Antithrombotic polypeptide PM4 and application thereof

Abstract

The invention belongs to the technical field of polypeptide preparation and biological medicine, and particularly relates to an antithrombotic polypeptide PM4 and application thereof. On the basis of original polypeptide obtained from transcriptome of salivary gland of poecilobdella manillensis, an amino acid fragment is intercepted from the original polypeptide, and {d-Phe}-PRP is added at the N end of the intercepted amino acid fragment through a synthetic strategy of hirudin analogue bivalirudin medicine; finally, the {d-Phe}-PRP and the intercepted amino acid fragment are connected through the connecting polypeptide to obtain the antithrombotic polypeptide PM4. The antithrombotic polypeptide PM4 can inhibit the activity of thrombin, is small in bleeding risk, shorter in in-vivo half-life period and higher in safety, and has remarkable resistance to thrombus including arterial thrombus; meanwhile, the sequence of the antithrombotic polypeptide PM4 is shorter than that of the original sequence, and the antithrombotic polypeptide PM4 is easier to synthesize.

Description

Technical Field

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

[0002] Thrombus refers to solid matter formed in blood vessels, usually composed of platelets, red blood cells, white blood cells and fibrin, etc. Thrombotic diseases such as deep vein thrombosis (DVT), pulmonary embolism (PE) and arterial thrombosis (such as myocardial infarction and stroke) are important public health problems worldwide.

[0003] Stroke is also known as apoplexy. It has a very high disability and mortality rate, which brings great burden to families and society. Stroke is clinically divided into ischemic stroke and hemorrhagic stroke. Ischemic stroke is also known as cerebral infarction, which is the main clinical type of stroke, accounting for about 80% of stroke.

[0004] Ischemic stroke is caused by the combined effects of multiple factors, and there is no particularly effective treatment drug so far. The commonly used treatment methods in clinical practice are: thrombolytic therapy (the treatment time window is 3-6 hours, and many patients cannot be treated in time), fibrinolytic therapy, antiplatelet therapy and anticoagulant therapy. Since thrombosis plays an important role in the onset 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 the risk of bleeding is relatively high, so more anticoagulant drugs need to be developed.

[0005] Leeches feed on the blood of other animals. The anticoagulant molecules in their saliva provide an important material basis for the development of anticoagulant drugs. Hirudin is a powerful anticoagulant that can effectively inhibit the activity of thrombin and prevent blood coagulation. It binds to thrombin and prevents it from converting fibrinogen into fibrin, thereby delaying the blood coagulation process. However, the bleeding risk of hirudin also limits its clinical application, so the development of hirudin-derived peptides or hirudin analogs is of great significance. Summary of the invention

[0006] The object 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, wherein the antithrombotic polypeptide PM4 is derived from an original polypeptide through a synthesis strategy of Bivalirudin, and 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 anti-thrombotic 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 effective 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 a pharmaceutically acceptable excipient.

[0017] Beneficial effects:

[0018] The invention provides an antithrombotic polypeptide PM4. The 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 the synthesis strategy of a hirudin analog Bivalirudin drug, and finally the {d-Phe}-PRP and the extracted amino acid sequence are connected through a connecting polypeptide GGGG (SEQ ID NO:4) to obtain the antithrombotic polypeptide PM4. The {d-Phe}-PRP can specifically bind to thrombin, and a protease cleavage site is provided between RP, so that the {d-Phe}-PRP is easily cleared in the antithrombotic polypeptide PM4 in vivo, the effect is reversible, and the risk of bleeding is small. The antithrombotic polypeptide PM4 can inhibit the activity of thrombin, has a shorter half-life in vivo, and is more safe. Meanwhile, the sequence of the antithrombotic polypeptide PM4 is shorter than the original sequence, and is 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 to be used 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;

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

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

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

[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;

[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;

[0026] Figure 8 The bleeding risk test result of the anti-thrombotic polypeptide PM4 in Example 7;

[0027] Fig. 9 The results of the cerebral hemorrhage activity test of the antithrombotic polypeptide PM4 in Example 8;

[0028] Fig.10 The results are for determining the half-life of the antithrombotic polypeptide PM4 in vivo in Example 9. DETAILED DESCRIPTION

[0029] The present invention provides an antithrombotic polypeptide PM4, wherein the antithrombotic polypeptide PM4 is derived from an original polypeptide through a synthesis strategy of bivalirudin, and 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 Philippine 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 ENDF EEIPDEDIMN amino acid fragment. {d-Phe}-PRP can specifically bind to thrombin and there is a protease cleavage site between RP. Therefore, the polypeptide is easy to clear in the body, the action is reversible, and the risk of bleeding 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 number of PRPGGGGENDFEEIPDEDIMN is SEOID NO: 2. The antithrombotic polypeptide PM4 of the present invention not only has the function of inhibiting thrombin activity, reversible activity, relatively short half-life and low bleeding risk, thereby achieving the antithrombotic effect, but also has a short sequence of the antithrombotic polypeptide PM4, which 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 antithrombotic drugs. As an embodiment, the antithrombotic drug includes a drug for anti-arterial thrombosis; as another embodiment, the anti-arterial thrombosis drug includes a drug for anti-stroke; as another embodiment, the anti-stroke drug includes a drug for anti-ischemic stroke. As an embodiment, the antithrombotic drug includes a drug with the function of inhibiting thrombin activity and / or preventing and treating cerebral hemorrhage; as another embodiment, the prevention and treatment of cerebral hemorrhage includes reducing cerebral hemorrhage activity.

[0032] The present invention also provides an antithrombotic drug, wherein the active ingredient of the antithrombotic drug includes the antithrombotic polypeptide PM4 described in the above technical solution. As an embodiment, the antithrombotic polypeptide PM4 is the only active ingredient in the antithrombotic drug. As an embodiment, the antithrombotic drug includes a pharmaceutically acceptable excipient. The present invention does not specifically limit the type of the excipient, and a conventional selection can be made according to 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 in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope 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 polypeptide PM4 is {d-Phe}-PRPGGGGENDFEEIPDEDIMN, which is synthesized by Hangzhou Gutuo Biotechnology Co., Ltd. through solid phase synthesis.

[0037] Example 2

[0038] The inhibitory activity of the antithrombotic polypeptide PM4 on thrombin in Example 1 was tested in the following steps:

[0039] The activity of α-thrombin was determined using a luminescent substrate: thrombin (50 nM, Enzyme Research Laboratories) and antithrombotic peptide PM4 (0-500 μg / mL, specifically 0, 4, 20, 100 and 500 μg / mL) were pre-incubated at 37°C for 10 min. After incubation, 0.5 mM luminescent substrate (S-2238, Chromogenix) was added to the 96-well plate to initiate the reaction, and 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 1 and Figure 2 As shown, in Figure 1 A 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 (P < 0.05) and (P < 0.001), respectively.

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

[0041] Example 3

[0042] The antithrombotic polypeptide PM4 in Example 1 is used to measure the thrombin inhibition constant. The test steps are 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, and then the peptide concentration was used as the horizontal axis and the inverse of the reaction rate was used as the vertical axis to plot. 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 through 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 in Figure 3 shown.

[0044] Depend on Figure 3 It can be concluded that the antithrombotic peptide PM4 has a strong inhibitory effect on thrombin, and the inhibition constant (Ki) is 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 according to the manufacturer's instructions using kits (TC0306, Leagene, for APTT; GMS10176, Genmed, for PT). 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 is 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 min 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 filter paper filled with 10% ferric chloride solution was attached to the exposed carotid artery, the mouse developed 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 is carried out in the following steps:

[0055] Male C57BL / 6 mice (6-8 weeks) were anesthetized with sodium pentobarbital (80 mg / kg). After complete anesthesia, the mice were fixed in the supine position and fixed on a constant temperature pad. The neck skin was prepared and disinfected with iodine. After the midline skin of the neck was incised, a 1 cm length of epidermis was cut in the middle of the neck triangle to expose the subcutaneous tissue. The sternocleidomastoid muscle was bluntly separated using curved forceps to expose the left common carotid artery (CCA), external carotid artery (ICA) and internal carotid artery. The tissues adhering to the blood vessels were gently peeled off to fully separate each blood vessel. Clamp the proximal common carotid artery and external carotid artery with vascular clamps, 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 along the direction of the blood vessel at the incision, and tighten the slipknot line, 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. The right cerebral artery is occluded for about 60 minutes.

[0056] The experiment was divided into sham operation group, saline group, antithrombotic peptide PM4 group (0.05, 0.1 and 1 mg / kg) and bivalirudin group (positive control: 1 mg / kg), and the drugs were injected into the tail vein 10 minutes before perfusion. Reperfusion was performed 60 minutes later, the common carotid artery was clamped with a vascular clamp, the tie line was loosened, the nylon monofilament was slowly removed, and then the vascular opening was ligated again. The bleeding was observed during the activity of the mice, and the wound was sutured after bleeding was absent.

[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 tissues were 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 detection of the anti-thrombotic polypeptide PM4 in Example 1 is carried out in the following steps:

[0061] Tail bleeding activity assay: Male C57BL / 6J mice (6-8 weeks) were injected with different doses of antithrombotic peptide PM4 (10, 2 and 0.4 mg / kg) through the tail 10 minutes before surgery, with saline as a negative control and bivalirudin (10, 2 and 0.4 mg / kg) as a positive control. Subsequently, 2 mm was cut off from the tail and carefully immersed in 20 mL of 37°C saline, and the bleeding time was recorded until the bleeding stopped. If bleeding occurred again within 30 seconds, this time would also be counted as the bleeding time in this example. The results are shown in Table 1. Figure 8 Shown in the middle left picture.

[0062] This example also measured the hemoglobin content of blood collected in 37°C constant temperature physiological saline. The blood collected from each group (200 μL) was incubated with 20 μL 1% (v / v) TritonX-100 (Abcone) at 37°C for 3 min. After incubation, centrifugation was performed at 1000 rpm for 5 min, 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 anti-thrombotic 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 is determined in the following steps:

[0066] C57BL / 6J mice (6-8 weeks) were anesthetized and fixed on a stereotaxic apparatus (RWD Life Science, China), and the body temperature of the mice was maintained with a constant temperature heating pad throughout the whole process. The scalp, periosteum, and other related 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 (about 1 mm in diameter) was carefully drilled on the skull (AP: -0.5, ML: -2.2, DV: -3.5) with a skull drill (RWD Life Science, China). The microinjection device was controlled by a microinjection pump (STOELTINGCO, USA) to regulate the inhalation and injection of collagenase (Sigma-Aldrich, USA). Collagenase (0.6 mL, 0.075 U) was injected into the brain area at the target coordinates at a rate of 0.06 μL / min using a 10 μL Hamilton syringe. After each injection, the needle tip was slowly withdrawn for 10 min to prevent collagenase from overflowing. 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 cut into 2 mm slices. The bleeding in the brain was recorded and photographed. The results are shown in Fig. 9 shown.

[0067] Depend on Fig. 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 (20-22g) at 10 mg / kg, and blood was collected from the orbital vein of 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, 3500rpm 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 centrifugation at 4°C, 13000rpm 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: Fig.10 shown.

[0071] Depend on Fig.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 risk of bleeding 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 has high safety.

[0073] 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. An antithrombotic polypeptide PM4, characterized in that: The antithrombotic polypeptide PM4 is derived from the original polypeptide through the synthesis strategy of bivalirudin, and the amino acid sequence of the original polypeptide is shown in SEQ ID NO:

1.

2. The antithrombotic polypeptide PM4 according to claim 1, characterized in that: The antithrombotic polypeptide PM4 includes the amino acid sequence shown in SEO ID NO: 2, and has d-phenylalanine modification at the N-terminus of the amino acid sequence shown in SEO ID NO:

2.

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

4. The use according to claim 3, characterized in that: The antithrombotic drugs include drugs for preventing arterial thrombosis.

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

6. The use according to claim 5, characterized in that: The anti-stroke drugs include anti-ischemic stroke drugs.

7. The use according to any one of claims 3 to 6, characterized in that: The anti-thrombotic drugs include drugs that have the function of inhibiting thrombin activity and / or preventing and treating cerebral hemorrhage.

8. An antithrombotic drug, characterized in that: The effective ingredient of the antithrombotic drug includes the antithrombotic polypeptide PM4 according to claim 1 or 2.

9. The antithrombotic drug according to claim 8, characterized in that: The antithrombotic polypeptide PM4 is the only active ingredient in the antithrombotic drug.

10. The antithrombotic drug according to claim 8 or 9, characterized in that: The antithrombotic drug comprises pharmaceutically acceptable excipients.

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