Polypeptide inhibitors of coagulation factor FXIa and their use in anticoagulation therapy

Abstract

The application discloses a polypeptide FXIa inhibitor FXIapepin with high inhibition and high specificity. Since FXIa can be used as a low hemorrhage risk anti-thrombus treatment target, the polypeptide inhibitor can be used for preparing a drug for developing a low hemorrhage risk treatment or prevention of a thrombotic disease, and has a wide application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and particularly relates to a polypeptide inhibitor of coagulation factor FXIa and its application in anticoagulation therapy. BACKGROUND

[0002] In recent years, cardiovascular diseases have seriously threatened people's health. Under normal circumstances, the coagulation and anticoagulation systems in the human body are in dynamic balance, and coagulation dysfunction is one of the important reasons for various thrombotic diseases. Thrombosis can occur at any site of the blood vessels, and is divided into arterial thrombosis, venous thrombosis, microvascular thrombosis and disseminated intravascular coagulation (DIC) according to the location. After the formation of in situ thrombus, it partially or completely occludes the in situ blood vessels, limits blood flow, and can cause ischemia or blood stasis and other symptoms. If the thrombus is detached, it can flow to the distant organs such as the heart, brain and lungs, which can lead to serious consequences such as myocardial infarction, stroke and pulmonary embolism. The risk of thrombosis often increases with age, and with the aging of the population, cardiovascular and cerebrovascular diseases show high incidence, high disability rate, high mortality and poor treatment effect, and the trend of younger patients is obvious, especially thrombotic diseases have become one of the important problems that need to be solved for the health of the Chinese people.

[0003] Under normal circumstances, the coagulation and anticoagulation systems in the human body are in dynamic balance, and coagulation dysfunction is one of the important reasons for various thrombotic diseases, so the intrinsic coagulation pathway is considered as an antithrombotic target with low risk of bleeding. Among them, FXI is a key factor for controlling the amount of thrombin generation, which can effectively balance the antithrombotic effect and bleeding side effects. FXI is a plasma serine protease that plays a key role in the initiation and expansion of thrombosis. In the intrinsic pathway, FXI is activated to FXIa by FXIIa, which in turn activates FIX and ultimately leads to the generation of a large amount of thrombin. At the same time, thrombin catalyzes the activation of FXI, which expands the generation of thrombin exponentially. However, this amplification of FXI-induced thrombin generation does not exist in the process of normal hemostasis, and FXI only plays a supporting role in hemostasis. FXI is a bridge between TF-initiated thrombin generation and the kinin kallikrein system (KKS). In addition, FXI does not participate in the activation of FIX by TF-FVIIa in the extrinsic coagulation pathway, so it does not interfere with the hemostatic coagulation caused by tissue damage. The development of polypeptide FXIa inhibitors is still in its infancy, and there are very limited potential FXIa polypeptide inhibitors, and no polypeptide drug targeting FXIa has entered clinical research.

[0004] In summary, there is an urgent need in the clinic to develop a polypeptide FXIa target antithrombotic drug with low risk of bleeding, low cost, high specificity, and both effectiveness and safety. It can not only provide a safe and efficient drug lead for anticoagulant therapy of thrombotic diseases, but also explore the application prospect of polypeptide drugs in targeted FXIa anticoagulant therapy. SUMMARY

[0005] In view of the problem of bleeding risk in the treatment and prevention of antithrombotic diseases, the present application utilizes the characteristic that tissue factor pathway inhibitor 2 (TFPI-2) has coagulation factor XIa (FXIa) inhibitory activity, takes the first Kunitz domain (KD1) of TFPI-2 as a template, optimizes the sequence of KD1 based on the structure, and designs a polypeptide FXIa inhibitor FXIapepin, thereby improving the inhibitory activity and specificity of KD1 to FXIa, and providing a safe and efficient drug lead for clinical targeted FXIa anticoagulant therapy.

[0006] To achieve the above object, the present application adopts the following technical scheme:

[0007] The present application discloses a polypeptide FXIapepin with anticoagulant effect, and the protein sequence of the FXIapepin is shown as SEQ ID NO. 1.

[0008] The above-mentioned FXIa inhibitor FXIapepin is used for preparing a coagulation factor XIa inhibitor, an anticoagulant, and a drug for preventing and treating thrombotic diseases. It utilizes the characteristic that FXIapepin has coagulation factor XIa (FXIa) inhibitory activity, and takes FXIapepin as a drug active ingredient to prepare any kind of pharmaceutically acceptable dosage form.

[0009] The pharmaceutically acceptable dosage form includes tablets, capsules, granules, oral liquids or injections. Through biological activity determination of FXIapepin, it is found that FXIapepin has good inhibitory activity to FXIa, and the Ki value of FXIapepin to FXIa is 0.97 nM, and the Ki value to fibrinolysin is 86.2 nM. In the in-vitro simulation thrombosis experiment, FXIapepin keeps a significant effect of prolonging plasma coagulation in the normal coagulation group and the FXIa-induced coagulation group. The ferric trichloride (FeCl3)-induced carotid artery thrombosis animal model experiment result shows that FXIapepin can significantly improve the thrombosis of mice; the bleeding risk evaluation experiment result shows that FXIapepin tail vein administration does not prolong the tail bleeding time of mice and does not affect the overall coagulation of mice, and therefore, FXIapepin or its pharmaceutically acceptable salt can be used as a FXIa inhibitor for preparing a drug for treating or preventing thrombotic diseases.

[0010] Due to the use of the above technical solutions, the present application has the advantages of:

[0011] 1. FXIapepin has significant anticoagulant effect, small risk of bleeding, reduces postoperative systemic massive hemorrhage, and high safety.

[0012] 2. FXIapepin shows significant effect in antithrombosis.

[0013] 3. FXIapepin has strong inhibitory activity on FXIa, and shows high specificity for FXIa among various serine proteases.

[0014] 4. The FXIa inhibitor FXIapepin can be applied to the preparation of a drug for treating thrombotic diseases, including acute myocardial infarction, acute pulmonary embolism, stroke, and venous thrombosis. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 : pPICZαA-FXIapepin plasmid map.

[0016] Figure 2 : SDS-PAGE electrophoresis diagram of FXIapepin superdex 75 purification, wherein 1 is Marker, and 2 is FXIapepin.

[0017] Figure 3 : In vitro anticoagulant activity of FXIapepin.

[0018] Figure 4 : Effect of FXIapepin on FXIa-induced plasma coagulation.

[0019] Figure 5 : FXIapepin on FeCl3-induced carotid artery thrombosis time chart.

[0020] Figure 6 : FXIapepin causes bleeding risk chart. DETAILED DESCRIPTION

[0021] In order to better understand the content of the present application, the present application will be further described below in combination with the drawings and specific examples of the specification, but the scope of the present application claimed is not limited to the scope expressed by the examples. Unless otherwise specified, the reagents and materials used in the following examples are commercially available. In the following examples, various processes and methods that are not described in detail are conventional methods known in the art.

[0022] The recombinant FXIapepin used is obtained by conventional molecular cloning and protein expression technology based on KD1.

[0023] The protein sequence of FXIapepin is SEQ ID NO. 1:

[0024] DAAQEPTGNNAEICLLPLDDGPCRALLLRYYYDRYTQSCRQFLYGGCEGNANNFYTWEACDDACWRIEKVHHHHHHH.

[0025] Example 1: Construction, expression and purification of FXIapepin

[0026] (1) Construction of recombinant plasmid pPICZαA-FXIapepin

[0027] As shown in Figure 1 , the FXIapepin fragment is inserted after the α-factor signal peptide "LEKR" sequence in the pPICZαA vector, and the amino acid sequence is:

[0028] DAAQEPTGNNAEICLLPLDDGPCRALLLRYYYDRYTQSCRQFLYGGCEGNANNFYTWEACDDACWRIEKV, and 6 His are added at the C-terminal of the target fragment for purification. The recombinant plasmid is transformed into E. coli DH5α by heat shock method, plated, and single colony strains are picked for gene sequencing. The glycerol bacteria of the strain with correct sequencing and strong signal peak are stored for cryopreservation at -80°C. The DH5α strain containing the correct FXIapepin sequence is expanded, and the pPICZαA-FXIapepin plasmid is extracted using Hlingene high-purity plasmid small amount rapid extraction kit for use in the following experiments. The pPICZαA-FXIapepin plasmid map is shown in Figure 1 .

[0029] (2) Expression and purification of FXIapepin

[0030] The small amount of expressed FXIapepin is inoculated into YPD culture medium, expanded at 28°C for 12 h, then inoculated into 500 mL BMMY medium, and 1% methanol is added for induction expression for five consecutive days, and the induction expression is performed at 28°C for 5 times. The bacterial solution is centrifuged at 7000 rpm for 30 min, and the supernatant is reserved. The centrifuged supernatant is filtered with a 0.22 μm filter membrane on ice. The filtered supernatant containing the target protein is passed through a balanced nickel column or mixed with balanced nickel filler for 1-2 h, and the nickel filler containing the target protein is collected in the column. Then, the impurities are washed with a washing buffer, and the flow-through is detected with Coomassie brilliant blue until no color change occurs. The target protein is eluted with an elution buffer, and the collected eluted protein solution is placed on ice or at 4°C. The separated and purified protein is identified by SDS-PAGE electrophoresis, as shown inFigure 2 The purified recombinant protein FXIapepin band was single, with a molecular weight of 9 kDa. Dialysis was performed in a refrigerator at 4°C for 4 h to remove imidazole. The target protein after dialysis was further purified by an AKTA protein purification system. According to the size of the protein molecular weight, we selected a gel chromatography column (Superdex 75 Increase 10 / 300 GL) to separate and purify the target protein, and collected the target protein at the target peak with an equilibration buffer. The target protein was concentrated to the appropriate concentration with a 3 kDa concentrator tube, aliquoted, and stored in a -80°C refrigerator.

[0031] Example 2: Detection of the enzyme kinetic activity of FXIapepin

[0032] Enzyme activity determination was performed by a small molecule chromogenic substrate method to determine whether FXIapepin had inhibitory activity on proteases. The inhibitory effects on FXIa, plasmin, trypsin, thrombin, uPA, and tPA enzymes were detected (substrates were S-2366, S-2302, S-2366, S-2288, S-2288, and S-2288, respectively). Specifically, in a 100 μL reaction system (containing 30 mM Tris-HCl pH 7.4, 150 mM NaCl), 10 nM FXIa or other proteases were pre-incubated with 0-2 μM FXIapepin at a final concentration for 10 minutes, then 400 μM luminescent substrate was added, mixed immediately, and then placed in a microplate reader. The absorbance was detected at 405 nm, measured every 30 s, and detected for 30 min. The obtained data were calculated and fitted by GraphPad 8.0.

[0033] As shown in Table 1, the enzyme activity determination results showed that the effects of different concentrations of FXIapepin on the activities of FXIa, plasmin, trypsin, thrombin, uPA, and tPA serine proteases were evaluated. The inhibition constant of FXIapepin on FXIa was Ki = 0.97 nM; the inhibition constant on plasmin was Ki = 86.2 nM; and the inhibition constant on trypsin was Ki = 14.59 nM. FXIapepin had no significant inhibitory activity on serine proteases such as thrombin, uPA, and tPA (Ki > 2000). Therefore, FXIapepin had high and specific inhibitory activity on FXIa.

[0034] Table 1

[0035] serine protease <![CDATA[Inhibition constant K i (nM)]]> coagulation factor XIa 0.97 trypsin 75.32 tPA >2000 uPA >2000 thrombin >2000 plasmin 86.2

[0036] Example 3: In vitro anticoagulant activity experiment of FXIapepin

[0037] The healthy volunteer's venous plasma was treated to obtain platelet-poor plasma (PPP), 25 μL of PPP was added to a sterile 96-well plate, 65 μL of FXIapepin was added to each well, and the final concentration was 2 μM. The control group was replaced with the same volume of balanced buffer, and incubated at room temperature for 10 min. After adding 10 μL of CaCl2 with a final concentration of 10 mM to each well, the ultraviolet-visible absorbance at 405 nm was detected by an enzyme marker, the detection temperature was set to 37°C, and the data was read every 30 s, a total of 60 min. The obtained data was statistically analyzed by Graphpad 8.0, and the area under the curve (AUC) was calculated. The effect on FXIa-induced plasma coagulation was also determined: 15 μL of human FXIa with a final concentration of 20 nM was added to the PPP before measurement, and 50 μL of FXIapepin was added to mix, so that the final concentration was 2 μM.

[0038] As shown in Figure 3 , after the addition of Ca 2+ , the turbidity of the control group began to rise at 8 min and reached a plateau at about 20.38 min. The sample containing 2 μM FXIapepin began to coagulate at 27 min and reached a plateau at an average of 55.38 min, which was 35 min longer than the control group. This indicates that the inhibitory effect of FXIapepin on in vitro coagulation has been improved. The area under the curve (AUC) of each group of reactions was summarized, and the AUC of the FXIapepin group was significantly reduced, indicating that FXIapepin has an antithrombotic effect.

[0039] To study the specific inhibitory effect of FXIapepin on FXIa, as shown in Figure 4 , a final concentration of 20 nM of FXIa was added, the plasma turbidity of the control group began to rise at 6 min, and reached a plateau after an average of 15.75 min. In the FXIapepin group, fibrin began to form at 29 min, and reached a plateau at an average of 54.38 min, which was not different from the case of measuring the coagulation time of normal plasma. The antithrombotic function was not disturbed by the addition of FXIa, indicating that FXIapepin has a specific antithrombotic effect on FXIa.

[0040] Example 4: Effect of FXIapepin on FeCl3-induced arterial thrombosis

[0041] ICR mice were randomly divided into 2 groups, each group containing 6 mice, first tail vein administration, in which the amount of FXIapepin administration is 5mg / kg, the saline administration group as a control. 10 min after administration, the mice were anesthetized (1.5% sodium pentobarbital, 30mg / kg, intraperitoneal injection) and the left common carotid artery was exposed, and a piece of filter paper (2*2mm) soaked with 15% FeCl3 was carefully placed below it 2 ). The blood flow of the carotid artery of the mice was monitored and recorded by laser speckle blood flow imaging instrument, the blood flow rate was recorded every 5s, and the average time for the formation of occlusive thrombus (occlusion rate reached 95%) in the carotid artery was recorded.

[0042] As shown in Figure 5 , the carotid artery of the mice in the saline group was occluded within 175 seconds after 15% FeCl3 stimulation, and the 5mg / kg FXIapepin group prolonged the occlusion time of the blood vessel to 351 seconds. FXIapepin showed significant inhibition of thrombus formation.

[0043] Example 5: Evaluation of the risk of bleeding caused by FXIapepin by tail cutting experiment

[0044] Group administration is similar to the method in Example 4, ICR mice were randomly divided into 2 groups, each group containing 6 mice, first tail vein administration, in which the amount of FXIapepin administration is 5mg / kg, the saline administration group as a control. 10 min after administration, the mice were anesthetized (1.5% sodium pentobarbital, 30mg / kg, intraperitoneal injection), and the mouse tail was cut off 10mm from the tip, and immediately immersed in 37℃ 10mL isotonic saline, and the bleeding time was recorded after stopping bleeding. The amount of blood loss was quantified by measuring the hemoglobin content collected in 10mL isotonic saline, and the red blood cells were collected after centrifugation at 1500rpm, lysed with 2mL lysis buffer (8.3g / L NH4Cl, 1g / L KHCO3, 0.037g / L EDTA), and finally the absorbance of each sample at 570nm was determined by a microplate reader.

[0045] As shown in Figure 6 , compared with the saline group, the amount of bleeding in the 5mg / kg FXIapepin group did not increase significantly, and the experimental results showed that FXIapepin did not cause the risk of bleeding.

[0046] The above only describes the preferred embodiments of the present application, and any equivalent changes and modifications made within the scope of the patent application of the present application shall be included in the scope of the present application.

Claims

1. A polypeptide FXIapepin having an anticoagulant effect, characterized in that, The protein sequence of the FXIapepin is shown as SEQ ID NO.

1.

2. The use of the polypeptide FXIapepin in claim 1 in the preparation of a factor XIa inhibitor.

3. Use according to claim 2, characterized in that, The inhibitor is prepared into a medicament with the FXIapepin as a pharmaceutically active ingredient.

4. The use of the polypeptide FXIapepin in claim 1 in the preparation of an anticoagulant.

5. Use according to claim 4, characterized in that, The anticoagulant is prepared into a medicament with the FXIapepin as a pharmaceutically active ingredient.

6. The use of the polypeptide FXIapepin in claim 1 in the preparation of a drug for preventing and treating thrombotic diseases.

7. Use according to claim 6, characterized in that, The drug for preventing and treating thrombotic diseases is prepared into a medicament with the FXIapepin as a pharmaceutically active ingredient.

8. Use according to claim 3, 5 or 7, characterized in that, The medicament includes tablets, capsules, granules, oral liquids or injections.

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