Application of polypeptide in preparation of disseminated intravascular coagulation drug

Abstract

The invention belongs to the technical field of medicines, and provides application of polypeptide in preparation of a disseminated intravascular coagulation drug. The amino acid sequence of the polypeptide is as follows: Ala-Glu-Pro-Val-Pro-Leu. Experiments show that the polypeptide can obviously prolong the survival time of a diffuse intravascular coagulation model rat to a certain extent, reduce the bleeding score of visceral organs, especially lungs, of the diffuse intravascular coagulation model rat, reduce pulmonary alveolar wall capillary thrombosis and inflammatory cell infiltration, and reduce the proportion of large and medium pulmonary artery thrombosis, so that the polypeptide can be used for treating pulmonary thrombosis. And alveolar fusion is reduced, and the compound has a certain application prospect in preparation of dispersive intravascular coagulation drugs.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular, relates to the application of polypeptides in the preparation of drugs for treating disseminated intravascular coagulation. Background Art

[0002] Disseminated intravascular coagulation (DIC) is a clinical syndrome of systemic coagulation activation. The International Society on Thrombosis and Hemostasis (ISTH) defines it as "an acquired coagulation disorder syndrome". DIC is a clinical syndrome characterized by microcirculatory failure and severe bleeding caused by pathogenic factors that damage the microvascular system during the development of many underlying diseases, leading to activation of the coagulation system, formation of extensive microvascular thrombi in capillaries, venules, and arterioles, and secondary hyperfibrinolysis due to massive consumption of coagulation factors. It can lead to organ dysfunction and endanger the patient's life.

[0003] Clinically, the underlying diseases that cause DIC are relatively complex, mainly including infectious diseases such as sepsis, solid tumors, blood cancers, severe obstetric complications such as amniotic fluid embolism or placental abruption, and severe poisoning or immune reactions. Among them, infection is the main cause of DIC, and 30%-51% of infected patients will have DIC symptoms. Studies have also found that 20%-30% of patients infected with the new coronavirus (Corona Virus Disease 2019, COVID-19) will also have abnormal coagulation function, and the incidence of DIC in severe patients is significantly higher than that in non-severe patients. Among the deaths, 71.4% have DIC. It is inferred that DIC is an important factor in the death of COVID-19 patients.

[0004] At present, the drugs commonly used in clinical treatment of DIC mainly include the following categories, including ISTH-recommended heparin, recombinant human activated protein C, antithrombin, recombinant human thrombomodulin drugs, and TF inhibitors and FXa inhibitors. In addition, some compound Chinese medicines and preparations are also widely used in the treatment of DIC patients in my country. However, it is difficult for the above drugs to achieve the expected results in actual use, and the overall efficacy is still controversial. There is even an increased risk of severe bleeding. Clinical trials have shown that they cannot significantly improve survival rates. Summary of the invention

[0005] In view of the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide an application of a polypeptide in the preparation of a drug for treating disseminated intravascular coagulation; the amino acid sequence of the polypeptide is: Ala-Glu-Pro-Val-Pro-Leu. Experiments show that the polypeptide can significantly prolong the survival time of rats with a disseminated intravascular coagulation model to a certain extent, reduce the bleeding scores of organs, especially the lungs, of rats with a disseminated intravascular coagulation model, reduce alveolar wall capillary thrombosis and inflammatory cell infiltration, and reduce the proportion of large and medium pulmonary artery thrombosis, reduce alveolar fusion, and has a certain application prospect in the preparation of drugs for treating disseminated intravascular coagulation.

[0006] In order to achieve the above object, the solution adopted by the present invention is:

[0007] The invention relates to an application of a polypeptide in the preparation of a drug for treating disseminated intravascular coagulation, wherein the amino acid sequence of the polypeptide is: Ala-Glu-Pro-Val-Pro-Leu.

[0008] Furthermore, in a preferred embodiment of the present invention, disseminated intravascular coagulation includes: sepsis complicated by disseminated intravascular coagulation.

[0009] Furthermore, in a preferred embodiment of the present invention, the dosage form of the drug includes one of oral solution, tablet, granule, capsule, pill, injection and in vivo implant preparation.

[0010] The beneficial effect of the use of the polypeptide provided by the present invention in the preparation of a drug for treating disseminated intravascular coagulation is that a disseminated intravascular coagulation model rat model is successfully established under the experimental conditions of Example 2 of the present application. The experimental results show that the polypeptide can significantly improve the survival time of rats with disseminated intravascular coagulation model to a certain extent, reduce the bleeding scores of organs, especially the lungs, of rats with disseminated intravascular coagulation model, reduce alveolar wall capillary thrombosis and inflammatory cell infiltration, and reduce the proportion of large and medium pulmonary artery thrombosis, reduce alveolar fusion, and has a certain application prospect in the preparation of drugs for treating disseminated intravascular coagulation. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 A schematic diagram of the steps of solid phase synthesis of polypeptides is shown;

[0012] Figure 2 A bar graph showing the proportion of thrombosis in large and medium pulmonary arteries in Example 2 (animal survival is less than 7 hours);

[0013] Figure 3 A bar graph showing the proportion of thrombosis in pulmonary arterioles and alveolar capillaries in Example 2 (animal survival less than 7 hours);

[0014] Figure 4A bar graph of the proportion of pulmonary hemorrhage in Example 2 (animals surviving less than 7 h) is shown. DETAILED DESCRIPTION

[0015] The following is an explanation of the present invention in combination with specific experiments, which is not intended to limit the scope of protection of the present invention.

[0016] Table 1 English names or abbreviations of reagents and solvents used in writing

[0017]

[0018] Example 1: Chemical synthesis of polypeptides

[0019] The synthesis of polypeptide compounds adopts conventional solid phase synthesis methods, and goes through multiple cycles of resin swelling, substitution, deprotection, washing, amino acid dissolution, amino acid activation and condensation, washing, re-deprotection, and finally cleavage and side chain deprotection.

[0020] Schematic diagram of peptide solid phase synthesis steps. Figure 1 . Figure 1 In the figure, Cl-2-Cl-Resin represents 2-chlorotrityl chloride resin (2-Chlorotrityl Chloride Resin); Fmoc-Aa(n) and the like represent 9-fluorenylmethoxycarbonyl amino acids; DIPEA is N,N-diisopropylethylamine; DCM is dichloromethane; PIP is piperidine; DMF is N,N-dimethylformamide; HOBt is 1-hydroxybenzotriazole; DIC is N,N'-diisopropylcarbodiimide; TFA is trifluoroacetic acid; and TIPS is triisopropylsilane.

[0021] The following is a method for the synthesis and purification of a polypeptide, comprising the steps of:

[0022] 1. Preparation of fully protected peptide resin; 2. Cleavage and deprotection; 3. Purification (salt exchange) and freeze-drying.

[0023] Step 1. Preparation of fully protected peptide resin

[0024] (1) Resin swelling: Weigh 2.0192 g of 2-Chlorotrityl Chloride Resin (S = 0.73 mmol / g), add it into a synthesis tube with a sieve plate, swell it with 40 ml of dichloromethane for 30 min, and remove the dichloromethane by suction filtration.

[0025] (2) Preparation of Fmoc-Asp(OtBu)-resin: Weigh Fmoc-Asp(OtBu)-OH and DIPEA at a molar ratio of 1:1.5:1.65, dissolve in 20 ml of dichloromethane, and add to a synthesis tube. Bubble with N2 at room temperature for 1-3 hours, directly add 2 ml of methanol to the reaction solution to seal for 30 minutes. Then wash with dimethylformamide 4 times, 25 ml / time, and drain the resin.

[0026] (3) Removal of Fmoc protecting group: 20 ml of 20% piperidine-DMF (v / v) solution was added to the reactor, N2 was bubbled for 20 min, and then the mixture was drained; then the mixture was washed with dimethylformamide for 6 times, 25 ml / time, 3 min / time, and the removal of Fmoc was detected by the ninhydrin method.

[0027] (4) Amino acid preactivation: Add 4.38 mmol of Fmoc-protected amino acid, 5.26 mmol of HOBt, and 4.60 mmol of DIC into a 250 ml round-bottom flask, dissolve in 20 ml of 1:1 DCM-DMF (v / v), and preactivate for 30-60 min under stirring at -5-0°C ice bath.

[0028] (5) Amino acid connection: Pour the activated protected amino acid solution into the reactor and add an appropriate amount of DCM to clean the utensils. Bubble N2 at room temperature for 1-3 hours. Use the ninhydrin method to check whether the amino acid connection is complete. If complete, drain. Wash the resin with dimethylformamide 4 times, 25 ml / time, 3 min / time, and drain. The amount of each amino acid, condensation agent and specific reaction time are shown in Table 2.

[0029] (6) After the first amino acid is condensed, steps (3) to (5) are repeated to extend the peptide chain in the order of amino acids until the last amino acid is coupled.

[0030] Table 2 Amount of amino acid and condensing agent

[0031] Amino acid name AA / eq Amino acid dosage / g HOBt / g DIPEA / g DIC / g Fmoc-L-Asp(OtBu)-OH 2.19 0.90 0 0.31 0 Fmoc-L-Gln(Trt)-OH 4.38 2.67 0.71 0.57 0.58 Fmoc-L-Lys(Boc)-OH 4.38 2.05 0.71 0.57 0.58 Fmoc-L-Val-OH 4.38 1.49 0.71 0.57 0.58 Fmoc-L-Leu-OH 4.38 1.55 0.71 0.57 0.58 Fmoc-L-Pro-OH 4.38 1.48 0.71 0.57 1.16 Fmoc-L-Val-OH 4.38 1.49 0.71 0.57 0.58 Fmoc-L-Pro-OH 4.38 1.48 0.71 0.57 1.16 <![CDATA[Fmoc-L-Glu(OtBu)-OH·H2O]]> 4.38 1.94 0.71 0.57 1.16 <![CDATA[Fmoc-L-Ala-OH·H2O]]> 4.38 1.44 0.71 0.57 1.16 <![CDATA[Fmoc-L-Ala-OH·H2O]]> 4.38 1.44 0.71 0.57 1.16 Fmoc-L-Pro-OH 4.38 1.48 0.71 0.57 1.16

[0032] Step 2: Cutting and deprotection

[0033] (1) Add 50 ml of cutting agent (TFA:TIPS:H2O=95:2.5:2.5, v / v) to the synthesis tube of step 1, and allow N2 to bubble for reaction for 1.5-3 hours.

[0034] (2) After the cleavage reaction is completed, the cleavage agent is filtered into a 250 ml round-bottom flask. After vacuum concentration to one-third of the original volume of the cleavage agent, 10 times the current volume of methyl tert-butyl ether is added and stirred for 30 minutes. The obtained mixed solvent is filtered and washed three times with 30 ml of methyl tert-butyl ether respectively, and the obtained crude peptide product is placed in a sand core funnel and blown dry with N2 in a fume hood to evaporate the solvent until the crude peptide is in powder form. 1.87 g of crude peptide is obtained, with a crude yield of 85.1%.

[0035] Step 3: Purification (salt exchange) and freeze-drying

[0036] The crude peptide obtained in step 2 was purified by HPLC using the following chromatographic parameter condition A. Specifically, the crude peptide obtained in step 2 was dissolved in water and / or acetonitrile and filtered through a 0.45 μm filter membrane; injected; gradient eluted with an acetonitrile-water mobile phase; the target peptide eluate was collected; and finally concentrated by rotary evaporation.

[0037] Chromatographic parameter conditions A:

[0038] Chromatographic column: YMC-Actus Triart C18 30*250mm;

[0039] Eluent A: 0.1% (v / v) TFA / H2O;

[0040] Eluent B: acetonitrile;

[0041] Flow rate: 25 ml / min;

[0042] UV detection wavelength 220nm;

[0043] Table 3 Gradient elution conditions

[0044] Time / min Eluent A (%) Eluent B (%) 0 90 10 30 75 25

[0045] Then, the product obtained in the above steps was salt-exchanged by HPLC using the following chromatographic parameter condition B to obtain the final polypeptide. Specifically, a 95% A1+5% B equilibrated chromatographic column was used; then the sample was injected; then a 95% A2+5% B equilibrated chromatographic column was used; gradient elution was performed using A1 and B; the target peptide eluate was collected; finally, the evaporation was concentrated by rotary evaporation and freeze-dried to obtain a polypeptide (purification yield 71.7%, purity 100%). The structure of the polypeptide was confirmed by MS and 1H-NMR.

[0046] Chromatographic parameter condition B:

[0047] Chromatographic column: YMC-Actus Triart C18 30*250mm

[0048] Eluent A1: 0.1 M acetic acid

[0049] Eluent A2: 0.025M acetic acid + 0.1M ammonium acetate

[0050] Eluent B: acetonitrile

[0051] Flow rate 25ml / min

[0052] UV detection wavelength 220nm

[0053] Table 4 Gradient elution conditions

[0054] Time / min Eluent A1 (%) Eluent B (%) 0 95 5 5 95 5 35 70 30

[0055] Table 5 Synthesized peptides

[0056] Compound sequence Amount of crude product(g) Purification yield purity MS Ala-Glu-Pro-Val-Pro-Leu 2.7 71.7 100％ 625.1

[0057] Peptide:Ala-Glu-Pro-Val-Pro-Leu acetate

[0058] 1 H NMR(600MHz,DMSO)δ8.29(d,J=7.1Hz,1H),7.89(d,=8.6Hz,1H),7.79(d,J=8.0Hz,1H), 4.54(d,J=5.4Hz,1H),4.42-4.38(m,1H),4.37-4.28(m,2H),4.09(dd,J=14.2,8.5Hz,1 H),3.66-3.46(m,5H),2.28(t,J=7.4Hz,2H),2.13-1.71(m,16H,AcOH),1.71-1.58(m,2 H),1.53-1.32(m,2H),1.18(d,J=6.9Hz,3H),0.90-0.83(m,9H),0.80(d,J=6.6Hz,3H).

[0059] Example 2

[0060] Experimental animals: 30 female Wistar rats, SPF grade, room-fed, 6-7 weeks old, 240-260 g; Source: Zhejiang Weitonglihua Experimental Animal Technology Co., Ltd., Experimental Animal Production License: SCXK (Zhejiang) 2019-0001, Experimental Animal Quality Certificate Number: 110324221101323851.

[0061] Model establishment: Weigh 10 mg of lipopolysaccharide (LPS) (based on purity), add 10 mL of normal saline and sonicate until no particles are visible to the naked eye, to obtain a solution with a concentration of 1 mg / mL, store at 4°C, and use within one month. Experimental animals are continuously infused with 20 mg / kg of lipopolysaccharide through the femoral vein within 4 hours to induce experimental disseminated intravascular coagulation.

[0062] Grouping and administration: Group design: model group, heparin sodium group, polypeptide group, 10 mice / group.

[0063] Preparation of polypeptide group: weigh 100 mg of polypeptide, add 31.133 mL of physiological saline for injection, vortex mix and sonicate until no particles are visible to the naked eye, then 3 mg / mL is obtained, which is prepared before use.

[0064] Animals were given medication via the right femoral vein 1 hour before the model was established. The experimental animals were continuously infused with 20 mg / kg lipopolysaccharide via the left femoral vein within 4 hours to induce experimental disseminated intravascular coagulation. If the animals survived until 20 hours after the model was established, they were euthanized and the dead animals were not perfused.

[0065] The test groups, test dosing and test procedures are shown in Table 6:

[0066] Table 6 Experimental groups, experimental medication and experimental procedures

[0067]

[0068] Detection indicators:

[0069] (1) Changes in animal survival time

[0070] Detection time: 24h;

[0071] Detection indicators: survival rate within 24 hours;

[0072] Detection method: Animals were observed for 24 hours, and every 4 hours in the first 12 hours. The animal status and death time were recorded in detail. If animal death was observed in the last 12 hours, the death time was recorded.

[0073] (2) Organ bleeding score

[0074] The lungs and small intestines were dissected and gross images were taken. Scores were given based on the bleeding conditions of the lungs and small intestines. The scoring criteria are shown in Table 7.

[0075] Table 7 Organ bleeding scores

[0076] Degree of bleeding none Point Focal Flake Diffuse score 0 1 2 3 4

[0077] (3) Lung H&E

[0078] According to the KCI pathology SOP standard, the left lung tissue was dehydrated, embedded and sectioned as a whole, and H&E staining was performed for pathological analysis.

[0079] (4) Principles of data analysis and elimination

[0080] Data were collated in Office Excel 2013 and GraphPad Prism 9.0, and data were expressed as Mean ± SEM (standard error). One-way ANOVA was used for analysis, and Tukey's analysis method was used for inter-group comparison and significance test; T-test was used for two-tailed test between two groups when two groups were compared. When p < 0.05, there was a significant difference between the two groups. When the data were statistically analyzed, all relevant data of animals excluded from the efficacy experiment would not be included in the statistical analysis. When there were a few large differences in the data within the group, GraphPad Prism 9.0 could be used for identifyoutliters data analysis to exclude the values ​​with large differences; when the data were statistically analyzed, all relevant data of animals excluded from the efficacy experiment would not be included in the statistical analysis.

[0081] Test results:

[0082] (1) Changes in animal survival time during the experiment

[0083] The changes in animal survival time are shown in Table 8:

[0084] Table 8 Changes in animal survival time (Mean ± SEM)

[0085] Group quantity Animal survival time (h) Model Group 10 5.67±1.40 Heparin sodium group 10 6.59±1.38 Peptide Group 10 <![CDATA[17.78±1.56 * ]]>

[0086] Note: *, P<0.05, vs model group.

[0087] During the experiment, the experimental animals were injected with LPS into the femoral vein within 4 hours to induce the DIC model. The survival time of the animals in the model group was 5.67±1.406h. After 1 hour of administration, LPS was continuously injected into the femoral vein for 4 hours. The survival time of the animals in the sodium heparin group was increased to 6.59±1.38h, but there was no statistical difference compared with the model group. The survival time of the animals in the polypeptide group was significantly increased to 17.78±1.56h, but there was a statistical difference compared with the model group. It shows that the polypeptide provided in this application can significantly improve the survival time of rats in the disseminated intravascular coagulation model.

[0088] (2) Lung and small intestinal bleeding scores

[0089] At the end of the test, gross images of the lungs and small intestines of the test animals were taken, and lung and small intestinal bleeding scores were performed.

[0090] Table 9 Lung and small intestinal bleeding scores

[0091] Group quantity Pulmonary hemorrhage score Small Intestinal Bleeding Score Model Group 10 2.55±0.28 1.64±0.36 Heparin sodium group 10 2.36±0.27 1.36±0.43 Peptide Group 10 1.25±0.27 0.85±0.22

[0092] Note: *, P<0.05, vs model group.

[0093] The results in Table 9 show that compared with the model group, the heparin sodium group had a slight downward trend in the scores of pulmonary bleeding and small intestinal bleeding, but there was no statistical difference. The peptide group had a significant downward trend in the scores of pulmonary bleeding and small intestinal bleeding compared with the model group, with statistical difference.

[0094] (3) Changes of pathological damage in the left lung

[0095] The model group and the polypeptide group were selected for observation and comparison. The subjects were rats with a survival time of less than 7 hours. The changes in pathological damage in the left lung were compared and analyzed.

[0096] Figure 2 A bar graph showing the proportion of thrombosis in large and medium pulmonary arteries (animal survival less than 7 h); Figure 3 A bar graph showing the proportion of thrombosis in pulmonary arterioles and alveolar capillaries (animals surviving less than 7 h); Figure 4 A bar graph of the proportion of pulmonary hemorrhage (animals surviving less than 7 h) is shown;

[0097] Depend on Figure 2-4 It can be seen that the model group animals with survival time less than 7h had a large number of thrombosis in the alveolar wall capillaries, small, medium and large pulmonary arteries in the lungs, and the number of animals with thrombosis accounted for 60% of the total number, the number of animals with thrombosis in large and medium pulmonary arteries accounted for 40% of the total number, and 20% had pulmonary hemorrhage, and no emphysema or alveolar fusion and inflammation + infiltration were observed. Compared with the model group, the proportion of animals with thrombosis in the polypeptide group was reduced, and the number of animals with thrombosis accounted for 10% of the total number, and there was no thrombosis in large and medium arteries, and no cases of pulmonary hemorrhage were observed.

[0098] In summary, experiments using the polypeptide provided by the present invention have shown that the polypeptide can, to a certain extent, significantly prolong the survival time of rats with a disseminated intravascular coagulation model, reduce the bleeding scores of organs, especially the lungs, of rats with a disseminated intravascular coagulation model, reduce alveolar wall capillary thrombosis and inflammatory cell infiltration, and reduce the proportion of large and medium-sized pulmonary artery thrombosis, and reduce alveolar fusion, and has certain application prospects in the preparation of drugs for the treatment of disseminated intravascular coagulation.

[0099] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Use of a polypeptide in the preparation of a drug for treating disseminated intravascular coagulation, characterized in that: The amino acid sequence of the polypeptide is: Ala-Glu-Pro-Val-Pro-Leu.

2. Use of the polypeptide according to claim 1 in the preparation of a drug for treating disseminated intravascular coagulation, characterized in that: The disseminated intravascular coagulation includes: sepsis complicated by disseminated intravascular coagulation.

3. Use of the polypeptide according to claim 1 in the preparation of a drug for treating disseminated intravascular coagulation, characterized in that: The dosage form of the medicine includes one of oral liquid, tablet, granule, capsule, dripping pill, injection and in vivo implant preparation.

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