Polypeptide PM7 and application thereof in prevention and treatment of thrombus

Abstract

The invention belongs to the technical field of polypeptide synthesis and biological medicine, and particularly relates to a polypeptide PM7 and application thereof in prevention and treatment of thrombus. An original polypeptide is obtained from a transcriptome of salivary glands of poecilobdella manillensis, a truncated polypeptide is intercepted from the original polypeptide, and the truncated polypeptide can be combined with a binding site of fibrinogen of thrombin to intervene the activity of thrombin, so that {d-Phe}-PRP is added at the N end of the truncated polypeptide, and the activity of the thrombin is further intervened. The {d-Phe}-PRP and the truncated polypeptide are connected through a connecting polypeptide, and the polypeptide PM7 is obtained; the polypeptide PM7 has the activity of inhibiting thrombin, the half-life period in vivo is shorter, the safety is high, and the effect of preventing and treating thrombus is achieved.

Description

Technical Field

[0001] The present invention belongs to the field of polypeptide synthesis and biomedicine technology, and specifically relates to a polypeptide PM7 and its application in preventing and treating thrombosis. Background Art

[0002] Thrombi are small pieces of blood formed on the surface of the peeling or repairing of the inner surface of the cardiovascular system. In the variable fluid-dependent type, thrombi are composed of insoluble fibrin, deposited platelets, accumulated white blood cells and trapped red blood cells. Thrombotic diseases such as deep vein thrombosis (DVT), pulmonary embolism (PE) and arterial thrombosis (such as myocardial infarction and stroke).

[0003] Stroke, also known as apoplexy, has a high incidence rate. Stroke can be divided into ischemic stroke and hemorrhagic stroke, and ischemic stroke is the main clinical type of stroke, accounting for about 80% of stroke. Because ischemic stroke is caused by the combined action of multiple factors, there is no particularly effective treatment drug so far. Currently, the commonly used treatment methods in clinical practice are: thrombolytic therapy, 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 common anticoagulants heparin and warfarin currently used are not very effective in treating ischemic stroke, and the risk of bleeding is relatively high, so more anticoagulant drugs need to be developed.

[0004] The anticoagulant molecules contained in the saliva of blood-sucking leeches 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 high risk of bleeding caused by hirudin also limits its clinical application. Summary of the invention

[0005] The purpose of the present invention is to provide a polypeptide PM7 and its application in preventing and treating thrombosis, wherein the polypeptide PM7 has a good preventive and therapeutic effect on thrombosis and has a low risk of bleeding.

[0006] The present invention provides a polypeptide PM7, which comprises {d-Phe}-PRP, a connecting polypeptide and a truncated polypeptide, wherein the {d-Phe}-PRP, the connecting polypeptide and the truncated polypeptide are sequentially connected from the N-terminus to the C-terminus, and the truncated polypeptide comprises the amino acid sequence shown in SEQID NO:3.

[0007] Preferably, the amino acid sequence of the connecting polypeptide is as shown in SEQ ID NO:4.

[0008] The present invention also provides the use of the polypeptide PM7 described in the above technical solution in the preparation of medicines for preventing and treating thrombosis.

[0009] Preferably, the drug for preventing and treating thrombosis includes a drug having the function of inhibiting thrombin activity and / or preventing and treating cerebral hemorrhage.

[0010] Preferably, the drug for preventing and treating thrombosis includes a drug for preventing and treating arterial thrombosis.

[0011] Preferably, the medicine for preventing and treating arterial thrombosis includes medicine for preventing and treating cerebral stroke.

[0012] Preferably, the drugs for preventing and treating stroke include drugs for preventing and treating ischemic stroke.

[0013] The present invention also provides a medicine for preventing and treating thrombosis, comprising the polypeptide PM7 described in the above technical solution.

[0014] Preferably, the polypeptide PM7 is the only active ingredient in the medicine.

[0015] Preferably, the drug comprises pharmaceutically acceptable excipients.

[0016] Beneficial effects:

[0017] The present invention provides a polypeptide PM7, which comprises {d-Phe}-PRP, a connecting polypeptide and a truncated polypeptide, wherein the {d-Phe}-PRP, the connecting polypeptide and the truncated polypeptide are sequentially connected from the N-terminus to the C-terminus, and the truncated polypeptide comprises the amino acid sequence shown in SEQID NO: 3. The present invention obtains an original polypeptide from the transcriptome of the salivary gland of the Philippine leech, and truncates the original polypeptide, wherein the truncated polypeptide can bind to the binding site of the fibrinogen of thrombin and thus interfere with the activity of thrombin, and then {d-Phe}-PRP is added to the N-terminus of the truncated polypeptide, and the {d-Phe}-PRP is connected to the truncated polypeptide by a connecting polypeptide to obtain the polypeptide PM7; the polypeptide PM7 of the present invention has the activity of inhibiting thrombin, has a shorter half-life in the body, and has high safety, so as to achieve the effect of preventing and treating thrombosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] 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.

[0019] Figures 1-2 The results of the determination of the inhibitory activity of the polypeptide PM7 on thrombin in Example 2;

[0020] Figure 3 The results of the determination of the thrombin inhibition constant of the polypeptide PM7 in Example 3;

[0021] Figure 4 The results of the determination of the inhibitory effect of the polypeptide PM7 on the activated partial thromboplastin time (APTT) in Example 4;

[0022] Figure 5 The results of the determination of the inhibitory effect of the polypeptide PM7 on the prothrombin time (PT) in Example 4;

[0023] Figure 6 The results of the determination of the inhibitory effect of polypeptide PM7 on FeCl3-induced carotid artery thrombosis in Example 5;

[0024] Figure 7 The results of the determination of the inhibitory effect of the polypeptide PM7 on ischemic stroke in mice in Example 6;

[0025] Figure 8 The bleeding risk test result of polypeptide PM7 in Example 7;

[0026] Fig. 9 The results of the cerebral hemorrhage activity test of the polypeptide PM7 in Example 8;

[0027] Fig.10 The results are for determining the in vivo half-life of the polypeptide PM7 in Example 9. DETAILED DESCRIPTION

[0028] The present invention provides a polypeptide PM7, which comprises {d-Phe}-PRP, a connecting polypeptide and a truncated polypeptide, wherein the {d-Phe}-PRP, the connecting polypeptide and the truncated polypeptide are sequentially connected from the N-terminus to the C-terminus, and the truncated polypeptide comprises the amino acid sequence shown in SEQID NO:3.

[0029] The truncated polypeptide of the present invention is truncated from the original polypeptide SEQ ID NO: 1 obtained from the transcriptome of the salivary gland of the Philippine leech, and the amino acid sequence of the SEQ ID NO: 1 is specifically: CICVSQAAFYTQCTASGQNLCICEGDNVCTGGNRCVLGSSTSENRCIKGEGTP KPNQVQNDFEDFPEDAIEKK; the amino acid sequence of the truncated polypeptide is: NDFEDFPEDAIEKK; the truncated polypeptide can bind to the fibrinogen binding site of thrombin and thereby interfere with the activity of thrombin. On this basis, the present invention synthesizes the polypeptide PM7 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 truncated polypeptide, {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 action is reversible, and the risk of bleeding is small; further, {d-Phe}-PRP is connected to the truncated polypeptide through a connecting polypeptide to obtain the polypeptide PM; as an embodiment, the amino acid sequence of the connecting polypeptide is as shown in SEQ ID NO:4, specifically GGGG; when the connecting polypeptide is GGGG, the amino acid sequence of the polypeptide PM7 is: {d-Phe}-PRPGGGGNDFEDFPEDAIEKK, wherein the amino acid sequence of PRPGGGGNDFEDFPEDAIEKK is numbered as SEQ ID NO: 2. The polypeptide PM7 of the present invention has the function of inhibiting thrombin activity, reversible activity, short half-life and low bleeding risk, thereby achieving an anti-thrombotic effect. At the same time, the polypeptide PM7 sequence is short and easy to chemically synthesize.

[0030] Based on the above advantages, the present invention also provides the use of the polypeptide PM7 described in the above technical solution in the preparation of a drug for preventing and treating thrombosis. As an embodiment, the drug for preventing and treating thrombosis of the present invention can be a drug with the function of inhibiting thrombin activity and / or preventing and treating cerebral hemorrhage. As an embodiment, the drug for preventing and treating thrombosis can be a drug for preventing and treating arterial thrombosis; as another embodiment, the drug for preventing and treating arterial thrombosis can be a drug for preventing and treating carotid artery thrombosis; as another embodiment, the drug for preventing and treating arterial thrombosis can be a drug for preventing and treating cerebral stroke; as another embodiment, the drug for preventing and treating cerebral stroke can be a drug for preventing and treating ischemic stroke.

[0031] The present invention also provides a drug for preventing and treating thrombosis, comprising the polypeptide PM7 described in the above technical solution. As an embodiment, the polypeptide PM7 is the only active ingredient in the drug. As an embodiment, the drug includes pharmaceutically acceptable excipients. The present invention does not specifically limit the type of the excipient, and it can be conventionally selected according to the dosage form of the drug.

[0032] 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.

[0033] In the following examples, the solvents used to dissolve the polypeptide PM7 and bivalirudin were both physiological saline.

[0034] Example 1

[0035] A polypeptide PM7, {d-Phe}-PRPGGGGNDFEDFPEDAIEKK, was synthesized by Hangzhou Gutuo Biotechnology Co., Ltd. through solid phase synthesis.

[0036] Example 2

[0037] The detection of the inhibitory activity of the polypeptide PM7 on thrombin in Example 1 is carried out in the following steps:

[0038] The activity of α-thrombin was detected using a luminescent substrate. Thrombin (50 nM, Enzyme Research Laboratories) and peptide PM7 (0-500 μg / mL, specifically 0, 4, 20, 100 and 500 μg / mL) were pre-incubated at 37°C for 10 min, and then 0.5 mM substrate (S-2238, Chromogenix) was added to start 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 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), P<0.01 and P<0.001, respectively.

[0039] Depend on Figure 1 and Figure 2 It can be concluded that the peptide PM7 can inhibit the activity of thrombin in a concentration-dependent manner.

[0040] Example 3

[0041] The determination of the thrombin inhibition constant of the polypeptide PM7 in Example 1 is carried out in the following steps:

[0042] The luminescent substrate (S-2238) was set to different concentrations of 50 μg / mL and 100 μg / mL, and the inhibitory activity of peptide PM7 on thrombin was detected. The curve was drawn using the peptide concentration as the horizontal axis and the inverse of the reaction rate as the vertical axis. The inhibition constant Ki of the derivative peptide on thrombin was calculated using the Dixon plot method, and two curves were fitted by regression analysis. The x value at the intersection of the two curves is the inhibition constant of the peptide on thrombin. The results are shown in Figure 2. Figure 3 shown.

[0043] Depend on Figure 3 It can be concluded that the peptide PM7 has a strong inhibitory effect on thrombin, and the inhibition constant (Ki) is 3.25 μg / mL (1105.2 nM).

[0044] Example 4

[0045] The inhibitory effect of the polypeptide PM7 in Example 1 on activated partial thromboplastin time (APTT) and prothrombin time (PT) was tested in the following steps:

[0046] Coagulation function tests included APTT and PT tests, which were performed using kits (TC0306, Leagene, for APTT; GMS10176, Genmed, for PT) according to the manufacturer's instructions, and the absorbance was measured at 650 nm. The results were as follows: Figure 4 and Figure 5 shown.

[0047] Depend on Figure 4 and Figure 5 It can be concluded that peptide PM7 can inhibit the intrinsic coagulation pathway and the extrinsic coagulation pathway respectively.

[0048] Example 5

[0049] The inhibitory effect of the polypeptide PM7 in Example 1 on FeCl3-induced carotid artery thrombosis was tested in the following steps:

[0050] Male C57BL / 6 mice (6-8 weeks) were divided into peptide PM7 administration groups (1, 0.1 and 0.05 mg / kg), normal saline (negative control), and bivalirudin (1 mg / kg, positive control). The mice were anesthetized with sodium pentobarbital (80 mg / kg) and kept on a heating pad during the operation. The different administration groups were injected through the tail vein 10 minutes before the operation. 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 thrombosis, and a Doppler microvascular probe (RWD) was placed on the exposed artery to monitor the blood flow in the blood vessels. The results are shown in Figure 2. Figure 6 shown.

[0051] Depend on Figure 6 It can be concluded that the PM7 peptide has a significant inhibitory effect on FeCl3-induced carotid artery thrombosis, and further concluded that the PM7 peptide has a good anti-arterial thrombotic effect.

[0052] Example 6

[0053] The inhibitory effect of the polypeptide PM7 in Example 1 on ischemic stroke in mice was tested in the following steps:

[0054] Male C57BL / 6 mice (6-8 weeks) were divided into PM7-administered groups (1, 0.1 and 0.05 mg / kg), saline (negative control), bivalirudin (1 mg / kg, positive control) and sham-operated groups. Mice were anesthetized with sodium pentobarbital (80 mg / kg), and fixed in a supine position after complete anesthesia, and fixed on a constant temperature pad. The neck was prepared for skin disinfection with iodine tincture. 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 tissue adhering to the blood vessels was gently peeled off, and each blood vessel was fully peeled off. 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.

[0055] 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 tie was loosened, the nylon monofilament was slowly removed, and then the vascular opening was ligated again. The mouse was observed for bleeding during activity, and the wound was sutured if there was no bleeding.

[0056] 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 staining solution (TTC, Sigma) was prepared in advance, the cut mouse brain tissues were placed in 2 mL TTC, placed in a 37 °C incubator away from light, stained, and waited for 15 to 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 shown as follows: Figure 7 shown.

[0057] Depend on Figure 7 It can be concluded that the peptide PM7 has a significant inhibitory effect on ischemic stroke in mice.

[0058] Example 7

[0059] The bleeding risk detection of the polypeptide PM7 in Example 1, the experimental steps are as follows:

[0060] Tail bleeding activity assay: Male C57BL / 6 mice (6-8 weeks) were divided into PM7 polypeptide administration groups (10, 2 and 0.4 mg / kg), saline (negative control), and bivalirudin (10, 2 and 0.4 mg / kg, positive control). Different administration groups were injected through the tail 10 minutes before surgery. Subsequently, 2 mm was cut off from the tail and then carefully immersed in 20 mL of 37°C saline. 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 the figure. Figure 8 Middle left picture.

[0061] This example also measured the hemoglobin content in saline solution at 37°C. 200 μL of blood collected from each group was incubated with 20 μL of 1% (v / v) TritonX-100 (Abcone) at 37°C for 3 min. After incubation, the samples were centrifuged 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 Middle right picture.

[0062] Depend on Figure 8 The results show that the risk of bleeding is significantly reduced when compared with Bivalirudin at the same concentration.

[0063] Example 8

[0064] The cerebral hemorrhage activity of the polypeptide PM7 in Example 1 was determined by the following test steps:

[0065] 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 (~1 mm) 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, PM7 (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.

[0066] Depend on Fig. 9 It can be concluded that the risk of cerebral hemorrhage in PM7 is also significantly lower than that in bivalirudin.

[0067] Example 9

[0068] The in vivo half-life of the polypeptide PM7 in Example 1 was determined by the following experimental steps:

[0069] The peptides (Bivalirudin and Peptide PM7) 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, after which the supernatant plasma was drawn 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, 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 Peptide PM7) dissolved in plasma. The results are shown as follows: Fig.10 shown.

[0070] Depend on Fig.10 It can be concluded that the half-life of peptide PM7 (20.17 min) is longer than that of Bivalirudin (16.04 min), but both can be degraded by more than half in half an hour, so the risk of bleeding is smaller.

[0071] It can be concluded from the above examples that the polypeptide PM7 has a good preventive and therapeutic effect on thrombosis and has a low risk of bleeding.

[0072] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A polypeptide PM7, characterized in that: The polypeptide PM7 includes {d-Phe}-PRP, a connecting polypeptide and a truncated polypeptide, wherein the {d-Phe}-PRP, the connecting polypeptide and the truncated polypeptide are sequentially connected from the N-terminus to the C-terminus, and the truncated polypeptide includes the amino acid sequence shown in SEQ ID NO:

3.

2. The polypeptide PM7 according to claim 1, characterized in that The amino acid sequence of the connecting polypeptide is shown in SEQ ID NO:

4.

3. Use of the polypeptide PM7 according to claim 1 or 2 in the preparation of medicines for preventing and treating thrombosis.

4. The use according to claim 3, characterized in that The medicines for preventing and treating thrombosis include medicines that have the function of inhibiting thrombin activity and / or preventing and treating cerebral hemorrhage.

5. The use according to claim 3, characterized in that The medicines for preventing and treating thrombosis include medicines for preventing and treating arterial thrombosis.

6. The use according to claim 5, characterized in that The medicines for preventing and treating arterial thrombosis include medicines for preventing and treating cerebral stroke.

7. The use according to claim 6, characterized in that The medicines for preventing and treating stroke include medicines for preventing and treating ischemic stroke.

8. A drug for preventing and treating thrombosis, characterized in that: Comprising the polypeptide PM7 described in claim 1 or 2.

9. The medicine according to claim 8, characterized in that The polypeptide PM7 is the only active ingredient in the medicine.

10. The medicine according to claim 8 or 9, characterized in that: The medicine includes pharmaceutically acceptable excipients.

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