, thrombin titration was performed. Observe whether a clot was formed within 1 minute and record the number of additions of the thrombin standard solution.

[0053] like Figure 3 As shown in Figure 2, at 0 hours, the supramolecular anticoagulant peptide had almost no anticoagulant activity, but as time went by, ( L -RADA)8-B solution and ( D -RADA)8-B solution's anticoagulant activity increased continuously and reached its maximum after 12 hours of incubation. L -RADA)8-B and ( D -RADA)8-B had no significant difference in the recovery rate of biological activity. D -RADA)8-GB had almost no anticoagulant activity after incubation with FXa.

[0054] 3.( L -RADA)8-B and ( D In vitro characterization of 8-B hydrogels

[0055] The hydrogel was prepared by using the salt ion triggered supramolecular anticoagulant peptide self-assembly method. (RADA)8-B was dissolved in 0.9% sodium chloride solution and allowed to stand at room temperature for 10 minutes to form a hydrogel. The final concentration of (RADA)8-B was 1% (w / v). Figure 4 As shown, there is no liquid flow in both L-type and D-type (RADA) 8-B, indicating that a stable hydrogel has been formed. The microstructure of (RADA) 8-B hydrogel was observed by transmission electron microscopy and cryo-scanning electron microscopy. Transmission electron microscopy images show that L-type and D-type hydrogels form uniform nanofibers, which are entangled with each other to form a network structure. Scanning electron microscopy images show that ( L -RADA)8-B hydrogel and ( D -RADA) The porous structure inside the 8-B hydrogel.

[0056] The mechanical properties of the two (RADA)8-B hydrogels were measured using a rheometer in a dynamic time sweep mode, as shown in Figure 2. Figure 5 The storage modulus G' of the two (RADA)8-B hydrogels was higher than the loss modulus G", indicating the successful preparation of the hydrogels; and ( D -RADA)8-B hydrogel has higher storage modulus and loss modulus than ( L -RADA)8-B hydrogel, description ( D- RADA)8-B hydrogel has strong mechanical capacity.

[0057] 4.( L -RADA)8-B and ( D In vitro anticoagulant activity assay of 8-B hydrogel

[0058] The in vitro anticoagulant activity of the two hydrogels was tested by whole blood incubation experiments. L -RADA)8-B hydrogel, ( D -RADA)8-B hydrogel or Biva solution, and add freshly collected whole blood to each well. Whole blood and Biva solution, ( L -RADA)8-B hydrogel, ( D -RADA)8-B hydrogel did not solidify after incubation ( Figure 6 ), while the blood in the physiological salt group was completely coagulated. After incubation for 1 hour, the uncoagulated blood was removed and fresh blood was added to continue incubation for another 1 hour to detect ( L -RADA)8-B hydrogel, ( D -RADA)8-B hydrogel anticoagulant effect is exhausted. In the second incubation, L -RADA)8-B hydrogel, ( D-RADA)8-B hydrogel pre-treated blood remained in solution, while the blood in the Biva-treated group coagulated due to the exhaustion of the anticoagulant Biva solution. D -RADA)8-B hydrogel remains in liquid form when in contact with blood, while ( L -RADA)8-B hydrogel pre-treated wells have coagulated. D -RADA)8-B hydrogel compared with ( L -RADA)8-B hydrogel has a longer-lasting anticoagulant activity.

[0059] 5.( L -RADA)8-B hydrogel and ( D In vitro degradation of 8-B hydrogels

[0060] The presence of proteolytic enzymes in the dermis and subcutaneous tissue is the main reason for the poor bioavailability of protein drugs administered via intradermal and subcutaneous routes. In order to investigate the degradation effect of proteolytic enzymes on two different chiral hydrogels, L -RADA)8-B hydrogel and ( D -RADA)8-B hydrogel was placed at the bottom of a glass bottle and 1 mL of proteinase K solution (5 U / mL) was added. Incubate with proteinase K at 37°C. After a predetermined time, the supernatant was removed and an equal volume of fresh proteinase K solution was added. The results were recorded and photographed. L -RADA)8-B and ( D -RADA)8-B hydrogel in vitro degradation optical image. Figure 7 As shown, ( L -RADA)8-B hydrogel degraded significantly faster than ( D -RADA)8-B, which suggests that the D-amino acids in the assembly motif confer D -RADA)8-B hydrogel has higher stability.

[0061] The above experimental results show that ( D -RADA)8-B hydrogel exhibited strong mechanical strength, in vitro anticoagulant activity and stability.

[0062] 6.( L -RADA)8-B hydrogel and ( D -RADA)8-B hydrogel in vivo residence time investigation

[0063] Biva or (RADA) 8-B was dissolved in PBS (pH 7.4) solution and reacted with 10 equivalents of Cy5.5-NHS at room temperature for 2 hours. The reaction solution was placed in a dialysis bag and dialyzed against PBS (pH 7.4) solution for 24 hours to remove unreacted fluorescent dye, thereby obtaining a fluorescently labeled protein solution. Fluorescently labeled hydrogels were prepared according to the above method. Mice were subcutaneously injected with Cy5.5-labeled ( L -RADA)8-B and ( D -RADA)8-B hydrogel (0.0136 mmol / kg), and the images of mice were captured at predetermined time points by the in vivo imaging system IVIS.

[0064] like Figure 8 As shown in the figure, the fluorescence signal of Cy 5.5 in the Cy5.5-Biva solution group decayed rapidly over time and was almost undetectable on the second day. This is presumably because Biva has a short half-life in the body and is quickly cleared after subcutaneous injection. L -RADA)8-B hydrogel group mice injected with Cy5.5 fluorescence signal can be maintained for more than 4 days, while Cy5.5-( D -RADA)8-B group can maintain for 7 days, indicating that the hydrogel can effectively prolong the retention time of Biva. D -RADA)8-B hydrogel has strong mechanical strength and stability, which effectively prolongs the retention time of the hydrogel under the skin.

[0065] 7.( L -RADA)8-B and ( D -RADA)8-B hydrogel in vivo anticoagulant activity

[0066] By establishing a ferric chloride-induced carotid artery thrombosis model in mice, we investigated ( L -RADA)8-B hydrogel and ( D -RADA)8-B hydrogel in vivo anticoagulant effect. First, rhodamine 6G solution (0.5 mg / kg) was injected into the tail vein. After anesthetizing the C57BL / 6 mice, the left carotid artery of the mice was surgically isolated. The carotid artery was covered with filter paper soaked in 5% ferric chloride solution for 1 minute to induce carotid thrombosis. The carotid thrombosis of the mice was observed under a fluorescence microscope. Ferric chloride-induced carotid artery injury will lead to the aggregation of rhodamine 6G-labeled platelets. The area and intensity of the red fluorescent signal indicate the degree of thrombosis. Subcutaneous injection of normal saline, Biva solution, Met-biva solution, ( L -RADA)8-B hydrogel and ( D-RADA) 8-B hydrogel (0.0136mmol / kg). After ferric chloride induction, obvious red fluorescence signals were observed in the carotid arteries of the saline group and the Met-biva group within 20 minutes. In Biva-treated mice, almost no thrombosis was formed 0.5 hours after administration. However, due to the rapid clearance of Biva solution in the body, thrombosis was not inhibited 1 day after administration, indicating that Biva solution can only provide a short-term thrombosis inhibition effect ( Figure 9 ). ( L -RADA)8-B and ( D -RADA)8-B group began to effectively inhibit thrombosis 2 hours after administration ( Figure 10 ) and continued to exert anticoagulant effect within 1 day. In addition, the in vivo antithrombotic activity of (RADA)8-B was monitored for one week. Figure 11 As shown. ( D -RADA)8-B group showed almost no red fluorescence signal within 7 days, compared with ( L -RADA)8-B showed a more persistent antithrombotic effect, indicating that ( D -RADA)8-B hydrogel can effectively inhibit thrombosis within one week.

[0067] 8.( D -RADA)8-B hydrogel in vivo safety evaluation

[0068] For the bleeding time test, mice were intravenously injected with saline, Biva and ( D -RADA)8-B solution (0.0073mmol / kg), and 10 minutes after administration, the tail of the mice was transected and the bleeding time was recorded. Figure 12 As shown, the Biva treatment group resulted in prolonged bleeding time, whereas ( D There was no significant difference in bleeding time between the RADA8-B solution group and the normal saline group, indicating that ( D -RADA)8-B circulates in an inert form, shielding the anticoagulant activity of Biva and thus reducing the risk of bleeding.

Claims

1. A supramolecular anticoagulant peptide, characterized in that: The supramolecular anticoagulant peptide comprises a peptide thrombin inhibitor, a self-assembly motif and a response motif of an enzyme related to thrombosis; The self-assembly motif is a self-assembling peptide.

2. The supramolecular anticoagulant peptide according to claim 1, characterized in that The supramolecular anticoagulant peptide is sequentially composed of a self-assembly motif, a response motif of a thrombosis-related enzyme, and a peptide thrombin inhibitor from the N-terminus to the C-terminus.

3. The supramolecular anticoagulant peptide according to claim 1 or 2, characterized in that The peptide thrombin inhibitor is selected from the group consisting of lepirudin, desirudin, bivalirudin, tsetse fly salivary gland extract, rhodniin, ornithodorin, variegin, and heamadin.

4. The supramolecular anticoagulant peptide according to claim 1 or 2, characterized in that The self-assembling peptide is selected from the following sequences: (RADA) n, n=4-20; (EAK) n, n = 4-20; AEAEAKAKAEAEAKAK; [(ME)n(MK)m]k, n=1-8, m=1-8, k=1-10; VKVKVKVKV D PPTKVKVKVKV; K(SL)6KGPRKLYDY; KLDLPVGLIGKLDL; VEVSVSVEV D PPTEVSVEVEVGGGGRGDV; CGELENEVAQLEREVRSLEDEAAELEQKVSRLKNEIEDLKAE; KSLSLSLRGSSLSLLKGKLTWQELYQLKYKGI; GKYGFYTHVFRLKKWIQKVIDQFGE.

5. The supramolecular anticoagulant peptide according to claim 1 or 2, characterized in that The thrombosis-related enzyme is coagulation factor VIIa, coagulation factor IXa, coagulation factor Xa, coagulation factor XIa, coagulation factor XIIa, coagulation factor XIIIa or thrombin.

6. Use of the supramolecular anticoagulant peptide according to any one of claims 1 to 5 in the preparation of anticoagulant products.

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