A Derivative Peptide PM6 and Its Application in Antithrombosis

Abstract

This invention belongs to the field of polypeptide synthesis and biomedical technology, specifically relating to a derived polypeptide PM6 and its application in antithrombosis. The invention obtains the original polypeptide SEQ ID NO:1 from the transcriptome of the salivary glands of *Hirudo medicinalis*, and then obtains a truncated polypeptide as shown in SEQ ID NO:3. Based on this, using a synthetic strategy similar to that of the hirudin analog *Bivalirudin*, {d-Phe}-PRP is added to the N-terminus of the truncated amino acid sequence. The {d-Phe}-PRP is then linked to the truncated polypeptide using a linker polypeptide to obtain the derived polypeptide PM6. The derived polypeptide PM6 of this invention exhibits good antithrombotic activity, and its bleeding risk is comparable to that of the positive control drug bilivadin.

Description

Technical Field

[0001] This invention belongs to the field of polypeptide synthesis and biomedicine technology, specifically relating to a derived polypeptide PM6 and its application in antithrombosis. Background Technology

[0002] A thrombus is a small clot that forms on the surface of a blood vessel in the cardiovascular system at a site of rupture or repair. It is composed of insoluble fibrin, deposited platelets, and accumulated white blood cells and red blood cells. Normally, blood has coagulation properties, helping the body to coagulate and stop bleeding in cases of injury or bleeding. However, if the balance of the coagulation-anticoagulation mechanism is disrupted during the coagulation process, thrombus formation can occur. Thrombotic diseases such as deep vein thrombosis (DVT), pulmonary embolism (PE), and arterial thrombosis (such as myocardial infarction and stroke) have become one of the most important disease types of concern globally.

[0003] Hirudin, secreted by blood-sucking leeches, is a potent anticoagulant that effectively inhibits thrombin activity, preventing blood clotting. It works by binding to thrombin, preventing the conversion of fibrinogen into fibrin, thereby delaying the blood clotting process. However, some existing studies have found that hirudin carries a bleeding risk, thus limiting its clinical application. Therefore, finding a substance that can replace the function of hirudin while having a lower bleeding risk is an urgent problem to be solved in this field. Summary of the Invention

[0004] The purpose of this invention is to provide a derived polypeptide PM6 and its application in antithrombosis, wherein the derived polypeptide PM6 has a significant antithrombotic effect and the bleeding risk is comparable to that of the positive drug bilivadin.

[0005] The present invention provides a derived polypeptide PM6, which is derived from the truncated polypeptide shown in SEQ ID NO:3 through a bivalirudin synthesis strategy.

[0006] Preferably, the derived polypeptide PM6 comprises, from the N-terminus to the C-terminus, a {d-Phe}-PRP, a linker polypeptide, and a truncated polypeptide linked sequentially; the amino acid sequence of the truncated polypeptide is shown in SEQ ID NO:3.

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

[0008] This invention also provides the application of the derived polypeptide PM6 described in the above technical solution in the preparation of antithrombotic products.

[0009] Preferably, the antithrombotic product includes products that treat arterial thrombosis.

[0010] Preferably, the anti-arterial thrombosis product includes products for treating carotid artery thrombosis.

[0011] Preferably, the antithrombotic product includes products that inhibit thrombin activity and / or prevent cerebral hemorrhage.

[0012] Preferably, the product includes pharmaceuticals.

[0013] The present invention also provides an antithrombotic drug, the active ingredient of which includes the derivative polypeptide PM6 described in the above technical solution.

[0014] Preferably, the only active ingredient in the antithrombotic drug is the derived polypeptide PM6.

[0015] Preferably, the antithrombotic drug includes pharmaceutically acceptable excipients.

[0016] Beneficial effects:

[0017] This invention provides a derived polypeptide PM6, which is derived from the truncated polypeptide shown in SEQ ID NO:3 using a bivalirudin synthesis strategy. The invention obtains the original polypeptide SEQ ID NO:1 from the transcriptome of the salivary glands of *Hirudo medicinalis*, and obtains the truncated polypeptide shown in SEQ ID NO:3 on the original polypeptide. Based on this, a {d-Phe}-PRP is added to the N-terminus of the truncated amino acid sequence using a bivalirudin analogue synthesis strategy. The {d-Phe}-PRP is then linked to the truncated polypeptide using a linker polypeptide to obtain the derived polypeptide PM6. The derived polypeptide PM6 of this invention exhibits good antithrombotic activity, and its bleeding risk is comparable to that of the positive control drug bivalirudin. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0019] Figures 1-2 The results show the assay results of the inhibitory activity of the derived polypeptide PM6 against thrombin in Example 2;

[0020] Figure 3 The results of the determination of the thrombin inhibition constant of the derived polypeptide PM6 in Example 3;

[0021] Figure 4 The results show the inhibitory effect of the derived polypeptide PM6 in Example 4 on activated partial thromboplastin time (APTT).

[0022] Figure 5 The results show the inhibitory effect of the derived polypeptide PM6 on prothrombin time (PT) in Example 4.

[0023] Figure 6 The results show the inhibitory effect of the derived polypeptide PM6 in Example 5 on FeCl3-induced carotid artery thrombosis.

[0024] Figure 7 This is a graph showing the bleeding risk detection results of the derived polypeptide PM6 in Example 6. Detailed Implementation

[0025] The present invention provides a derived polypeptide PM6, which is derived from the truncated polypeptide shown in SEQ ID NO:3 through a bivalirudin synthesis strategy.

[0026] This invention obtains the original polypeptide SEQ ID NO:1 from the transcriptome of the salivary glands of *Hirudo medicinalis*, specifically SVSSENISKMFSLKLFGVFLAVCICVSQAMRYTACTESGQNQCICEGNDVCGQ GRNCQFDSSGKKCVEGEGTHKPQNKGQNDFEPIPEDYFS. Based on this original polypeptide, a portion of the peptide is truncated to obtain a shortened polypeptide, the amino acid sequence of which is shown in SEQ ID NO:3, specifically: NDFEPIPEDYFS. This shortened polypeptide can bind to the fibrinogen binding site of thrombin, thereby interfering with thrombin activity. Based on this, the present invention synthesizes the derived polypeptide PM6 using the synthetic strategy of the hirudin analog Bivalirudin (reference [Warkentin, TE, A. Greinacher, and A. Koster, Bivalirudin. Thromb Haemost, 2008. 99(5): p. 830-9.]). Specifically, two fragments {d-Phe}-PRP are added to the N-terminus of the NDFEPIPEDYFS amino acid fragment. The {d-Phe}-PRP can specifically bind to thrombin, and the RPs are protease cleavage sites. Therefore, the polypeptide is easily cleared in vivo, its effects are reversible, and the risk of bleeding is low. Finally, a linker polypeptide is used to connect {d-Phe}-PRP and NDFEPIPEDYFS to form the derived polypeptide PM6. In one embodiment, the amino acid sequence of the linker polypeptide is as shown in SEQ ID NO:4, specifically GGGG. When the amino acid sequence of the linker polypeptide is GGGG, the amino acid sequence of the derived polypeptide PM6 is specifically {d-Phe}-PRPGGGGNDFEPIPEDYFS, where the amino acid sequence number of PRPGGGGNDFEPIPEDYFS is SEQ ID NO:2. The antithrombotic polypeptide of this invention not only inhibits thrombin activity, has reversible activity, and a relatively short half-life, thus achieving an antithrombotic effect, but its bleeding risk is comparable to that of the positive control drug bivalirudin. Furthermore, the derived polypeptide PM6 has a short sequence, making it easy to synthesize chemically.

[0027] Based on the above advantages, the present invention also provides the application of the derived polypeptide PM6 described in the above technical solution in the preparation of antithrombotic products. As one embodiment, the antithrombotic product can be an anti-arterial thrombotic product; as another embodiment, the anti-arterial thrombotic product can be an anti-carotid artery thrombotic product. As one embodiment, the antithrombotic product can be a product with the function of inhibiting thrombin activity and / or preventing cerebral hemorrhage. As one embodiment, the product includes a pharmaceutical product.

[0028] This invention also provides an antithrombotic drug, the active ingredient of which includes the derived polypeptide PM6 described in the above-mentioned technical solution. In one embodiment, the sole active ingredient in the antithrombotic drug is the derived polypeptide PM6. In another embodiment, the antithrombotic drug includes pharmaceutically acceptable excipients. This invention does not specifically limit the type of excipients; conventional selection based on the drug's dosage form is sufficient.

[0029] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0030] In the following examples, the solvent used to dissolve the derived polypeptide PM6 and bivalirudin was physiological saline.

[0031] Example 1

[0032] A derivative polypeptide PM6, {d-Phe}-PRPGGGGNDFEPIPEDYFS, was obtained by Hangzhou Gutuo Biotechnology Co., Ltd. via solid-phase synthesis.

[0033] Example 2

[0034] The determination of the inhibitory activity of the derived polypeptide PM6 against thrombin in Example 1 was performed as follows:

[0035] The inhibitory activity of the derived peptide PM6 against α-thrombin was determined using a luminescent substrate. The derived peptide PM6 (0–500 μg / mL, specifically 0, 4, 20, 100, and 500 μg / mL) was pre-incubated with α-thrombin (50 nM, Enzyme Research Laboratories) at 37 °C for 10 min. Then, 0.5 mM of substrate (S-2238, Chromogenix) was added to 96-well plates to initiate the reaction. The reaction was continuously monitored and recorded at 405 nm using an Epoch microplate reader (BioTek) for 30 min. Results are shown below. Figure 1 and Figure 2 As shown, in Figure 1 In the ordinate of A t A0 and A1 represent the absorbance value at a certain time point and the initial absorbance value, respectively. Figure 2 In the ordinate, A 30 A0 and A0 represent the absorbance value at 30 min and the initial absorbance value, respectively; meanwhile, in Figure 2 In the text, “*”, “**” and “***” represent significant differences, P<0.05, P<0.01 and P<0.001, respectively.

[0036] Depend on Figure 1 and Figure 2It can be concluded that the derived polypeptide PM6 can inhibit thrombin activity in a concentration-dependent manner.

[0037] Example 3

[0038] The determination of the thrombin inhibition constant of the derived polypeptide PM6 in Example 1 was performed as follows:

[0039] To calculate the inhibitory constant Ki of the derived peptide on thrombin, the Dixon plot was used, with peptide concentration on the x-axis and the reciprocal of the reaction rate on the y-axis. Regression analysis was performed to fit two curves, and the x-value at the intersection of the two curves was calculated as the inhibitory constant Ki of the peptide on thrombin. The reaction rate was obtained by detecting the inhibitory activity of the derived peptide PM6 on thrombin at different concentrations (50 μg / mL and 100 μg / mL) of the luminescent substrate (S-2238), using a 405 nm wavelength curve. The results are shown below. Figure 3 As shown.

[0040] Depend on Figure 3 It can be concluded that the derived polypeptide PM6 has a strong inhibitory effect on thrombin, with an inhibition constant (Ki) of 1.15 μg / mL (523.2 nM).

[0041] Example 4

[0042] The inhibitory effect of the derived polypeptide PM6 in Example 1 on activated partial thromboplastin time (APTT) and prothrombin time (PT) was investigated using the following experimental procedures:

[0043] Coagulation function tests include APTT and PT measurements. Following the manufacturer's instructions, the absorbance was measured at 650 nm using the kits (TC0306, Leagene, for APTT detection; GMS10176, Genmed, for PT detection). Results are as follows: Figure 4 and Figure 5 As shown.

[0044] Depend on Figure 4 and Figure 5 It can be concluded that the derived polypeptide PM6 can inhibit both the intrinsic and extrinsic coagulation pathways.

[0045] Example 5

[0046] The inhibitory effect of the derived polypeptide PM6 on FeCl3-induced carotid artery thrombosis in Example 1 is illustrated by the following steps:

[0047] Ten minutes before surgery, mice were administered different drug groups via tail vein injection, including the derivative peptide PM6 (1, 0.1, and 0.05 mg / kg), a saline group (negative control), and a bivalirudin group (1 mg / kg, positive control). Male C57BL / 6 mice (6–8 weeks old) were anesthetized with sodium pentobarbital (80 mg / kg), and the procedure was performed on a heated pad. The neck of the mice was cut to expose the carotid artery, which was then separated from the vagus nerve and surrounding tissues. After a 2 × 2 mm sheet of 10% ferric chloride solution-filled filter paper was applied to the exposed carotid artery, thrombosis occurred in the mice. A Doppler microvascular probe (RWD) was placed on the exposed artery to measure vascular blood flow, and the results were as follows: Figure 6 As shown.

[0048] Depend on Figure 6 It can be concluded that the derived polypeptide PM6 has a significant inhibitory effect on FeCl3-induced carotid artery thrombosis, thus indicating that the derived polypeptide PM6 has good resistance to carotid artery thrombosis.

[0049] Example 6

[0050] The bleeding risk detection of the derived polypeptide PM6 in Example 1 is performed as follows:

[0051] Tail hemorrhage activity assay: Male C57BL / 6J mice (6–8 weeks old) were injected via tail injection 10 minutes before surgery with different doses of the derived polypeptide PM6 (10, 2, and 0.4 mg / kg). Physiological saline was used as a negative control, and bivalirudin (10, 2, and 0.4 mg / kg) was used as a positive control. Subsequently, a 2 mm section was cut from the tail and carefully immersed in 20 mL of 37°C physiological saline. The bleeding time was recorded until blood flow stopped. If bleeding resumed within 30 seconds, this time was also included in the bleeding time. The results are as follows: Figure 7 As shown in the left figure.

[0052] This embodiment also determined the hemoglobin content of blood collected in a constant-temperature physiological saline solution at 37°C. Each group of collected blood (200 μL) was incubated with 20 μL of 1% (v / v) Triton X-100 (Abcone) at 37°C for 3 min. After incubation, the sample was centrifuged at 1000 rpm for 5 min, and the absorbance of the supernatant was measured at 540 nm. The results are shown below. Figure 7 As shown in the figure on the right.

[0053] Depend on Figure 7 The results show that the bleeding risk of the derived polypeptide PM6 is comparable to that of the positive control group bivalirudin.

[0054] From the above examples, it can be concluded that the derived polypeptide PM6 described herein has a significant antithrombotic effect, and the bleeding risk is comparable to that of the positive drug bismuth subtilisin.

[0055] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A derived polypeptide PM6, characterized in that, The derived polypeptide PM6 is {d-Phe}-PRPGGGGNDFEPIPEDYFS.

2. The use of the derived polypeptide PM6 according to claim 1 in the preparation of antithrombotic drugs.

3. The application as described in claim 2, characterized in that, The antithrombotic drugs include those that prevent arterial thrombosis.

4. An antithrombotic drug, characterized in that, The active ingredient includes the derivative polypeptide PM6 as described in claim 1.

5. The antithrombotic drug as described in claim 4, characterized in that, The only active ingredient in the aforementioned antithrombotic drug is the derived polypeptide PM6.

6. The antithrombotic drug as described in claim 4 or 5, characterized in that, The antithrombotic drug includes pharmaceutically acceptable excipients.

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