Antithrombotic polypeptide PM4 and application thereof

Abstract

The present application belongs to the technical field of polypeptide preparation and biological medicine, and particularly relates to an antithrombotic polypeptide PM4 and application thereof. The present application is based on an original polypeptide obtained from a transcriptome of a leech salivary gland and an amino acid fragment cut from the original polypeptide, increases {d-Phe}-PRP at an N terminal of the cut amino acid fragment through a synthesis strategy of a hirudin analogue drug bivalirudin, and finally connects the {d-Phe}-PRP and the cut amino acid fragment through a connecting polypeptide to obtain the antithrombotic polypeptide PM4. The antithrombotic polypeptide PM4 can inhibit the activity of thrombin, has a small risk of bleeding, a shorter half-life in the body, higher safety, and significant resistance to thrombosis including arterial thrombosis. Meanwhile, the sequence of the antithrombotic polypeptide PM4 is shorter than the original sequence and is easier to synthesize.

Description

Technical Field

[0001] The present invention belongs to the field of polypeptide preparation and biomedicine technology, and specifically relates to an anti-thrombotic polypeptide PM4 and an application thereof. Background Art

[0002] A thrombus is a solid mass formed within a blood vessel, typically composed of platelets, red blood cells, white blood cells, and fibrin. Thrombotic diseases such as deep vein thrombosis (DVT), pulmonary embolism (PE), and arterial thrombosis (such as myocardial infarction and stroke) are significant public health issues worldwide.

[0003] Stroke, also known as cerebral infarction, carries extremely high rates of disability and mortality, placing a significant burden on families and society. Clinically, stroke is primarily categorized as ischemic and hemorrhagic. Ischemic stroke, also known as cerebral infarction, is the predominant clinical type of stroke, accounting for approximately 80% of all strokes.

[0004] Because ischemic stroke is caused by the combined effects of multiple factors, there is still no particularly effective treatment drug. Currently, the commonly used treatment methods in clinical practice include: thrombolytic therapy (the treatment time window is 3-6 hours, and many patients cannot receive timely treatment), fibrinolytic therapy, antiplatelet therapy, and anticoagulant therapy. Because thrombosis plays an important role in the pathogenesis of ischemic stroke, anticoagulant therapy has received increasing attention in the treatment and prevention of ischemic stroke. The anticoagulants currently used are mainly heparin and warfarin, but these two anticoagulants are not very effective in treating ischemic stroke and have a high risk of bleeding. Therefore, there is a need to develop more anticoagulant drugs.

[0005] Leeches feed on the blood of other animals, and the anticoagulant molecules in their saliva provide an important material basis for the development of anticoagulant drugs. Hirudin is a potent anticoagulant that effectively inhibits thrombin activity and prevents blood clotting. It binds to thrombin, preventing it from converting fibrinogen to fibrin, thereby slowing the blood clotting process. However, the bleeding risk of hirudin also limits its clinical application. Therefore, the development of hirudin-derived peptides or hirudin analogs is of great significance. Summary of the Invention

[0006] The purpose of the present invention is to provide an antithrombotic polypeptide PM4 and its application. The antithrombotic polypeptide PM4 has significant antithrombotic efficacy, low bleeding risk, is easy to synthesize, and has high safety.

[0007] The present invention provides an antithrombotic polypeptide PM4, which is derived from an original polypeptide through a synthesis strategy of Bivalirudin. The amino acid sequence of the original polypeptide is shown in SEQ ID NO: 1.

[0008] Preferably, the antithrombotic polypeptide PM4 comprises the amino acid sequence shown in SEO ID NO: 2, and has a d-phenylalanine modification (d-Phe) at the N-terminus of the amino acid sequence shown in SEO ID NO: 2.

[0009] The present invention also provides the use of the antithrombotic polypeptide PM4 described in the above technical solution in the preparation of antithrombotic drugs.

[0010] Preferably, the antithrombotic drug includes an anti-arterial thrombotic drug.

[0011] Preferably, the anti-arterial thrombotic drug includes an anti-stroke drug.

[0012] Preferably, the anti-stroke drug includes an anti-ischemic stroke drug.

[0013] Preferably, the antithrombotic drug includes a drug having the function of inhibiting thrombin activity and / or preventing and treating cerebral hemorrhage.

[0014] The present invention also provides an antithrombotic drug, the active ingredient of which includes the antithrombotic polypeptide PM4 described in the above technical solution.

[0015] Preferably, the antithrombotic polypeptide PM4 is the only active ingredient in the antithrombotic drug.

[0016] Preferably, the antithrombotic drug comprises pharmaceutically acceptable excipients.

[0017] Beneficial effects:

[0018] The present invention provides an anti-thrombotic polypeptide PM4. The present invention obtains an original polypeptide SEQ ID NO:1 from the transcriptome of the salivary gland of a leech, and extracts an amino acid sequence ENDFEEIPDEDIMN (SEQ ID NO:3) from the original polypeptide. {d-Phe}-PRP is added to the N-terminus of the extracted amino acid sequence through a synthesis strategy of a hirudin analog Bivalirudin drug. Finally, the {d-Phe}-PRP and the extracted amino acid sequence are connected via a linker polypeptide GGGG (SEQ ID NO:4) to obtain the anti-thrombotic polypeptide PM4. The {d-Phe}-PRP can specifically bind to thrombin, and a protease cleavage site is provided between RP. Therefore, the {d-Phe}-PRP-containing anti-thrombotic polypeptide PM4 is easily cleared from the body, has a reversible effect, and has a low risk of bleeding. The anti-thrombotic polypeptide PM4 can inhibit the activity of thrombin, has a shorter half-life in the body, and is safer. Furthermore, the sequence of the anti-thrombotic polypeptide PM4 is shorter than the original sequence, making it easier to synthesize. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments are briefly introduced below.

[0020] Figures 1-2 The results of the determination of the inhibitory activity of the antithrombotic polypeptide PM4 on thrombin in Example 2 are as follows;

[0021] Figure 3 The results of the determination of the thrombin inhibition constant of the antithrombotic polypeptide PM4 in Example 3 are as follows;

[0022] Figure 4 The results of the inhibitory effect of the antithrombotic polypeptide PM4 on activated partial thromboplastin time (APTT) in Example 4 are shown;

[0023] Figure 5 The results of the inhibitory effect of the antithrombotic polypeptide PM4 on prothrombin time (PT) in Example 4 are shown;

[0024] Figure 6 The results of the determination of the inhibitory effect of the antithrombotic polypeptide PM4 on FeCl3-induced carotid artery thrombosis in Example 5 are as follows;

[0025] Figure 7 The results of the determination of the inhibitory effect of the antithrombotic polypeptide PM4 on ischemic stroke in mice in Example 6 are as follows;

[0026] Figure 8 The bleeding risk test results of the antithrombotic polypeptide PM4 in Example 7;

[0027] Figure 9 The results of the cerebral hemorrhage activity test of the antithrombotic polypeptide PM4 in Example 8 are as follows;

[0028] Figure 10 This is the result of measuring the half-life of the antithrombotic polypeptide PM4 in vivo in Example 9. DETAILED DESCRIPTION

[0029] The present invention provides an antithrombotic polypeptide PM4, which is derived from an original polypeptide through a synthesis strategy of bivalirudin. The amino acid sequence of the original polypeptide is shown in SEQ ID NO: 1.

[0030] The original polypeptide of the present invention is obtained from the transcriptome of the salivary gland of the leech of the Philippine cattle leech, and the amino acid sequence of the original polypeptide is: VCICVSQAVSYTDCTSGQNYCLCGGNFCGGGKHCKMDGSGNQCVDGEGTPKPKSQTENDFEEIPDEDIMN (SEQ ID NO: 1). On the basis of the original polypeptide, an amino acid fragment ENDFEEIPDEDIMN (SEQ ID NO: 3) on the original polypeptide is intercepted. This amino acid sequence can bind to the fibrinogen binding site of thrombin and thus interfere with the activity of thrombin. On this basis, the present invention synthesizes the anti-thrombotic polypeptide PM4 through the synthesis strategy of the hirudin analog Bivalirudin drug (reference [Warkentin, TE, A. Greinacher, and A. Koster, Bivalirudin. Thromb Haemost, 2008.99(5): p.830-9.], specifically: {d-Phe}-PRP is added to the N-terminus of the ENDFEEIPDEDIMN amino acid fragment. {d-Phe}-PRP can specifically bind to thrombin and there is a protease cleavage site between RP, so the polypeptide is easy to clear in the body, the effect is reversible, and the bleeding risk is low; the ENDFEEIPDEDIMN amino acid fragment and {d-Phe}-PRP are connected by a polypeptide GGGG (SEQ ID NO:4) to obtain the antithrombotic polypeptide PM4: {d-Phe}-PRPG GGGENDFEEIPDEDIMN, wherein the amino acid sequence of PRPGGGGENDFEEIPDEDIMN is numbered as SEOID NO:2. The antithrombotic polypeptide PM4 of the present invention not only has the effect of inhibiting thrombin activity, the activity is reversible, the half-life is relatively short, and the bleeding risk is reduced, thereby achieving an antithrombotic effect, but also has a short sequence and is easy to chemically synthesize.

[0031] Based on the above advantages, the present invention also provides the use of the antithrombotic polypeptide PM4 described in the above technical solution in the preparation of an antithrombotic drug. In one embodiment, the antithrombotic drug includes an anti-arterial thrombotic drug; in another embodiment, the anti-arterial thrombotic drug includes an anti-stroke drug; in another embodiment, the anti-stroke drug includes an anti-ischemic stroke drug. In one embodiment, the antithrombotic drug includes a drug that inhibits thrombin activity and / or prevents and treats cerebral hemorrhage; in another embodiment, the prevention and treatment of cerebral hemorrhage includes reducing cerebral hemorrhage activity.

[0032] The present invention also provides an antithrombotic drug, the active ingredient of which includes the antithrombotic polypeptide PM4 described in the above technical solution. In one embodiment, the antithrombotic polypeptide PM4 is the sole active ingredient in the antithrombotic drug. In one embodiment, the antithrombotic drug includes a pharmaceutically acceptable excipient. The present invention does not specifically limit the type of excipient; the excipient may be selected based on the dosage form of the drug.

[0033] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below with reference to the accompanying drawings and embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0034] In the following examples, the solvents used to dissolve the antithrombotic polypeptide PM4 and bivalirudin were both physiological saline.

[0035] Example 1

[0036] An antithrombotic peptide PM4, {d-Phe}-PRPGGGGENDFEEIPDEDIMN, was synthesized by Hangzhou Gutuo Biotechnology Co., Ltd. through solid-phase synthesis.

[0037] Example 2

[0038] The antithrombotic polypeptide PM4 in Example 1 was tested for its inhibitory activity against thrombin in the following steps:

[0039] α-thrombin activity was measured using a luminescent substrate: thrombin (50 nM, Enzyme Research Laboratories) and the antithrombotic peptide PM4 (0-500 μg / mL, specifically 0, 4, 20, 100, and 500 μg / mL) were preincubated at 37°C for 10 min. After incubation, 0.5 mM luminescent substrate (S-2238, Chromogenix) was added to a 96-well plate to initiate the reaction. The reaction was continuously monitored at a wavelength of 405 nm for 30 min using an Epoch microplate reader (BioTek) and recorded. The results are shown in Figure 2. Figure 1 and Figure 2 As shown, in Figure 1 A in the vertical coordinate t and A0 represent the absorbance value at a certain time point and the initial absorbance value, respectively. Figure 2 In the vertical axis, A 30 and A0 represent the absorbance value at 30 min and the initial absorbance value, respectively; Figure 2 “*” and “***” indicate significant differences at P < 0.05 and P < 0.001, respectively.

[0040] Depend on Figure 1 and Figure 2It can be concluded that the antithrombotic peptide PM4 can inhibit the activity of thrombin in a concentration-dependent manner.

[0041] Example 3

[0042] The antithrombotic polypeptide PM4 in Example 1 was used to determine the thrombin inhibition constant. The experimental steps were as follows:

[0043] The luminescent substrate (S-2238) was set to different concentrations (50 μg / mL and 100 μg / mL) and the enzymatic kinetic reaction of the antithrombotic peptide PM4 on thrombin was repeated. The peptide concentration was then used as the horizontal axis and the inverse of the reaction rate was used as the vertical axis to plot the reaction rate. The reaction rate was measured at a wavelength of 405 nm. In order to calculate the inhibition constant Ki of the derivative peptide on thrombin, the Dixon plotting method was used to fit two curves by regression analysis, and the x value at the intersection of the two curves was calculated as the inhibition constant of the peptide on thrombin. The results are shown as follows: Figure 3 shown.

[0044] Depend on Figure 3 It can be concluded that the antithrombotic peptide PM4 has a strong inhibitory effect on thrombin, with an inhibition constant (Ki) of 0.72 μg / mL (295.6 nM).

[0045] Example 4

[0046] The inhibitory effect of the antithrombotic polypeptide PM4 in Example 1 on activated partial thromboplastin time (APTT) and prothrombin time (PT) was tested in the following steps:

[0047] Coagulation function tests (APTT and PT) were performed using kits (TC0306, Leagene, for APTT; GMS10176, Genmed, for PT) according to the manufacturer's instructions. The absorbance was measured at 650 nm. The results were shown in Table 1. Figure 4 and Figure 5 shown.

[0048] Depend on Figure 4 and Figure 5 It can be concluded that the antithrombotic peptide PM4 can inhibit the intrinsic coagulation pathway and the extrinsic coagulation pathway respectively.

[0049] Example 5

[0050] The inhibitory effect of the antithrombotic polypeptide PM4 on FeCl3-induced carotid artery thrombosis in Example 1 was carried out as follows:

[0051] Male C57BL / 6 mice (6-8 weeks) were anesthetized with sodium pentobarbital (80 mg / kg) and kept on a heating pad during the operation. Different doses of the antithrombotic peptide PM4 (1, 0.1 and 0.05 mg / kg) were injected through the tail vein 10 minutes before the operation. Normal saline was used as a negative control, and bivalirudin (1 mg / kg) was used as a positive control. The neck of the mouse was incised to expose the carotid artery, which was then separated from the vagus nerve and surrounding tissues. After a 2×2 mm piece of filter paper filled with 10% ferric chloride solution was attached to the exposed carotid artery, the mouse developed a thrombus, and a Doppler microvascular probe (RWD) was placed on the exposed artery to measure vascular blood flow. The results are shown in Figure 2. Figure 6 shown.

[0052] Depend on Figure 6 It can be concluded that the antithrombotic peptide PM4 has a significant inhibitory effect on FeCl3-induced carotid artery thrombosis, which further indicates that the antithrombotic peptide PM4 has good resistance to carotid artery thrombosis.

[0053] Example 6

[0054] The inhibitory effect of the antithrombotic polypeptide PM4 on ischemic stroke in mice in Example 1 was determined by the following steps:

[0055] Male C57BL / 6 mice (6-8 weeks) were anesthetized with sodium pentobarbital (80 mg / kg) and immobilized in the supine position on a thermostatic pad. The neck was prepared and disinfected with iodine. After midline skin incision, a 1-cm cut was made in the epidermis at the center of the cervical triangle to expose the subcutaneous tissue. The sternocleidomastoid muscle was bluntly dissected with curved forceps to expose the left common carotid artery (CCA), external carotid artery (ICA), and internal carotid artery. Tissue adhering to the vessels was gently peeled off to fully separate the vessels. Clamp the proximal common carotid artery and external carotid artery with a vascular clamp, tie surgical lines to the distal and proximal ends of the internal carotid artery respectively, tie a dead knot at the distal end of the internal carotid artery and a slipknot at the proximal end, use surgical scissors to cut an incision between the dead knot and the slipknot, insert a standard silicone rubber-coated nylon monofilament into the incision along the direction of the blood vessel, and tie the slipknot line tightly, cut the remaining blood vessels at the incision of the internal carotid artery, remove the vascular clamps of the common carotid artery and external carotid artery, flip the nylon monofilament and slowly insert it into the right internal carotid artery, and stop at the landmark. Occlude the right cerebral artery for about 60 minutes.

[0056] The experiment was divided into sham-operated groups, saline-treated groups, antithrombotic peptide PM4 groups (0.05, 0.1, and 1 mg / kg), and bivalirudin groups (positive control: 1 mg / kg). Drugs were injected into the tail vein 10 minutes before reperfusion. Reperfusion was performed 60 minutes later. The common carotid artery was clamped with a vascular clamp, the ligature was loosened, the nylon monofilament was slowly removed, and the vessel opening was ligated again. The mice were observed for bleeding during activity. If no bleeding occurred, the wound was sutured.

[0057] 24 hours after ischemia-reperfusion, mice were anesthetized with sodium pentobarbital, brain tissues were taken from mice, brain tissues were sliced ​​with brain slice molds, 2 mm thick coronal brain slices were collected, 2% 2,3,5-triphenyltetrazolium chloride (TTC, Sigma) staining solution was prepared in advance, the cut mouse brain tissue was placed in 2 mL 2% TTC staining solution, protected from light, placed in a 37 ° C incubator for staining, and waited for 15-25 minutes. After staining, the brain tissue was removed, the infarcted tissue was pale, and the normal tissue was dark red. Then a digital camera was used to take pictures of the slices to obtain the relative infarct volume rate. The results are as follows: Figure 7 shown.

[0058] Depend on Figure 7 It can be concluded that the anti-thrombotic peptide PM4 has a significant inhibitory effect on ischemic stroke in mice.

[0059] Example 7

[0060] The bleeding risk test of the antithrombotic polypeptide PM4 in Example 1 is performed as follows:

[0061] Tail bleeding activity assay: Male C57BL / 6J mice (6-8 weeks) were injected with different doses of the antithrombotic peptide PM4 (10, 2, and 0.4 mg / kg) via the tail 10 minutes before surgery. Normal saline was used as a negative control, and bivalirudin (10, 2, and 0.4 mg / kg) was used as a positive control. Subsequently, 2 mm of the tail was excised and carefully immersed in 20 mL of 37°C normal saline. Bleeding time was recorded until bleeding ceased. If bleeding resumed within 30 seconds, this time was also included in the bleeding time in this example. The results are shown in Figure 2. Figure 8 As shown in the middle left picture.

[0062] This example also measured the hemoglobin content of blood collected in 37°C constant temperature physiological saline. Blood (200 μL) from each group was incubated with 20 μL of 1% (v / v) Triton X-100 (Abcone) at 37°C for 3 minutes. After incubation, the cells were centrifuged at 1000 rpm for 5 minutes, and the absorbance of the supernatant was measured at 540 nm. The results are shown in Table 1. Figure 8 As shown in the middle right picture.

[0063] Depend on Figure 8It can be concluded that the antithrombotic peptide PM4 has a significantly lower risk of bleeding than bivalirudin at the same concentration.

[0064] Example 8

[0065] The cerebral hemorrhage activity of the antithrombotic polypeptide PM4 in Example 1 was determined in the following steps:

[0066] C57BL / 6J mice (6-8 weeks) were anesthetized and fixed in a stereotaxic apparatus (RWD Life Science, China), with body temperature maintained on a constant-temperature heating pad. The scalp, periosteum, and other associated soft tissues on the skull were removed. The skull was cleaned with 3% hydrogen peroxide solution and PBS. After the skull was dried, a circular opening (approximately 1 mm in diameter) was carefully drilled in the skull using a skull drill (RWD Life Science, China) (AP: -0.5, ML: -2.2, DV: -3.5). A microinjection device was controlled by a microinjection pump (STOELTINGCO, USA) to regulate the aspiration and injection of collagenase (Sigma-Aldrich, USA). Collagenase (0.6 mL, 0.075 U) was injected into the brain region at the target coordinate using a 10 μL Hamilton syringe at a rate of 0.06 μL / min. After each injection, the needle tip was slowly withdrawn to prevent collagenase spillage. After surgery, the antithrombotic peptide PM4 (10, 2, and 0.4 mg / kg), saline (negative control), and bivalirudin (10, 2, and 0.4 mg / kg, positive control) were injected through the tail vein. 24 hours later, the mice were deeply anesthetized (sodium pentobarbital, 80 mg / kg) and the brains of the mice were isolated and sliced ​​into 2 mm sections. The brain hemorrhage was recorded and photographed. The results are shown in Figure 2. Figure 9 shown.

[0067] Depend on Figure 9 It can be concluded that the risk of cerebral hemorrhage with the antithrombotic peptide PM4 is also significantly lower than that with bivalirudin.

[0068] Example 9

[0069] The steps for determining the in vivo half-life of the antithrombotic polypeptide PM4 in Example 1 are as follows:

[0070] The peptides (bivalirudin and antithrombotic peptide PM4) were administered intravenously to C57 male mice (20-22 g) at 10 mg / kg, and blood was collected from the orbital vein of the C57 male mice at different time points after the injection. The blood was placed in a centrifuge tube containing EDTA-K2 and centrifuged at 4°C, 3500 rpm for 10 minutes, and then the supernatant plasma was aspirated into a new centrifuge tube. Acetonitrile 6 times the volume of the plasma sample was added for protein precipitation, followed by vortexing for 2 minutes and centrifuging at 4°C, 13000 rpm for 15 minutes. LC-MS / MS (Waters, USA) was used for analysis, and the half-life was calculated using PKSolver based on the standard curve prepared based on different concentrations of peptides (bivalirudin and antithrombotic peptide PM4) dissolved in plasma. The results are shown as follows: Figure 10 shown.

[0071] Depend on Figure 10 It can be concluded that the half-life measured under the same conditions shows that the half-life of the antithrombotic peptide PM4 (9.12 min) is shorter than that of bivalirudin (16.04 min), so the bleeding risk is lower and the safety is better.

[0072] It can be concluded from the above examples that the antithrombotic polypeptide PM4 has significant antithrombotic efficacy, low bleeding risk, is easy to synthesize, and is highly safe.

[0073] Although the above embodiment provides a detailed description of the present invention, 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 scope of protection of the present invention.

Claims

1. An antithrombotic polypeptide PM4, characterized in that: The antithrombotic polypeptide PM4 is {d-Phe}-PRPGGGGENDFEEIPDEDIMN.

2. Use of the antithrombotic polypeptide PM4 according to claim 1 in the preparation of antithrombotic drugs.

3. The use according to claim 2, characterized in that The antithrombotic drugs include anti-arterial thrombotic drugs.

4. The use according to claim 3, characterized in that The anti-arterial thrombotic drugs include anti-ischemic stroke drugs.

5. The use according to any one of claims 2 to 4, characterized in that The anti-thrombotic drugs include drugs for preventing and treating cerebral hemorrhage.

6. An antithrombotic drug, characterized in that: The active ingredient of the antithrombotic drug includes the antithrombotic polypeptide PM4 according to claim 1.

7. The antithrombotic drug according to claim 6, characterized in that The antithrombotic polypeptide PM4 is the only active ingredient in the antithrombotic drug.

8. The antithrombotic drug according to claim 6 or 7, characterized in that: The antithrombotic drug includes pharmaceutically acceptable excipients.

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