Anti-platelet aggregation transmembrane polypeptide and application thereof

Abstract

The application discloses a kind of anti-platelet aggregation transmembrane polypeptide Palmitoyl-DD1-P and its application, belong to medical biotechnology field.The transmembrane polypeptide of the present application is composed of 8 amino acids, has the sequence of polypeptide of SEQ ID NO:1, molecular weight is 1112.20, sequence Arg-Ala-Ser-Asp-Gln-Arg-Asp-Leu, and the polypeptide is modified by palmitoyl.The polypeptide is not limited to inhibiting collagen-induced platelet aggregation, can also act on its influence on platelet-related functions with other platelet activators (arachidonic acid, ADP, adrenaline, restomycin, etc.), and can also be used for monitoring existing anti-platelet therapy.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical biotechnology, and relates to a polypeptide, specifically a transmembrane polypeptide that inhibits platelet aggregation, its preparation method, a pharmaceutical composition composed of the polypeptide as an active ingredient, and its application in the preparation of antiplatelet drugs. Background Technology

[0002] Platelets play a crucial role in arterial thrombosis. Classic antiplatelet drugs like aspirin and novel drugs such as abciximab, which block the binding of αIIbβ3 to fibrinogen, have played important clinical roles, but they also carry potential risks such as gastrointestinal reactions and bleeding. Developing antiplatelet drugs with better safety and efficacy has significant clinical and social value.

[0003] Damage to vascular endothelial cells or plaque rupture exposes subendothelial collagen, initiating acute thrombosis. Collagen, along with platelet surface glycoprotein VI and integrin-α2β1, activates the integrin αIIbβ3 activation pathway, leading to platelet activation and aggregation. Thrombin and other activating substances released during platelet activation further promote platelet aggregation, ultimately resulting in thrombus formation. Currently, there are no inhibitors of platelet surface glycoprotein VI used clinically.

[0004] Currently, there are no reports in existing technologies regarding the antiplatelet aggregation transmembrane peptide Palmitoyl-DD1-P and its activity. Summary of the Invention

[0005] The purpose of this invention is to provide a transmembrane polypeptide Palmitoyl-DD1-P that inhibits platelet aggregation, its preparation method, a pharmaceutical composition using it as an active ingredient, and its application in pharmaceutical manufacturing.

[0006] To achieve the above-mentioned objectives of the present invention, the present invention provides the following technical solution:

[0007] A transmembrane polypeptide Palmitoyl-DD1-P that inhibits platelet aggregation consists of the 8 amino acid sequences shown in Arg-Ala-Ser-Asp-Gln-Arg-Asp-Leu, has the amino acid sequence described in SEQ ID NO:1 in the sequence listing, has a molecular weight of 1112.20, and is modified with palmitoyl.

[0008] This invention also provides the application of the aforementioned anti-platelet aggregation transmembrane polypeptide Palmitoyl-DD1-P in the preparation of formulations that inhibit platelet aggregation.

[0009] And the application of the aforementioned antiplatelet aggregation transmembrane polypeptide Palmitoyl-DD1-P in the preparation of antiplatelet aggregation drugs.

[0010] Furthermore, the application of the antiplatelet aggregation transmembrane polypeptide Palmitoyl-DD1-P in the preparation of drugs for treating cardiovascular diseases.

[0011] According to the application of the membrane-penetrating peptide Palmitoyl-DD1-P in the preparation of drugs for antiplatelet aggregation or treatment of cardiovascular diseases, the membrane-penetrating peptide Palmitoyl-DD1-P can achieve antiplatelet aggregation effect by significantly inhibiting human platelet aggregation induced by collagen and GPVI specific activator Convulxin.

[0012] The application of the anti-platelet aggregation transmembrane polypeptide Palmitoyl-DD1-P in the preparation of platelet activators.

[0013] The application of the anti-platelet aggregation transmembrane peptide Palmitoyl-DD1-P in monitoring the inhibition of platelet aggregation induced by collagen and the GPVI-specific activator Convulxin.

[0014] The present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of an antiplatelet aggregation transmembrane polypeptide Palmitoyl-DD1-P and a pharmaceutically acceptable carrier.

[0015] The membrane-penetrating polypeptide Palmitoyl-DD1-P involved in this invention consists of 8 amino acids with a molecular weight of 1112.20 and the sequence Arg-Ala-Ser-Asp-Gln-Arg-Asp-Leu. Experiments show that DD1-P significantly inhibits collagen and GPVI-specific activator Convulxin-induced human platelet aggregation. Therefore, DD1-P can be used in the development of antiplatelet drugs. DD1-P can also be prepared in combination with one or more pharmaceutically acceptable carriers for the preparation of antiplatelet and antithrombotic drugs.

[0016] When the membrane-penetrating polypeptide Palmitoyl-DD1-P of the present invention is used as a drug, it can be used directly or in the form of a pharmaceutical composition. The pharmaceutical composition contains 0.1-99%, preferably 0.5-90%, of the compound of the present invention, with the remainder being pharmaceutically acceptable, non-toxic, and inert pharmaceutically acceptable carriers and / or excipients for humans and animals.

[0017] The pharmaceutical carrier or excipient is one or more solid, semi-solid, and liquid diluents, fillers, and pharmaceutical excipients. The pharmaceutical composition of the present invention is used in a dose per unit body weight. The drug of the present invention can be administered by injection (intravenous or intramuscular) and orally.

[0018] Compared with the prior art, the advantages of this invention are:

[0019] 1. This invention has invented the membrane-penetrating polypeptide Palmitoyl-DD1-P and explored its medical applications, thus opening up a new field of application.

[0020] 2. The membrane-penetrating polypeptide Palmitoyl-DD1-P of the present invention is inexpensive, has no toxic side effects, has a simple preparation process, and can be made into oral dosage forms, injection dosage forms, tablets, etc., which are convenient to use.

[0021] 3. The membrane-penetrating polypeptide Palmitoyl-DD1-P of this invention can significantly inhibit collagen- and convulxin-induced platelet aggregation. It can effectively inhibit platelet aggregation and can be effectively used as an antiplatelet aggregation drug. Attached Figure Description

[0022] Figure 1 The high-performance liquid chromatography (HPLC) chromatogram of the polypeptide provided by this invention shows that peak C represents the purified Palmitoyl-DD1-P.

[0023] Figure 2 for Figure 1 Secondary mass spectrometry identification of peak C (Palmitoyl-DD1-P).

[0024] Figure 3 A bar chart showing the effect of the transmembrane peptide Palmitoyl-DD1-P on collagen-induced platelet aggregation;

[0025] Figure 4 A bar chart showing the effect of the transmembrane peptide Palmitoyl-DD1-P on Convulxin-induced platelet aggregation;

[0026] Figure 5 A bar chart showing the effect of the transmembrane peptide Palmitoyl-DD1-P on thrombin-induced platelet aggregation. Detailed Implementation

[0027] The specific embodiments of the present invention will be described in more detail below with reference to the accompanying drawings and specific examples. However, this is not intended to limit the scope of the invention.

[0028] Example 1

[0029] Following the method reported in the literature (La Face DM, Couture C, Anderson K, et al. Differential T cell signaling induced by antagonist peptide-MHC complexes and the associated phenotypic responses. J Immunol. 1997 Mar 1;158 (5):2057–2064.), the transmembrane peptide Palmitoyl-DD1-P was synthesized using a solid-phase peptide synthesis method and then modified with palmitoyl. The synthesized peptide was purified by high-performance liquid chromatography (HPLC) to a concentration ≥95% ( Figure 1 Subsequently, the amino acid sequence and molecular weight were determined by mass spectrometry (MS), confirming that the synthesized polypeptide was Palmitoyl-DD1-P (…). Figure 2 ).

[0030] DD1-P inhibits thrombin-induced human platelet aggregation in vitro.

[0031] (1) The platelets in the experimental subjects were obtained from healthy volunteers who signed informed consent forms for blood donation and were given certain nutritional subsidies. Venous whole blood was collected, and apheresis platelets were separated by the Kunming Blood Center and collected at the Department of Hematology of the First Affiliated Hospital of Kunming Medical University.

[0032] (2) The specific implementation method is as follows:

[0033] Step 1: Dissolve and adjust the concentrations of DD1-P, palmitoyl-modified DD1-P (Palmitoyl-DD1-P), and palmitoyl (Palmitoyl) to 500 mM using DMSO.

[0034] Step 2, wash platelets. Take 1 mL of human apheresis platelets stored at 25℃ with shaking and add them to a 1.5 mL centrifuge tube. Add 5 mM EDTA and 0.1 U / mL Apyrase (prepared with physiological saline to prevent platelet aggregation during centrifugation). Centrifuge at 400g for 10 minutes at room temperature. Discard the supernatant after centrifugation. Add 1 mL Tyrode's Buffer B (137 mM NaCl, 27 mM KCl, 1 mM MgCl2, 0.42 mM NaH2PO4, 5.5 mM Glucose, 5.55 mM HEPES, 0.25% Bovine Serum Albumin, pH 6.5), 5 mM EDTA, and 0.1 U / mL Apyrase to the cell pellet. Gently mix and centrifuge again at 400g for 10 minutes at room temperature. The platelets were resuspended after centrifugation in a solution of KCl (1 mM), MgCl2 (0.42 mM), NaH2PO4 (5.5 mM), Glucose (5.55 mM), HEPES (0.25%), and Bovine Serum Albumin (pH 7.4). The platelet count was then adjusted to 150-250 × 10⁻⁶. 9 / L. Store at 70rpm and 25℃ with shaking. Use washed platelets within 1 hour.

[0035] Step 3: Take 400 μL of washed platelet resuspension and preheat at 37°C for 5 min. Add DD1-P, Palmitoyl, and Palmitoyl-DD1-P to the experimental group at a final concentration of 0.1 mM. Add an equal volume of DMSO to the positive control group (Vehicle). Incubate at 37°C for 10 min. Turn on the platelet aggregator, set the parameters as required, place a magnetic stir bar in the incubated washed platelets, insert the platelet reaction cup with a magnetic stir bar into the detection port of the machine, place it in the test area of ​​the platelet aggregator, zero it, and add activators such as collagen. Observe the effect of DD1-P on platelet aggregation induced by the activator at 37°C and 1200 rpm for 5-10 min. Plot the corresponding bar graph based on the platelet aggregation curve recorded by the platelet aggregator. Perform statistical analysis on the experimental data using GraphPadPrism 8.0 software. Comparison between groups was performed using analysis of variance. A p-value < 0.05 indicates that the difference is statistically significant.

[0036] manual Figure 3-5As shown: Human washed platelets were incubated at 37°C with 100 μM DD1-P, Palmitoyl-DD1-P, Palmitoyl, or an equal volume of DMSO for 10 minutes. Collagen (5 μg / mL), thrombin (0.05 U / mL), and Convulxin (2.5 ng / mL) were added to activate the platelets. Platelet aggregation curves were plotted and recorded using a platelet aggregator. The ordinate represents the real-time platelet aggregation rate plotted by the platelet aggregator, and the abscissa represents the experimental group name. A bar chart comparing the platelet aggregation peak values ​​of the Vehicle group and the 100 μM DD1-P, Palmitoyl-DD1-P, and Palmitoyl groups is also shown. Each experiment was repeated four times for both the Vehicle and experimental groups. Platelet aggregation peak values ​​are expressed as mean ± standard error. * in the figure indicates a statistically significant difference, where **** represents p ≤ 0.0001 compared to the Vehicle group. Figures 3-5 It can be seen that Palmitoyl-DD1-P can significantly inhibit platelet aggregation induced by collagen and the GPVI-specific activator Convulxin, while DD1-P and Palmitoyl have no significant effect on platelet aggregation induced by collagen, thrombin, and the GPVI-specific activator Convulxin. At the same time, Palmitoyl-DD1-P also has no significant effect on thrombin-induced platelet aggregation, suggesting that the inhibitory effect of Palmitoyl-DD1-P on platelet aggregation has a certain degree of selectivity.

[0037] Formulation Examples

[0038] In the following formulation examples, conventional reagents were selected and the formulations were prepared according to existing conventional methods. This application example only demonstrates the preparation of different formulations of the transmembrane polypeptide Palmitoyl-DD1-P described in this invention, and does not specifically limit the specific reagents and operations.

[0039] 1. The membrane-penetrating peptide Palmitoyl-DD1-P is dissolved in DMSO, then mixed with water for injection according to conventional methods, filtered, filled, and sterilized to prepare an injection solution with a concentration of 0.5~5 mg / mL.

[0040] 2. The membrane-penetrating peptide Palmitoyl-DD1-P was dissolved in DMSO, then dissolved in sterile water for injection. The solution was stirred until dissolved, filtered through a sterile vacuum funnel, then sterilely filtered again, dispensed into ampoules, freeze-dried at low temperature, and then sterilely sealed to obtain a powder for injection.

[0041] 3. Add the membrane-penetrating peptide Palmitoyl-DD1-P to the excipient at a mass ratio of 9:1 to prepare a powder.

[0042] 4. Add the membrane-penetrating peptide Palmitoyl-DD1-P to the excipient at a mass ratio of 5:1, then granulate and compress into tablets.

[0043] 5. The membrane-penetrating polypeptide Palmitoyl-DD1-P was prepared into an oral liquid using conventional oral liquid preparation methods.

[0044] 6. Add the membrane-penetrating peptide Palmitoyl-DD1-P to the excipient at a mass ratio of 5:1 to make capsules.

[0045] 7. Add the membrane-penetrating peptide Palmitoyl-DD1-P to the excipient at a mass ratio of 5:1 to prepare granules.

[0046] 8. Take the membrane-penetrating polypeptide Palmitoyl-DD1-P, add it to the excipient at a weight ratio of 3:1, and make capsules.

[0047] The scope of protection of this invention is not limited to the above embodiments. The above embodiments are only for the purpose of explaining and illustrating this invention, and are not intended to limit the scope of protection of this invention. Any design that is the same as the design of this invention or that is an equivalent substitution falls within the scope of protection claimed by this invention.

Claims

1. An antiplatelet aggregation polypeptide Palmitoyl-DD1-P, consisting of an 8-amino acid sequence as shown in Arg-Ala-Ser-Asp-Gln-Arg-Asp-Leu, with a molecular weight of 1112.20, and modified with palmitoyl.

2. The use of the antiplatelet aggregation polypeptide Palmitoyl-DD1-P according to claim 1 in the preparation of antiplatelet aggregation reagents.

3. The application of the polypeptide Palmitoyl-DD1-P according to claim 2 in the preparation of reagents for antiplatelet aggregation, characterized in that: The peptide Palmitoyl-DD1-P can significantly inhibit human platelet aggregation through collagen and the GPVI-specific activator Convulxin, thereby achieving an anti-platelet aggregation effect.

4. The application of the polypeptide Palmitoyl-DD1-P according to claim 2 in the preparation of reagents for antiplatelet aggregation, characterized in that: The peptide Palmitoyl-DD1-P significantly inhibits collagen-induced platelet aggregation.

5. The application of the polypeptide Palmitoyl-DD1-P according to claim 2 in the preparation of reagents for antiplatelet aggregation, characterized in that: The peptide Palmitoyl-DD1-P significantly inhibits platelet aggregation induced by the GPVI-specific activator Convulxin.

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