CN117883468A

CN117883468A - New coronavirus resistant application of sulfonated polysaccharide

Info

Publication number: CN117883468A
Application number: CN202211262590.6A
Authority: CN
Inventors: 刘昌胜; 王晓刚; 王靖
Original assignee: East China University of Science and Technology
Current assignee: East China University of Science and Technology
Priority date: 2022-10-14
Filing date: 2022-10-14
Publication date: 2024-04-16
Also published as: WO2024078312A1

Abstract

The invention discloses an application of sulfonated polysaccharide, which has strong capability of inhibiting new coronavirus from infecting host cells and can be used for preparing medicines for treating and/or preventing new coronavirus pneumonia.

Description

New coronavirus resistant application of sulfonated polysaccharide

Technical Field

The invention relates to the field of biological medicine, in particular to application of sulfonated polysaccharide.

Background

Coronavirus disease in 2019 induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to a global health crisis. The U.S. government has emergency authorities authorized the use of Remdesivir (remdesivir), hydroxychloroquine (hydroxychloroquine), the combination of Nemactevir/ritonavir (Paxlovid) and Mo Nupi of the same (Monnpiravir). Hydroxychloroquine was de-authorized for lack of effectiveness in clinical use, and the world health organization also showed a lack of substantial efficacy in mid-term experiments with adefovir. Whereas mutation of the novel coronavirus Spike protein (Spike, S) resulted in a mutant strain with greater infectivity. The rapid spread of new coronavirus variants suggests that there are significant limitations to the availability of currently approved anti-new coronadrugs, neutralizing antibodies, and new coronavaccines. Despite the current new use of a number of older drugs, such as alzvudine, there is still a need for effective new drugs or new strategies for treating and suppressing new crown infections.

For severe and critical patients with symptoms such as thrombus after infection of the new coronavirus, clinical report that the use of high-dose heparin can promote the healing of the patients with the new coronavirus, however, the high-dose heparin is accompanied with the risk of massive hemorrhage, which greatly limits the research of heparin in the field of new coronavirus treatment. The novel coronavirus requires that the receptor binding domain (Receptor Binding Domain, RBD) of the viral surface S protein bind to the host cell surface receptor Angiotensin converting enzyme 2 (Angiotenin-Converting Enzyme, ACE 2). Anti-neocoronavirus monoclonal antibodies (Monoclonal antibodies, mAbs) exert inhibitory effects against neocoronavirus infection by targeting the RBD of the S protein, the RBD-ACE2 binding site, or by effectively neutralizing the binding activity. However, monoclonal antibodies are used in relatively high doses, requiring 4-100mg/kg to be effective, storage and transport conditions are stringent. This greatly increases the cost of treatment, in addition to which the most important drawbacks of monoclonal antibodies are low efficacy against the variants, in particular the ommicon variant strain. Therefore, how to find antiviral drugs with broad spectrum is of great significance for suppressing rapid mutation of new coronaviruses.

Disclosure of Invention

The invention aims at providing application of sulfonated polysaccharide.

In a first aspect of the invention there is provided the use of a sulphonated polysaccharide for the manufacture of a medicament for the treatment and/or prophylaxis and alleviation of diseases caused by coronavirus infection.

In another preferred embodiment, the sulfonated polysaccharide has a molecular weight of 3000-350000Da and has a strong ability to inhibit infection of host cells by coronaviruses, in particular to inhibit/attenuate invasion of host cells by coronaviruses based on new coronaviruses.

In another preferred embodiment, the sulfonated polysaccharide is selected from the group consisting of: sulfonated chitosan, sulfonated dextran, heparin sulfate, cellulose sulfate, and chondroitin sulfate.

In another preferred example, the molecular weight of the sulfonated chitosan is 8000-35000Da and the sulfur content is 0.5% -20%.

In another preferred embodiment, the sulfonated dextran has a molecular weight of 15000-30000Da.

In another preferred embodiment, the molecular weight of chondroitin sulfate is 37000Da to 40000Da.

In another preferred embodiment, the coronavirus is SARS-CoV-2 or a variant strain thereof.

In another preferred embodiment, the variant strain is selected from the group consisting of: alpha strain, beta strain, gamma strain, delta strain, omicron strain.

In another preferred embodiment, the omacron strain is selected from the group consisting of: omicron BA2, BA4 and BA5.

The sulfonated polysaccharide can inhibit the infection of the host cells by the novel coronavirus; the method has broad spectrum and multiple effects. Broad spectrum is effective against a variety of novel crown variants (wild-type, delta and Omicron strains); the multiple efficacy includes: anti-inflammatory effects, alleviating viral inflammatory responses; improving the virus phagocytic capacity of immune cells, and being beneficial to rapidly removing viruses; can be used in combination with therapeutic drugs having heparin binding domain, such as interferon, monoclonal antibody, etc., to prolong half-life of the drug and enhance therapeutic effect.

In another preferred embodiment, the medicament is administered orally, by injection, by inhalation or by the luminal route.

In another preferred embodiment, the medicament further comprises pharmaceutically acceptable excipients selected from the group consisting of: solvents, diluents, disintegrants, precipitation inhibitors, surfactants, glidants, binders, lubricants, dispersants, suspending agents, isotonic agents, thickening agents, emulsifiers, preservatives, stabilizers, hydration agents, emulsification accelerators, buffers, absorbents, colorants, flavorants, sweeteners, ion exchangers, mold release agents, coating agents, flavoring agents, antioxidants, preservatives, carbohydrates, fats, vitamins, amino acids, trace elements, or proteins.

In another preferred embodiment, the disease caused by a coronavirus infection is a respiratory and/or digestive system new coronavirus infection.

In another preferred embodiment, the dosage form of the drug is selected from the group consisting of: capsules, tablets, granules, spray, gel, sustained release agents, oral liquid, dripping pills and nano preparations.

In another preferred embodiment, the sulfonated polysaccharide is dissolved in ultrapure water/physiological saline/phosphate buffer solution and then used, and the sulfonated polysaccharide is prepared into spray/gel to prevent the infection of host cells by the new coronavirus and accelerate the removal of the new coronavirus in vivo. In another preferred embodiment, the sulfonated polysaccharide solution has a concentration of 4.25nM to 400nM (187.5 ng/mL to 12000 ng/mL). The higher the concentration, the greater the ability of the sulfonated polysaccharide complex to inhibit infection of host cells by the new crown.

In another preferred embodiment, the invention provides the use of sulfonated chitosan for the preparation of a medicament for the treatment and/or prevention, alleviation of diseases caused by coronavirus infections, in particular diseases caused by Omicron BA2, BA4 and BA5 infections.

Experiments of molecular docking and protein interaction of the present invention show that sulfonated polysaccharides can inhibit infection of host cells by multiple novel coronavirus subtypes (e.g., omicron BA2, BA4, and BA 5). These results prove that the sulfonated polysaccharide can be used for scientific research and clinical application of new coronaviruses, and the sulfonated polysaccharide can be used for inhibiting the infection of the new coronaviruses to host cells, so that corresponding new coronavirus prevention and treatment strategies can be developed for inhibiting the prevention and treatment of the infection of the new coronaviruses and other various occasions.

It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. Each feature disclosed in the description may be replaced by alternative features serving the same, equivalent or similar purpose. And are limited to a space, and are not described in detail herein.

Drawings

FIG. 1 shows the result of the docking of sulfonated polysaccharide with Omicron BA2.2 RBD molecules, wherein the amino acids in the enlarged region are residues of the sulfonated polysaccharide having hydrogen bond interactions with proteins.

FIG. 2 shows the results of Omicron BA2 RBD interfacing with ACE2 molecules, cyan ACE protein; green Spike protein, the amino acids in the enlarged region are Omicron BA2 RBD and ACE2 binding residues.

FIG. 3 shows a sulfonated polysaccharide, omacron BA2.2 RBD and ACE2 trimer complex molecular docking model, cyan ACE2 protein; green Spike protein; yellow sulfonated polysaccharide complex, and the enlarged part is the binding site of sulfonated polysaccharide and RBD/ACE2 amino acid

FIG. 4 shows that sulfonated polysaccharide inhibits Omicron BA2 RBD protein and ACE2 protein IC ₅₀ Response curves.

FIG. 5 shows that sulfonated polysaccharide inhibits Omicron BA4/BA5RBD protein and ACE2 protein IC ₅₀ Response curves.

FIG. 6 is a graph showing the binding curves of Omacron BA4/BA5RBD proteins to ACE2 protein at various concentrations for SPR examination.

FIG. 7 is a graph of SPR examination of binding of Omacron BA4/BA5RBD protein to ACE2 protein at various concentrations under intervention of sulfonated polysaccharide (1 mg/mL).

FIG. 8 shows the results of an in vitro anticoagulation time test of sulfonated materials.

Detailed Description

The inventors of the present application have studied extensively and intensively, and found that sulfonated polysaccharides inhibit infection of host cells by various novel coronavirus subtypes (Omicron BA2, BA4 and BA 5), and as a result, they have shown that sulfonated polysaccharides can tightly bind to Omicron Spike (BA 2) RBD, inhibit binding of Omicron Spike (BA 2, BA4 and BA 5) RBD to ACE2, and can be used for treating and/or preventing, alleviating diseases caused by coronavirus infection. In addition, the sulfonated polysaccharide has no anticoagulation effect in the dosage range, and has no major bleeding risk in the process of promoting the cure of patients with new crown infection. On this basis, the present invention has been completed.

Terminology

Sulfonated polysaccharide

Polysaccharide (polysaccharide) is a polymeric sugar high molecular carbohydrate composed of sugar chains bonded by glycosidic bonds, and at least more than 10 monosaccharides. Including chitosan, starch, aminoglycans, and cellulose, etc.

The sulfonated polysaccharide is a product obtained by sulfonation reaction of polysaccharide.

Sulfonation is a process of introducing sulfonic acid groups (-SO) into an organic molecule ₃ H) Or sulfonyl chloride (-SO) ₃ Cl). As the sulfonating agent, sulfur trioxide, concentrated sulfuric acid, fuming sulfuric acid and the like are usually used. Chlorosulfonic acid, sulfur dioxide plus chlorine, sulfur dioxide plus oxygen, sodium sulfite, and the like are also sometimes used as sulfonating agents. The sulfonation method includes a liquid-phase sulfonation method and a gas-phase sulfonation method. The replacement of hydrogen on a carbon atom by a sulfonic acid group during sulfonation is referred to as direct sulfonation; the sulfonic acid group replaces the halogen or nitro group on the carbon atom, known as indirect sulfonation.

The sulfonated polysaccharide is one or more than two selected from sulfonated chitosan, sulfonated dextran, heparin, heparan sulfate and cellulose sulfate. The chitosan and glucan are used as raw materials by a method known in the art, and are prepared after sulfonation. The sulfonated chitosan and sulfonated dextran are prepared, for example, by chlorosulfonic acid sulfonation or concentrated sulfuric acid sulfonation (Jilin university journal, 2007,24 (2): 20. University of West China university journal, 2006,27 (1): 95).

The sulfur content of the sulfonated polysaccharide was determined by elemental analysis (Elementar, vario Macro company, germany).

The molecular weight of the sulfonated polysaccharide is 8000-350000Da, and the sulfur content is 0.5-20%. In another preferred embodiment, the sulfonated polysaccharide has a molecular weight of 10000-300000Da and a sulfur content of 1% -20%.

The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedures, which do not address the specific conditions in the examples below, are generally carried out under conventional conditions (e.g.those described in Sambrook et al, molecular cloning: A laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989)) or under conditions recommended by the manufacturer. Percentages and parts are weight percentages and parts unless otherwise indicated.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred methods and materials described herein are presented for illustrative purposes only.

Example 1

Preparation of sulfonated polysaccharide solution

Dissolving sulfonated chitosan with molecular weight of 30000Da and sulfur content of 13% in deionized water to prepare sulfonated chitosan aqueous solution.

Example 2

The purpose of this example was to demonstrate that the sulfonated polysaccharide solution of example 1 inhibited infection of host cells with the novel coronavirus (omacron BA 2).

The sulfonated polysaccharide solutions (833. Mu.g/mL, 416. Mu.g/mL, 208. Mu.g/mL, 104. Mu.g/mL, 52. Mu.g/mL, 26. Mu.g/mL, 13. Mu.g/mL, 6.5. Mu.g/mL, 3.25. Mu.g/mL, 1.625. Mu.g/mL) of the different concentrations formulated in example 1 were added to a solution containing Omicron Spike RBD (BA.2) 0.75. Mu.g/mL, the sulfonated polysaccharide solution was mixed with RBD solution in equal volumes, and the binding levels of ACE2 to RBD were detected using ELISA detection kit.

FIG. 4 shows the inhibition of Omicron BA2 RBD protein and ACE2 protein IC by sulfonated polysaccharide ₅₀ Response curve, ELISA experiments prove that the sulfonated polysaccharide can effectively inhibit Omicron Spike RBD (BA.2) from combining with ACE2, and IC ₅₀ 138.4 μg/mL.

The three-dimensional structure of the protein refers to the PDB database, encoding bit 7T9L. The sulfonated polysaccharide was plotted against Chemdraw. Molecular docking was performed using autodock4.2 software. Omicron Spike RBD (BA 2) was used for this molecular docking. The lowest energy conformation was selected for molecular docking and the interaction between ligand and protein was analyzed using pymol visualization.

The results in FIG. 1 show that sulfonated polysaccharide can form a large number of hydrogen bonds with the Omicron BA2 RBD binding interface, indicating that sulfonated polysaccharide can tightly bind to the Omicron Spike (BA 2) RBD.

FIG. 2 shows that Omacron Spike (BA 2) RBD binds tightly to ACE2 protein without intervention of sulfonated polysaccharide with free binding energy ΔG of-14.9 kcal/mol.

FIG. 3 shows that sulfonated polysaccharide has a binding energy ΔG of-8.9 kcal/mol between Omicron Spike (BA 2) RBD and ACE2 protein interface and between Omicron Spike (BA 2) RBD and ACE 2.

The above results demonstrate that sulfonated polysaccharide can bind tightly to Omicron Spike (BA 2) RBD and reduce Omicron Spike (BA 2) RBD binding to ACE2 by 40.3%.

Example 3

The purpose of this example was to demonstrate that the sulfonated polysaccharide solution of example 1 inhibited infection of host cells with the novel coronavirus (omacron BA4/BA 5).

Sulfonated polysaccharide solutions of different concentrations (833. Mu.g/mL, 416. Mu.g/mL, 208. Mu.g/mL, 104. Mu.g/mL, 52. Mu.g/mL, 26. Mu.g/mL, 13. Mu.g/mL, 6.5. Mu.g/mL, 3.25. Mu.g/mL, 1.625. Mu.g/mL) formulated as in example 1 were added to a solution containing Omicron Spike RBD (BA.4/BA.5) 0.75. Mu.g/mL, the sulfonated polysaccharide solution was mixed with the RBD solution in equal volumes, and the ACE2 binding content to the RBD was detected using ELISA detection kit.

FIG. 5 shows the inhibition of Omicron BA2 RBD protein and ACE2 protein IC by sulfonated polysaccharide ₅₀ Response curve, ELISA experiments prove that the sulfonated polysaccharide can effectively inhibit Omicron Spike RBD (BA.2) from combining with ACE2, and IC ₅₀ 108.9. Mu.g/mL.

Example 4

The purpose of this example was to demonstrate that the sulfonated polysaccharide solution of example 1 can reduce the affinity of the novel coronavirus (omacron BA 5) for host cells.

The binding constants between omacron Spike (BA 5) (6.25 nM, 12.5nM, 25nM, 50nM, 100nM, 200nM, 400 nM) and ACE2 proteins were measured at different concentrations using a surface plasmon resonance (Surface plasma resonance, SPR) instrument. At the same time, the binding constants between the various concentrations of Omicron Spike (BA 5) and ACE2 protein described above were examined with SPR under intervention of sulfonated polysaccharide solution (1 mg/mL).

FIG. 6 is a response curve of Omacron Spike (BA 5) and ACE2 protein without intervention of sulfonated polysaccharide, and quantification results show that the binding rate Ka 2.93×10 ⁴ Separation rate Kd 2.61×10 ^-4 Affinity constant was KD 8.90×10 ^-9 。

FIG. 7 is a response curve of Omacron Spike (BA 5) with ACE2 protein under sulfonated polysaccharide intervention, and quantification shows a binding rate Ka 1.79×10 ⁴ 、Kd 6.62×10 ^-4 、KD 3.70×10 ^-8 . The results demonstrate that the Ka binding constant of omacron Spike (BA 5) protein to ACE2 was reduced by 38.9% with sulfonated polysaccharide (1 mg/mL) intervention (binding becomes difficult); the dissociation constant Kd is improved by 60.7 percent (dissociation becomes easy); the affinity KD is reduced by 75.9% (reduced affinity).

The results indicate that the sulfonated polysaccharide effectively inhibits the binding of omacron Spike (BA 5) protein to ACE 2.

Example 5

This example demonstrates that the sulfonated polysaccharide does not have an anticoagulant effect.

Fresh rabbit blood was first collected in BD anticoagulation tube via rabbit ear vein, anticoagulation assay procedure as follows: 200. Mu.L of blood was added to a 1.5mL EP tube, and 20. Mu.L of 0.2M CaCl2 was added to neutralize sodium citrate in the blood. Then 20. Mu.L of the sulfonated chitosan of example 1 (2. Mu.g/mL in PBS) was added, and the blank was added with an equal volume of PBS. Immediately after the sample was thoroughly mixed, it was placed at 37 ℃ and timing was started, and after the blood was completely coagulated, the timing was stopped and the clotting time was recorded.

The results in fig. 8 show no significant difference between sulfonated chitosan SCS and PBS group, indicating that SCS does not have anticoagulant properties.

Example 6

Dissolving sulfonated chitosan with molecular weight of 50000Da and sulfur content of 15% in deionized water to prepare sulfonated chitosan water solution.

Dissolving sulfonated chitosan with molecular weight of 100000Da and sulfur content of 15% in deionized water to prepare sulfonated chitosan water solution.

The sulfonated chitosan with the molecular weight of 140000Da and the sulfur content of 16 percent is dissolved in deionized water to prepare the sulfonated chitosan aqueous solution.

Dissolving sulfonated chitosan with molecular weight of 200000Da and sulfur content of 19% in deionized water to prepare sulfonated chitosan water solution.

The sulfonated chitosan with molecular weight of 220000Da and sulfur content of 16% is dissolved in deionized water to prepare sulfonated chitosan aqueous solution.

The sulfonated chitosan with the molecular weight of 250000Da and the sulfur content of 18 percent is dissolved in deionized water to prepare the sulfonated chitosan aqueous solution.

The sulfonated chitosan solution was tested according to the methods of examples 2 to 6, and the results showed that the sulfonated chitosan could closely bind to Omicron Spike (BA 2) RBD and decreased the binding ability of Omicron Spike (BA 2) RBD to ACE 2; effectively inhibit the binding of Omicron Spike (BA 5) protein to ACE 2; and does not have anticoagulant properties.

Example 7

The solution of novel coronavirus (Wild type) was mixed homogeneously in a ratio of 1:4 by volume of the sulfonated polysaccharide solution of example 1. 25. Mu.L of pseudovirus product was dissolved in 75. Mu.L of sulfonated polysaccharide solutions of different concentrations (1000. Mu.g/mL, 500. Mu.g/mL, 250. Mu.g/mL, 125. Mu.g/mL, 62.5. Mu.g/mL, 31.25. Mu.g/mL, 15.625. Mu.g/mL, 7.8125. Mu.g/mL, 3.90625. Mu.g/mL, 1.953125. Mu.g/mL), respectively. The pseudovirus-containing solution is added into a human embryo kidney cell pore plate which is over-expressed with ACE2 protein after being evenly mixed for culturing for 48 hours. Subsequently, the cells were added with a fluorescence detection reagent and the fluorescence intensity was detected with a microplate reader. The test results show that the sulfonated polysaccharide can inhibit the infection of human embryo kidney cells by the novel coronavirus (Wild type).

Example 8

The solution of novel coronavirus (Delta) was mixed homogeneously in a ratio of 1:4 by volume of the sulfonated polysaccharide solution of example 1. 25. Mu.L of pseudovirus product was dissolved in 75. Mu.L of sulfonated polysaccharide complex solutions of different concentrations (1000. Mu.g/mL, 500. Mu.g/mL, 250. Mu.g/mL, 125. Mu.g/mL, 62.5. Mu.g/mL, 31.25. Mu.g/mL, 15.625. Mu.g/mL, 7.8125. Mu.g/mL, 3.90625. Mu.g/mL, 1.953125. Mu.g/mL), respectively. The pseudovirus-containing solution is added into a human embryo kidney cell pore plate which is over-expressed with ACE2 protein after being evenly mixed for culturing for 48 hours. Subsequently, the cells were added with a fluorescence detection reagent and the fluorescence intensity was detected with a microplate reader. The test results show that the sulfonated polysaccharide can inhibit the infection of human embryo kidney cells by the novel coronavirus (Delta).

Example 9

The solution of novel coronavirus (omacron BA 2) was homogeneously mixed with the sulfonated polysaccharide solution of example 1 in a volume ratio of 1:4. 25. Mu.L of pseudovirus product was dissolved in 75. Mu.L of sulfonated polysaccharide complex solutions of different concentrations (1000. Mu.g/mL, 500. Mu.g/mL, 250. Mu.g/mL, 125. Mu.g/mL, 62.5. Mu.g/mL, 31.25. Mu.g/mL, 15.625. Mu.g/mL, 7.8125. Mu.g/mL, 3.90625. Mu.g/mL, 1.953125. Mu.g/mL), respectively. The pseudovirus-containing solution is added into a human embryo kidney cell pore plate which is over-expressed with ACE2 protein after being evenly mixed for culturing for 48 hours. Subsequently, the cells were added with a fluorescence detection reagent and the fluorescence intensity was detected with a microplate reader. The test results show that the sulfonated polysaccharide can inhibit the infection of human embryo kidney cells by the novel coronavirus (Omicron BA 2).

All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.

Claims

1. The use of a sulfonated polysaccharide for the preparation of a medicament for the treatment and/or prophylaxis and/or alleviation of diseases caused by coronavirus infections.

2. The use according to claim 1, wherein the sulfonated polysaccharide is selected from the group consisting of: sulfonated chitosan, sulfonated dextran, heparin sulfate, cellulose sulfate, and chondroitin sulfate.

3. The use according to claim 2, wherein the sulfonated chitosan has a molecular weight of 8000-35000Da and a sulphur content of 0.5% -20%.

4. The use according to claim 1, wherein the coronavirus is SARS-CoV-2 or a variant strain thereof.

5. The use of claim 4, wherein the variant strain is selected from the group consisting of: alpha strain, beta strain, gamma strain, delta strain, omicron strain.

6. The use according to claim 5, wherein said omacron strain is selected from the group consisting of: omicron BA2, BA4 and BA5.

7. The use according to claim 1, wherein the medicament is administered orally, by injection, by inhalation or via the luminal tract.

8. The use according to claim 1, wherein the medicament further comprises a pharmaceutically acceptable adjuvant selected from the group consisting of: solvents, diluents, disintegrants, precipitation inhibitors, surfactants, glidants, binders, lubricants, dispersants, suspending agents, isotonic agents, thickening agents, emulsifiers, preservatives, stabilizers, hydration agents, emulsification accelerators, buffers, absorbents, colorants, flavorants, sweeteners, ion exchangers, mold release agents, coating agents, flavoring agents, antioxidants, preservatives, carbohydrates, fats, vitamins, amino acids, trace elements, or proteins.

9. Use according to claim 1, wherein the disease caused by coronavirus infection is a respiratory and/or digestive system new coronavirus infection.

10. The use according to claim 1, wherein the pharmaceutical dosage form is selected from the group consisting of: capsules, tablets, granules, spray, gel, sustained release agents, oral liquid, dripping pills and nano preparations.