KR102641224B1 - Pharmaceutical compositions for preventing or treating SARS-CoV-2 infection - Google Patents

Abstract

The present invention relates to a composition for preventing or treating SARS-CoV-2 virus infection. It was completed by confirming that biopolymer nanoparticles with DNase-I protein bound to the surface exhibit excellent effects in treating SARS-CoV-2 virus infection. It has been done. As a result of treating the plasma of severe COVID-19 patients with the nanoparticles according to the present invention, it was confirmed that the activity of DNase-I was increased and the NETosis of neutrophils, the level of extracellular DNA, and the level of various inflammatory cytokines were decreased. A COVID-19-like animal model administered nanoparticles also confirmed that the production of various inflammatory factors and tissue damage were reduced as well as the survival rate was increased. In other words, the DNase-I nanoparticle according to the present invention can effectively treat SARS-CoV-2 virus infection by alleviating excessive inflammatory response caused by SARS-CoV-2 virus infection and increasing DNase-I activity, thereby preventing the new coronavirus. It is expected to be used as a treatment strategy.

Description

Pharmaceutical compositions for preventing or treating SARS-CoV-2 virus infection {Pharmaceutical compositions for preventing or treating SARS-CoV-2 infection}

The present invention relates to a composition for preventing or treating SARS-CoV-2 virus (Severe acute respiratory syndrome coronavirus 2) infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a new coronavirus that emerged in 2019, and infection is causing the coronavirus disease 2019 (COVID-19) pandemic. poses a serious threat to public health worldwide. According to recent statistics, there have been more than 100,000 deaths and approximately 2,000,000 confirmed cases in 180 countries around the world. The number of confirmed cases continues to increase, and experts predict that hundreds of thousands of deaths will occur worldwide.

SARS-CoV-2 infection causes abnormal adaptive immune responses and inflammatory innate immune responses. A rapid immune response can cause septic shock, increasing the patient's likelihood of death. Therefore, the development of new treatment strategies that can inhibit the progression of sepsis caused by SARS-CoV-2 infection is urgently needed.

Numerous research teams and global pharmaceutical companies are conducting clinical trials on SARS-CoV-2 treatment candidates. However, most of these drugs were not developed for the treatment of SARS-CoV-2 infection, but rather Ebola virus, human immunodeficiency virus (HIV), and severe acute respiratory syndrome. , SARS) virus, Middle East respiratory syndrome (MERS) virus, Influenza A virus, and ZIKA virus.

Various clinical studies have shown that COVID-19 patients have a particularly difficult time overcoming cytokine release syndrome (CRS) and severe respiratory damage. Recently, U.S. biotechnology company Genentech announced FDA approval for a phase 3 clinical trial of Tocilizumab (Actemra) to treat hospitalized patients with severe SARS-CoV-2 pneumonia. However, even if the viral infection is resolved, not only will the patient's tissue damage not fully recover, but more serious diseases such as acute respiratory distress syndrome (ARDS) or sepsis may develop, and in severe cases, death. It can reach.

Currently, the World Health Organization (WHO) does not recommend the use of corticosteroids in the treatment of patients with COVID-19, as corticosteroids may worsen SARS-CoV-2-related acute respiratory infections. Because. Because SARS-CoV-2 can cause a variety of acute complications and death, there is an urgent need for new strategies to rapidly resolve acute respiratory-related inflammatory responses.

Republic of Korea Patent Publication No. 10-1421343

The present invention was devised to solve the above problem, and the present inventors completed the present invention by confirming that melanin nanoparticles with DNase-I bound to the surface exhibit an excellent treatment effect for SARS-CoV-2 virus infection. .

Therefore, the purpose of the present invention is to provide a pharmaceutical composition for preventing or treating SARS-CoV-2 virus infection, which contains biopolymer nanoparticles with DNase-I protein bound to the surface as an active ingredient.

However, the technical problem to be achieved by the present invention is not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art from the description below. There will be.

The present invention provides a pharmaceutical composition for preventing or treating SARS-CoV-2 virus infection, which contains biopolymer nanoparticles with DNase-I protein bound to the surface as an active ingredient.

In one embodiment of the present invention, the biomaterial is melanin, chitosan, alginate, heparin, cellulose, gelatin, polyvinyl alcohol, poly Polyvinylpyrrolidione, collagen, gelatin, polycaprolactone, Poly(lactic-co-glycolic acid), polylactide, and It may be one or more selected from the group consisting of their derivatives, but is not limited thereto.

In another embodiment of the present invention, the biopolymer nanoparticle may have a surface coated with polyethylene glycol, but is not limited thereto.

In another embodiment of the present invention, the biopolymer nanoparticle with the DNase-I protein bound to its surface may be characterized by satisfying one or more of the following characteristics, but is not limited thereto:

(a) the average diameter of the nanoparticles is 100 to 300 nm;

(b) the surface charge of the nanoparticles is -20 mV to -0.1 mV;

(c) the polydispersity of the nanoparticles is 0.02 to 0.15; or

(d) The nanoparticles are spherical.

In another embodiment of the present invention, the binding ratio of DNase-I protein to the biopolymer nanoparticle may be 10 to 70% by weight, but is not limited thereto.

In another embodiment of the present invention, the SARS-CoV-2 virus infection includes coronavirus infection-19, sepsis, acute respiratory syndrome, pneumonia, cytokine storm, cytokine release syndrome, systemic inflammatory response syndrome, and multiple organ failure, but is not limited thereto.

In another embodiment of the present invention, the pharmaceutical composition is used for the treatment of individuals with increased levels of extracellular DNA and citrullinated histone H3 in the blood compared to normal people. It may be for, but is not limited to.

In another embodiment of the present invention, the pharmaceutical composition may be for the treatment of individuals with reduced activity or level of DNase-I compared to normal people, but is not limited thereto.

In another embodiment of the present invention, the pharmaceutical composition may increase the activity or level of DNase-I in the blood, but is not limited thereto.

In another embodiment of the present invention, the pharmaceutical composition promotes neutrophil proliferation, myeloid peroxidase activity, neutrophil elastase activity, neutrophil extracellular trap formation (NETosis), and neutrophil NF-κB activity. It is possible to suppress one or more selected from the group consisting of, but is not limited to this.

In another embodiment of the present invention, the pharmaceutical composition can inhibit the production of inflammatory cytokines, but is not limited thereto.

In another embodiment of the present invention, the inflammatory cytokine may be one or more selected from the group consisting of IL-1β, IL-6, IL-8, CCL2, IFN-γ, and TNF-α, but is not limited thereto. .

In addition, the present invention provides a method for preventing or treating SARS-CoV-2 virus infection, comprising administering biopolymer nanoparticles with the DNase-I protein bound to the surface to an individual in need thereof.

In addition, the present invention provides the use of biopolymer nanoparticles with the DNase-I protein bound to their surface for the manufacture of drugs for the treatment of SARS-CoV-2 virus infection.

In addition, the present invention provides the use of biopolymer nanoparticles with the DNase-I protein bound to their surface for the prevention or treatment of SARS-CoV-2 virus infection.

Additionally, the present invention provides a kit for preventing or treating SARS-CoV-2 virus infection, including the composition according to the present invention.

In addition, the present invention provides a quasi-drug composition for preventing or improving SARS-CoV-2 virus infection, which contains biopolymer nanoparticles with DNase-I protein bound to the surface as an active ingredient. In addition, the present invention provides a quasi-drug for preventing or improving viral infections comprising the quasi-drug composition.

In addition, the present invention provides a method for producing biopolymer nanoparticles with DNase-I protein bound to the surface, comprising the following steps:

(S1) preparing nanoparticles by adding an alkaline solution to the biopolymer solution; and

(S2) Adding the nanoparticles obtained in step (S1) to a buffer containing DNase-I.

In one embodiment of the present invention, the buffer solution containing DNase-I in step (S2) may further include polyethylene glycol, but is not limited thereto.

In another embodiment of the present invention, in step (S2), the nanoparticles and DNase-I may be mixed at a weight ratio of 1:0.2 to 2 (nanoparticles: DNase-I), but the mixture is not limited thereto.

The present invention relates to a composition for preventing or treating SARS-CoV-2 virus infection. It was completed by confirming that biopolymer nanoparticles with DNase-I protein bound to the surface exhibit excellent effects in treating SARS-CoV-2 virus infection. It has been done. As a result of treating the plasma of severe COVID-19 patients with the nanoparticles according to the present invention, it was confirmed that the activity of DNase-I was increased and the NETosis of neutrophils, the level of extracellular DNA, and the level of various inflammatory cytokines were decreased. A COVID-19-like animal model administered nanoparticles also confirmed that the production of various inflammatory factors and tissue damage were reduced as well as the survival rate was increased. In other words, the DNase-I nanoparticle according to the present invention can effectively treat SARS-CoV-2 virus infection by alleviating excessive inflammatory response caused by SARS-CoV-2 virus infection and increasing DNase-I activity, thereby preventing the new coronavirus. It is expected to be used as a treatment strategy.

Figure 1 shows lung CT scan results in patients with mild or severe COVID-19.
Figure 2 is a diagram showing a one-pot process for producing DNase-I pMNS.
Figure 3 is a graph showing the particle size distribution of bMNS, pMNS, and DNase-I pMNS (bMNS, bare melanin-like nanoparticles; pMNS, PEG-coated melanin-like nanoparticles; DNase -I pMNS, pMNS whose surface is coated with DNase-I; same hereinafter).
Figure 4 is a graph of the zeta potential of bMNS, pMNS, and DNase-I pMNS.
Figure 5 is an SEM image of bMNS, pMNS, and DNase-I pMNS. Scale bar: 500 nm.
Figure 6 shows the results of observing the migration profile of DNA after treatment with free DNase-I, bMNS, pMNS, or DNase-I pMNS at various concentrations.
Figure 7a shows the results of confirming the degree of DNA degradation over time after incubating long-acting DNase-I pMNS (1 μg) in PBS containing 10% FBS.
Figure 7b shows the results of confirming the degree of DNA degradation over time after incubating long-acting DNase-I pMNS (1 μg) in simple PBS.
Figure 8 shows the results of measuring changes in eDNA levels and DNase-I activity after treating the plasma of severe COVID-19 patients with pMNS, free DNase-I, or DNase-I pMNS.
Figure 9 shows changes in NET levels, MPO activity, and NE levels of neutrophils isolated from severe COVID-19 patients after treating the plasma of severe COVID-19 patients with pMNS, free DNase-I, or DNase-I pMNS. Shows one result.
Figure 10 shows changes in NF-κB activity in PBMCs of severe COVID-19 patients after treatment of plasma from severe COVID-19 patients with pMNS, free DNase-I, or DNase-I pMNS; and the results of measuring changes in the levels of inflammatory cytokines IL-1β, IL-6, IFN-γ, and TNF-α.
Figure 11 shows the results of confirming the survival rate (Kaplan-Meier curve) over time after treating CLP mice with PBS, PEG-Nano, free DNase-I, or DNase-I pMNS.
Figure 12 shows the results of confirming lung tissue changes in the control group and the DNase-I or DNase-I pMNS administered group 3 hours after CLP surgery in mice. Scale bar: 100 μm.
Figure 13 shows the results of confirming changes in neutrophils, NET, eDNA, DNase activity, NE activity, and MPO activity in the mouse abdominal cavity according to administration of PBS, pMNS, free DNase-I, or DNase-I pMNS after CLP surgery.
Figure 14 shows the results of confirming changes in sepsis markers (ALT, AST, BUN, creatine, LDH, and CRP) in mice according to administration of PBS, pMNS, free DNase-I, or DNase-I pMNS after CLP surgery.
Figure 15 shows the results of measuring the absolute neutrophil count, absolute white blood cell count, and total white blood cell count of mice following administration of PBS, pMNS, free DNase-I, or DNase-I pMNS after CLP surgery.
Figure 16 shows the results of mRNA analysis of inflammation-related genes in lung tissue according to the administration of DNase-I pMNS after CLP surgery in mice as a heatmap.
Figure 17 shows the results of comparing the degree of recovery of vascular barrier integrity in mice according to administration of PBS, pMNS, free DNase-I, or DNase-I pMNS after CLP surgery.
Figure 18 shows the results of confirming the survival rate (Kaplan-Meier curve) over time after treating CLP mice with DNase-I pMNS at various concentrations.

The present invention relates to a composition for preventing or treating SARS-CoV-2 virus infection. It was completed by confirming that biopolymer nanoparticles with DNase-I protein bound to the surface exhibit excellent effects in treating SARS-CoV-2 virus infection. It has been done.

Specifically, in one embodiment of the present invention, blood samples from COVID-19 patients were analyzed and CT scans were performed, and it was confirmed that severe sepsis appeared and the number of neutrophils was significantly increased in severe COVID-19 patients (Example 1).

In another embodiment of the present invention, as a result of analyzing blood samples from normal people, mild COVID-19 patients, and severe COVID-19 patients, NETosis markers eDNA (Extracellular DNA) and citrullinated histone H3 ( It was confirmed that the level of citrullinated histone H3) was increased, and in particular, the level of DNase-I was confirmed to be significantly reduced in severe COVID-19 patients (Example 2).

In another embodiment of the present invention, nanoparticles made of melanin, a biopolymer, are prepared, coated with polyethylene glycol, and then DNase-I is bound to produce biopolymer nanoparticles with DNase-I bound to the surface (DNase pMNS). ) were prepared, and the size and surface charge of the nanoparticles were confirmed (Example 3).

In another example of the present invention, when DNase pMNS was treated with the plasma of severe COVID-19 patients, it was confirmed that the level of eDNA in the plasma was decreased and DNase-I activity was increased, and the activity of NF-κB and various inflammatory cytokines were confirmed. It was confirmed that the level decreased (Example 4).

In another embodiment of the present invention, a COVID-19-like animal model was created using cecal ligation-perforation surgery and the in vivo effect of DNase pMNS was confirmed. As a result, mice administered DNase pMNS were treated with free DNase-I as well as the control group. It was confirmed that the survival rate and DNase-I activity were increased, tissue damage was reduced, and the levels of various inflammatory cytokines were reduced compared to the mice that received them (Example 5).

Hereinafter, the present invention will be described in detail.

The purpose of the present invention is to provide a pharmaceutical composition for preventing or treating SARS-CoV-2 virus infection, which contains biopolymer nanoparticles with DNase-I protein bound to the surface as an active ingredient.

In the present invention, “SARS-CoV-2 virus” refers to Severe acute respiratory syndrome coronavirus 2, a new coronavirus that occurred in December 2019. This is a positive-sense single-stranded RNA virus, and its genome sequence consists of approximately 30,000 bases. The virus is known to have a high infectivity in humans and is highly contagious and has a significant mutation rate.

In the present invention, “DNase-I (deoxyribonuclease-I)” refers to an endonuclease that hydrolyzes the phosphodiester bond inside the DNA molecule chain. In particular, DNase-I from Roche was used as DNase-I for preparing the pharmaceutical composition according to the present invention, but this is only used as a representative example of DNase-I enzyme and is not limited thereto, and other commercially available DNase-I enzymes are used. I, as well as native DNase-I, DNase-I expressed from recombinant DNA prepared by genetic engineering methods, or chemically synthesized DNase-I, etc. can be applied to the present invention. Additionally, the DNase-I enzyme according to the present invention may be isoform 1 or isoform 2. Specific information about DNase-I can be found at NCBI (National Center for Biotechnology Information) (Gene ID: 1773).

As described above, there is no limitation to the DNase-I according to the present invention as long as it is a protein that has the activity of decomposing the DNA molecular chain. For example, the DNase-I includes any one of the amino acid sequences represented by any one of SEQ ID NOs: 1 to 5. It can be done, and further, variants of the above amino acid sequence are included within the scope of the present invention. That is, the amino acid sequence of the present invention is a functional equivalent of the peptide molecule constituting it, for example, although some amino acid sequences of the peptide molecule have been modified by deletion, substitution, or insertion, the peptide molecule It is a concept that includes variants that can perform the same functional action. Specifically, the DNase-I protein contains at least 70%, more preferably at least 80%, even more preferably at least 90%, and most preferably at least 95% of the amino acid sequence represented by any one of SEQ ID NOs: 1 to 5. It may include an amino acid sequence having more than one sequence homology. For example, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85. %, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence homology. It contains an amino acid sequence having . The “% sequence homology” for an amino acid sequence is determined by comparing a comparison region with two optimally aligned sequences, and a portion of the amino acid sequence in the comparison region is a reference sequence (with additions or deletions) for the optimal alignment of the two sequences. may contain additions or deletions (i.e. gaps) compared to those that do not contain (i.e., gaps). However, as described above, this is only a representative example of DNase-I, and the type of DNase-I is not limited by the sequence.

In the present invention, the DNase-I protein is characterized in that it is bound, captured, linked, or immobilized on the surface of the biopolymer nanoparticle according to the present invention. That is, the DNase-I nanoparticle (DNase-I pMNS) according to the present invention is a sustained-release DNase-I in which DNase-I is supported or bound to a biopolymer nanoparticle, and has a lower half-life in the blood compared to free DNase-I. ) is longer and is characterized by high stability.

In the present invention, “biopolymer nanoparticles” refers to nanoparticles based on biopolymers. “Biopolymer” means a polymer or a variant or derivative thereof that can be produced by cells of a living organism. Biopolymers are characterized by biocompatibility, biodegradability, and low immunogenicity, making them suitable as nanoparticles for clinical applications. In the present invention, the biopolymers include melanin, chitosan, alginate, heparin, cellulose, gelatin, polyvinyl alcohol, and polyvinylpyrrolidone ( polyvinylpyrrolidione, collagen, gelatin, polycaprolactone, poly(lactic-co-glycolic acid), polylactide, and their derivatives. It may be selected from the group consisting of, but is not limited to this. Preferably, the biopolymer in the present invention is melanin or dopamine. More preferably, the biopolymer nanoparticles according to the present invention are obtained by polymerizing dopamine with a base.

In the present invention, the biopolymer nanoparticle may have a surface coated with polyethylene glycol. That is, the biopolymer nanoparticles according to the present invention are characterized by PEGylation, which serves to increase the in vivo delivery efficiency of the nanoparticles.

In the present invention, the average diameter of the biopolymer nanoparticles is 100 to 300 nm, 100 to 250 nm, 100 to 200 nm, 100 to 180 nm, 120 to 300 nm, 140 to 300 nm, 160 to 300 nm, and 120 to 120 nm. It may be 250 nm, 140 to 220 nm, or 150 to 200 nm, but is not limited thereto. Additionally, the specific shape of the biopolymer nanoparticles is not limited, but is preferably spherical.

In the present invention, the polydispersity index (PDI) of the biopolymer nanoparticles is 0.01 to 0.15, 0.01 to 0.12, 0.02 to 0.15, 0.04 to 0.15, 0.06 to 0.15, 0.08 to 0.15, 0.08 to 0.12, or 0.08. It may be from 0.1 to 0.1, but is not limited thereto.

In the present invention, the surface charge of the biopolymer nanoparticles is -20 mV to -0.1 mV, -18 mV to -0.1 mV, -16 mV to -0.1 mV, -14 mV to -0.1 mV, -12 mV to -0.1. mV, -20 mV to -0.2 mV, -20 mV to -1 mV, -20 mV to -2 mV, -20 mV to -4 mV, -20 mV to -6 mV, -20 mV to -8 mV, It may be -15 mV to -8 mV, or -12 mV to -9 mV, but is not limited thereto.

In the present invention, the binding ratio of DNase-I protein to the biopolymer nanoparticle is 10 to 70% by weight, 20 to 70% by weight, 30 to 70% by weight, 40 to 70% by weight, 50 to 70% by weight, 60% by weight. It may be from 70% by weight, 10 to 60% by weight, 20 to 50% by weight, 30 to 50% by weight, 35 to 50% by weight, or 40 to 50% by weight, but is not limited thereto.

DNase-I nanoparticles according to the present invention have preventive and/or therapeutic effects against SARS-CoV-2 virus infection. The above “SARS-CoV-2 virus infection” refers to a disease caused by infection with the SARS-CoV-2 virus. There is no limitation on the specific type of the SARS-CoV-2 virus infection, but it is preferably a severe inflammatory disease. More preferably, the SARS-CoV-2 virus infection is coronavirus disease-19 (COVID-19), sepsis, acute respiratory syndrome, pneumonia, cytokine storm, and cytokine release syndrome. , systemic inflammatory response syndrome, and multiple organ failure.

As a result of analyzing the blood of severe COVID-19 patients, the present inventors confirmed that the DNase-I level was decreased and the NETosis marker (blood extracellular DNA, Cit-His H3) was increased compared to normal people and mild COVID-19 patients. .

Therefore, the DNase-I nanoparticle according to the present invention can show an excellent therapeutic effect, especially in individuals with increased levels of extracellular DNA and/or citrullinated histone H3 in the blood compared to normal people. , it can show excellent therapeutic effects in individuals with reduced activity or level of DNase-I compared to normal people.

In the present invention, “Neutrophil Extracellular Trap (NET)” refers to a large network structure that exists outside the cell and is composed of cytoplasm, granule proteins, etc. centered on decondensed chromatin. NETs exert anti-infective effects by capturing pathogenic microorganisms, including viruses, preventing their spread, and inactivating pathogenic factors. However, an increase in NETs due to an excessive immune response may contribute to the development of immune-related diseases.

The NETs are created through an apoptosis process called NETosis. When NETosis progresses in neutrophils due to inflammation, infection, hypoxia, etc., nuclear dedifferentiation and nuclear membrane disassembly occur, and NETs are secreted through the process of cell depolarization and chromatin decondensation. Excessive NETosis and subsequent NET increase can lead to excessive immune response and tissue damage. Increased NETosis is characterized by increased myeloperoxidase (MPO) activity, increased neutrophil elastase (NE) activity, and increased levels of citrullinated histone H3 protein. The “citrullinated histone H3 (Cit-His H3)” refers to the citrullinated histone protein H3 family, and the protein is known to be involved in the formation of NETs in neutrophils.

In the present invention, the term “extracellular DNA (eDNA)” is used interchangeably with the term “cell-free DNA,” which refers to various types of DNA fragments that do not exist in cells and freely circulate in the bloodstream. do. Types of eDNA may include, but are not limited to, cell-free fetal DNA, cell-free tumor DNA, and cell-free circulating mitochondrial DNA. eDNA can exist alone in body fluids or in extracellular vesicles (exosomes).

In addition, in the present invention, there is no limit to the length (base pairs) of eDNA, but about 50 to 60 bp, 60 to 70 bp, 70 to 80 bp, 80 to 90 bp, 90 to 100 bp, 100 to 110 bp, 110 to 120 bp, 120 to 120 bp. 130bp, 130 to 140bp, 140 to 150bp, 150 to 160bp, 160 to 170bp, 170 to 180bp, 180 to 190bp, 190 to 200bp, 200 to 210 bp, 210 to 220bp, 220 to 230bp, 230 to 2 40bp, 240 to 250bp It can be. Additionally, in the present invention, eDNA may be single-stranded DNA or double-stranded DNA. Extracellular DNA is produced by wounds or tissue necrosis, and can also be released by NETosis. An excessive increase in extracellular DNA can induce an inflammatory response.

DNase-I nanoparticles according to the present invention are selected from the group consisting of neutrophil proliferation, myeloid peroxidase activity, neutrophil elastase activity, neutrophil extracellular trap formation (NETosis), and neutrophil NF-κB activity. It is characterized by suppressing one or more.

In addition, the DNase-I nanoparticle according to the present invention is characterized by inhibiting the production of inflammatory cytokines, and there is no limitation on the specific type of the inflammatory cytokine, but preferably IL-1β, IL-6, IL- 8, CCL2, IFN-γ, and TNF-α.

The content of the DNase-I nanoparticles (DNase-I pMNS) in the composition of the present invention can be appropriately adjusted depending on the symptoms of the disease, the degree of progression of the symptoms, the patient's condition, etc., for example, 0.0001 to 99.9 based on the total weight of the composition. It may be % by weight, or 0.001 to 50% by weight, but is not limited thereto. The content ratio is a value based on the dry amount with the solvent removed. Expressed differently, the content of the DNase-I nanoparticles (DNase-I pMNS) in the composition of the present invention is 1 to 200 units, 20 to 200 units, 40 to 200 units, 60 to 200 units, 80 to 200 units. Unit, 20 to 180 units, 40 to 160 units, 60 to 140 units, 80 to 120 units, or 90 to 110 units, but is not limited thereto. The unit refers to the concentration of enzyme at which enzyme activity appears, but is not limited thereto.

The pharmaceutical composition according to the present invention may further include appropriate carriers, excipients, and diluents commonly used in the preparation of pharmaceutical compositions. The excipient may be, for example, one or more selected from the group consisting of diluents, binders, disintegrants, lubricants, adsorbents, humectants, film-coating materials, and controlled-release additives.

The pharmaceutical composition according to the present invention can be prepared as powder, granules, sustained-release granules, enteric-coated granules, solutions, eye drops, ellipsis, emulsions, suspensions, spirits, troches, perfumes, and limonadese according to conventional methods. , tablets, sustained-release tablets, enteric-coated tablets, sublingual tablets, hard capsules, soft capsules, sustained-release capsules, enteric-coated capsules, pills, tinctures, soft extracts, dry extracts, liquid extracts, injections, capsules, perfusate, It can be formulated and used in the form of external preparations such as warning agents, lotions, pasta preparations, sprays, inhalants, patches, sterilized injection solutions, or aerosols, and the external preparations include creams, gels, patches, sprays, ointments, and warning agents. , it may have a dosage form such as lotion, liniment, pasta, or cataplasma.

Carriers, excipients, and diluents that may be included in the pharmaceutical composition according to the present invention include lactose, dextrose, sucrose, oligosaccharides, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, and calcium. These include phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat the disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the type, severity, drug activity, and It can be determined based on factors including sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, drugs used simultaneously, and other factors well known in the medical field.

The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. Considering all of the above factors, it is important to administer an amount that can achieve the maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art to which the present invention pertains.

The pharmaceutical composition of the present invention can be administered to an individual through various routes. All modes of administration are contemplated, including oral administration, subcutaneous injection, intraperitoneal administration, intravenous injection, intramuscular injection, paraspinal space (intrathecal) injection, sublingual administration, buccal administration, intrarectal injection, vaginal injection. It can be administered by internal insertion, ocular administration, ear administration, nasal administration, inhalation, spraying through the mouth or nose, dermal administration, transdermal administration, etc.

The pharmaceutical composition of the present invention is determined depending on the type of drug as the active ingredient along with various related factors such as the disease to be treated, the route of administration, the patient's age, gender, weight, and severity of the disease.

In the present invention, “individual” refers to a subject in need of treatment for a disease, and more specifically, human or non-human primates, mice, rats, dogs, cats, horses, cows, etc. refers to mammals of

In the present invention, “administration” means providing a given composition of the present invention to an individual by any appropriate method.

In the present invention, “prevention” refers to any action that suppresses or delays the onset of the desired disease, and “treatment” refers to the improvement or improvement of the desired disease and its associated metabolic abnormalities by administration of the pharmaceutical composition according to the present invention. It refers to all actions that are beneficially changed, and “improvement” refers to all actions that reduce parameters related to the target disease, such as the degree of symptoms, by administering the composition according to the present invention.

In addition, the present invention provides a quasi-drug composition for preventing or improving SARS-CoV-2 virus infection, which contains biopolymer nanoparticles with DNase-I protein bound to the surface as an active ingredient. In addition, the present invention provides a quasi-drug for preventing or improving viral infections comprising the quasi-drug composition.

In addition to the above ingredients, the quasi-drug composition of the present invention may further include pharmaceutically acceptable carriers, excipients, or diluents as needed. The pharmaceutically acceptable carriers, excipients, or diluents are not limited as long as they do not affect the effect of the present invention, and include, for example, fillers, extenders, binders, wetting agents, disintegrants, surfactants, lubricants, sweeteners, flavoring agents, and preservatives. It may include etc.

Representative examples of carriers, excipients or diluents acceptable for the quasi-drug composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, maltitol, starch, gelatin, glycerin, gum acacia, alginate, calcium phosphate, calcium. Carbonate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, propylene glycol, polyethylene glycol, vegetable oil. , injectable esters, Wethepsol, Macrogol, Tween 61, Kakaoji, Lauridge, etc.

When the composition of the present invention is provided as an external preparation, it is not limited thereto, but may be in the form of a solution, ointment, patch, gel, cream, or spray. According to one embodiment of the present invention, the quasi-drug of the present invention may include oral care products including toothpaste, mouthwash, and mouth spray, ointments, masks, poultices, patches, and transdermal absorbents. The formulation method, dosage, usage method, components, etc. of the quasi-drug may be appropriately selected from common techniques known in the technical field.

In addition, the present invention provides a method for producing biopolymer nanoparticles with DNase-I protein bound to the surface, comprising the following steps:

(S1) preparing nanoparticles by adding an alkaline solution to the biopolymer solution; and

(S2) Adding the nanoparticles obtained in step (S1) to a buffer containing DNase-I.

In one embodiment of the present invention, the production method may further include the step of purifying the nanoparticles after the (S1) step and/or (S2) step. The purification can preferably be performed through centrifugation. More preferably, the centrifugation is performed at 10,000 to 20,000 rpm, 12,000 to 20,000 rpm, 14,000 to 20,000 rpm, 16,000 to 20,000 rpm, 17,000 to 20,000 rpm, 12,000 to 19,000 rpm, 14,000 rpm. From 0 to 18,000 rpm, or from 16,000 to 18,000 rpm It can be done, but it is not limited to this.

In the present invention, there is no limitation on the specific type of the alkaline solution, but it is preferably a NaOH solution.

In the present invention, there is no limitation on the specific type of the buffer, but it is preferably Tris buffer.

In the present invention, in the step (S2), the nanoparticles and DNase-I are used at a weight ratio of 1:0.2 to 2, a weight ratio of 1:0.4 to 2, a weight ratio of 1:0.6 to 2, and a weight ratio of 1:0.8 to 2. , weight ratio of 1:1 to 2, weight ratio of 1:1.2 to 2, weight ratio of 1:1.5 to 2, 1:weight ratio of 0.2 to 1.8, 1:weight ratio of 0.4 to 1.6, weight ratio of 1:0.6 to 1.4, 1 : may be mixed at a weight ratio of 0.8 to 1.2, or 1 : 0.9 to 1.1 (nanoparticles: DNase-I).

Additionally, the buffer solution containing DNase-I in step (S2) may further include polyethylene glycol. Preferably, the nanoparticles and polyethylene glycol have a weight ratio of 1:0.1 to 1.5, 1:0.4 to 1.5, 1:0.6 to 1.5, 1:0.8 to 1.5, 1:0.8 to 1.3. It may be mixed at a weight ratio of 1:0.8 to 1.2, or 1:0.9 to 1.1 (nanoparticles: polyethylene glycol).

In the present invention, the production method is performed by mixing the mixed solution of nanoparticles, DNase-I, and/or polyethylene glycol in step (S2) for 1 to 6 hours, 1 to 5 hours, 1 to 4 hours, 1 to 4 hours. It may further include stirring for 3 hours, 2 to 5 hours, or 2.5 to 4.5 hours. Additionally, the stirring may be performed at 2 to 10°C, 2 to 6°C, 2 to 5°C, 3 to 6°C, or 3 to 5°C.

Below, preferred embodiments are presented to aid understanding of the present invention. However, the following examples are provided only to make the present invention easier to understand, and the content of the present invention is not limited by the following examples.

[Experimental materials and methods]

experiment material

Dopamine hydrochloride and poly(ethylene glycol) (PEG, four arm-amine termini, HCL salt) were purchased from Sigma (USA, PA) and JenKem (USA), respectively. , TX). NaOH (1 N) solution and Tris buffer (10 mM, pH 8.5) were purchased from Daejeong (Suwon, Korea) and Biosesang (Seoul, Korea), respectively. Recombinant DNase-I was purchased from Roche (Basel, Switzerland). Pierce BCA protein assay kit was purchased from Thermo Scientific (USA, MA). Dulbecco's phosphate-buffered saline (PBS) and fetal bovine serum (FBS) were purchased from Welgene (Gyeongsan, Korea) and Gibco (USA, CA), respectively.

plasma sample

At Yeungnam University Medical Center, whole blood samples were obtained from patients diagnosed with SARS-CoV-2 infection at a public health center in Daegu, South Korea. COVID-19 sepsis patients were defined according to the criteria established by the Sepsis Consensus Conference Committee ( JAMA 2016 , 315, 801). The human study protocol was approved by the Institutional Review Board of Yeungnam University Hospital, Daegu, Korea (YUH 2020-03-057, 2020-05-031-001).

Measurement of neutrophil count in COVID19 patients

Complete blood counts were analyzed from venous blood samples within 24 hours after the patient was admitted to the hospital. Neutrophil counts were measured using a Sysmex XE-2100 automated hematology system (TOA Medical Electronics; Kobe, Japan).

NET enzyme-linked immunosorbent assay (ELISA)

NET (Neutrophil Extracellular Trap) was incubated with phorbol-myristate acetate (PMA; 25 nM) (Sigma-Aldrich; USA, MO) or control medium (RPMI1640 containing glutamine, penicillin, and streptomycin). It was generated from freshly isolated neutrophils (1×10 ⁵ cells) using a stimulation method, and was analyzed using fluorescence measurement technology, referring to [ Nat Rev Gastroenterol Hepatol 2017 , 14]. NET production was measured in arbitrary fluorescent units (AFUs).

MPO ELISA and Cit-His H3 ELISA

Plasma samples were analyzed to measure the release of granule matrix proteins following degranulation of peripheral blood mononuclear cells (PBMCs) from SARS-CoV-2-infected patients and mice. For human samples, the MPO ELISA kit (Invitrogen; USA) was used, and for mouse samples, the mouse myeloperoxidase ELISA kit (MyBioSource; USA) was used.

Cit-His H3 concentration in cell culture medium or SARS-CoV-2 patient serum was measured using the human citrullinated histone H3 ELISA kit (MyBioSource).

Quantification of plasma extracellular DNA (eDNA)

To measure eDNA (i.e., cell-free DNA) in SARS-CoV-2-infected patient or mouse plasma, plasma samples were centrifuged at 16,800 × g for 10 minutes, and Qiagen QIAamp DNA Mini Blood Mini Kit (Qiagen; USA, CA) was used to extract DNA according to the manufacturer's instructions. Purified eDNA was quantified using a NanoDrop spectrophotometer.

ELISA for measuring DNase-I activity

Plasma samples were diluted 1:50 and analyzed using spiked digestion buffer with double-stranded DNA (1 μg/ml). Samples were stained using PicoGreen (Invitrogen) according to the manufacturer's instructions. After incubation at 37°C for 5 hours, the degree of reduction of PicoGreen staining (fluorescence emission, Em) was measured using a fluorometer.

Neutrophil isolation

For density gradient isolation of PBMC, Percoll (pH 8.5-9.5; Sigma-Aldrich, UK) was used, referring to [ Blood 2014 , 123, 239]. PBMCs (with 95% purity and 97% viability according to trypan blue exclusion) were suspended in RPMI1640 medium (Sigma-Aldrich). For discontinuous density gradient centrifugation of neutrophils, one-step polymorphs (Axis-Shield; Oslo, Norway) were used. To increase purity, neutrophils were purified using CD45 antibody-coupled magnetic beads and magnetic-activated cell sorting (MACS). As a result of trypan blue dye exclusion method, it was confirmed that the general survival rate of neutrophils was higher than 95%. To confirm the effect of DNase-I on cytokine production, neutrophils were isolated from COVID-19 patients and then treated with DNase-I MNS. DNase-I MNS was also administered to CLP-operated septic mice. The supernatant was used for cytokine analysis using ELISA, and cell lysates were used to measure NF-κb activity.

Measurement of NF-κb activity

After extracting nuclei from cells, TransAM analysis was performed according to [ J Mol Med 2014 , 92, 77]. The activity of each NF-κb subunit was measured by ELISA using the NF-κb family transcription factor assay kit (Active Motif; CA, USA). Briefly, nuclear extract (2 μg) was added to each well of an NF-κb consensus oligonucleotide-encoded 96-well plate, and the plate was incubated with NF-κb primary antibody followed by HRP-conjugated secondary antibody. Antibody binding was observed by incubation with antibodies. For analysis, optical density (OD) was measured at 450 nm using a Tecan Spark microplate reader (Tecan; Austria).

Cytokine ELISA

The levels of inflammatory cytokines IL-1β, IL-6, IFN-γ, and TNF-α in the supernatant were measured after treatment of PBMCs with pMNSs, Free-DNase-I, and pMNSs-DNase-I (100 units). Measurements were made using an ELISA kit (R&D Systems; MN, USA) according to the manufacturer's instructions.

animal testing

Animal experiments were conducted in accordance with the Institutional Animal Care and Use Committee of the Korea Research Institute of Bioscience and Biotechnology (IACUC no. 1305021) and Sungkyunkwan University College of Medicine (IACUC No. SKKU IACUC2020-06-29-2). It was performed according to a protocol approved by the Care and Use Committee (IACUC). Six to seven week old C57BL/6 male mice (18-20 g) were purchased from Orient Bio (Seongnam, Korea). Mice were used after an adaptation period of 12 days. Five mice per cage were maintained on a 12:12 h light/dark cycle at a controlled temperature of 20-25 °C and humidity of 40-45%. Mice were fed a typical rodent pellet diet and water was provided ad libitum .

Cecal ligation and perforation

A mouse model in which cecal ligation and puncture (CLP) was performed was created with reference to [ Nat Protoc 2009 , 4, 31]. Briefly, a 2 cm midline incision was made to expose the cecum and adjacent intestine. Next, the area 5.0 mm from the end of the cecum was tightly ligated using a 3.0-silk suture, a puncture was made with a 22-gauge needle, and stool was pushed out from the puncture site by gently squeezing. The cecum was then returned to the abdominal cavity and the laparotomy site was sutured using 4.0-silk. For the sham operation, the appendix was surgically exposed and placed back into the abdominal cavity without ligation or perforation.

In vivo Neutrophil migration assay

To evaluate the extent of neutrophil migration, 6 hours after CLP surgery, CLP-treated mice were treated with pMNS, free DNase-I, and pMNS-DNase-I (100 units). Afterwards, the mouse was euthanized and the abdominal cavity was washed with 5 mL of physiological saline. Neutrophil counts were measured using an automated hematology analyzer (Mindray, BC-5000 Vet). The results were expressed as neutrophils per abdominal cavity × 10 ⁶ .

RNA analysis

RNA was extracted from lung tissue using the RNeasy mini-kit (Qiagen Venlo, Netherlands) according to the manufacturer's protocol. RNA-seq libraries were generated using the TruSeq RNA Sample Prep kit v2 (Illumina) according to the manufacturer's protocol. RNA-seq libraries were paired-end sequenced on the Illumina Hi-seq 3000/4000 SBS kit v3 (MACROGEN Inc.). All RNA-seq data were mapped using the Tophat package against Affymetrix Human Gene 2.0 ST arrays (902136). The remaining mRNA was used for qPCR analysis. Fold-change was measured using the R package limma, and P-values were adjusted with Benjamini-Hochberg (BH). Analysis results can be found in NCBI's Gene Expression Omnibus (GEO) database (accession number: GSE101126).

Hematoxylin & eosin (H&E) staining and histopathological analysis

Male C57BL/6 mice were intravenously administered pMNS, free DNase-I, and pMNS-DNase-I (100 units) 12 or 24 hours after CLP. Mice were euthanized 72 hours after CLP surgery. Lung tissue was isolated from mice and phenotypic changes were analyzed. H&E staining was performed according to routine protocols.

Measurement of cytokine levels in plasma of septic mice

The levels of AST, ALT, BUN, creatinine, and LDH in mouse serum were measured using a biochemistry kit (MyBioSource). Accurate values were confirmed using an ELISA plate reader (Tecan; Austria).

statistical analysis

All in vitro and in vivo data were analyzed using two-tailed unpaired t-test using Graphpad prism 7 software, the sample size was n>=3, and P<0.05 was considered statistically significant. All data normalization procedures were performed according to the manufacturer's protocol.

[Example]

Example 1. Confirmation of increased neutrophil count and decreased lymphocyte count in severe COVID-19 patients

After performing computed tomography (CT), blood samples were analyzed at the time of hospitalization of COVID-19 patients. The CT scan results of mild or severe COVID-19 patients are shown in Figure 1, and the characteristics of COVID-19 patients are summarized in Table 1 below.

Total patients (n=60) Mild patients (n=40) Severely ill patients (n=20) P-value characteristic Age (y) 33.7 ± 16.4 55.4 ± 17.1 72.2 ± 13.1 0.014 ARDS 11 (18.3) 0 (0) 11 (55) <0.001 blood poisoning 13 (21.7) 0 (0) 13 (65) <0.001 Discharge 40 40 (100) 0 (0) <0.001 Dead 16 0 (0) 16 (80%) <0.001 complete blood count White blood cell count, ×10 ⁹ /L [normal range 4-10] 6.5 ± 3.4 5.9 ± 3.6 9.1 ± 2.6 0.005 Neutrophil count, ×10 ⁹ /L [normal range 3-7] 4.6 ± 3.4 4.1 ± 3.2 7.7 ± 3.3 <0.001 Lymphocyte count, ×10 ⁹ /L [normal range 1.5-4] 1.4 ± 0.7 1.5 ± 0.7 0.8 ± 0.3 <0.001 Hemoglobin, g/Dl [normal range 12-17] 13.0 ± 1.6 13.5 ± 1.7 13.0 ± 1.6 0.253 Platelets, ×10 ⁹ /L [normal range 140-400] 237.3 ± 104.9 245.0 ± 107.9 186.7 ± 64.9 0.061 Data are presented as mean ± SD (range) or number (percentage). Mild patients: non-ICU, no symptoms; Critically ill patients: ICU, ventilator, oxygen therapy, ARDS, and sepsis

CT scan results showed that patients with severe clinical symptoms had severe lung tissue damage, indicating increased severity of sepsis. Severe clinical symptoms such as ARDS (Acute respiratory distress syndrome) and sepsis mostly occurred in older patients (mean age 72.2 ± 13.1 years), who had increased neutrophil counts and lymphocytes compared to patients with mild symptoms. The number appeared to have decreased (Table 1).

Example 2. Confirmation of increase in NETosis marker level in severe COVID-19 patients

Extracellular DNA (eDNA), DNase-I, and citrullinated histone H3 in blood samples from 20 healthy control patients (normal group) and 60 COVID19 patients (including mild and severe patients). The level of Cit-His H3) was measured, and the results are shown in Table 2 below.

Normal people (n=20) Mild patients (n=40) Severely ill patients (n=20) NETosis eDNA [mg/mL] 0.41 ± 0.12 0.85 ± 0.27 2.83 ± 0.37*** DNase-I [mg/mL] 2.11 ± 0.38 3.11 ± 1.07 0.97 ± 0.30*** Cit-His H3 [mg/mL] 0.05 ± 0.01 0.30 ± 0.32 17.74 ± 2.91*** Data are presented as mean ± SD (range). ^*** P < 0.001. eDNA, extracellular DNA; NE, Neutrophil Elastase; MPO, Myeloperoxidase; Cit-His H3, citrullinated histone H3.

First, the median serum eDNA level in the normal group was 0.41 (0.29-0.53) μg/mL. In comparison, the serum eDNA level of 40 mild COVID-19 patients increased to 0.85 (0.58-1.12 μg/mL), and the serum eDNA level of 20 severe COVID-19 patients increased to 2.83 (2.46-3.20) μg/mL. It was confirmed that there was an even greater increase. The level of Cit-His H3 (citrullinated histone H3) showed an even greater difference. The median serum Cit-His H3 in the normal group was only 0.05 (0.04-0.06) μg/mL, but increased to 0.30 (0.02-0.62) μg/mL in patients with mild COVID-19, and serum Cit in patients with severe COVID-19. -His H3 median value was confirmed to have increased significantly to 17.74 (14.83-20.65) μg/mL. On the other hand, in the case of DNase-I, somewhat different results were obtained. In the normal group, the DNase-I level was 2.11 (1.73-2.49) μg/mL, and was found to be slightly increased to 3.11 (2.04-4.18) μg/mL in patients with mild COVID-19, but in patients with severe COVID-19, the level of DNase-I was 2.11 (1.73-2.49) μg/mL. DNase-I levels were found to be significantly reduced to 0.97 (0.67-1.27) μg/mL.

The results show that in COVID-19 patients, especially severe COVID-19 patients, the levels of NETosis markers eDNA and Cit-His H3 increase, and DNase-I levels decrease.

Example 3. Preparation and characterization of DNase-I pMNS

As confirmed in Example 2, in severe COVID-19 patients, NETosis-related factors increase and the level of DNase-I decreases, so there is a need to compensate for the loss of endogenous DNase-I in severe COVID-19 patients. However, even if DNase-I is injected into an individual, the half-life of DNase-I in the blood is very short. Accordingly, the present inventors used melanin to produce nanoparticles that can circulate in the body for a long time (i.e., long-acting DNase-I), and added polyethylene glycol to the melanin nanoparticles. After coating, DNase-I was adsorbed to prepare DNase-I melanin nanoparticles (DNase-I pMNS) that can be maintained and circulated in the blood for a long time.

3-1. Preparation of DNase-I pMNS

Uncoated melanin-like nanospheres (bMNSs) were synthesized with dopamine hydrochloride, referring to [ ACS Appl Mater Interfaces 2016 , 8, 7739]. Dopamine hydrochloride (10 mg) was dissolved in deionized water (DW; 50 mL), and then NaOH solution (50 μL, 1 N) was added to the dopamine hydrochloride solution. The reaction was stirred for 24 hours, and it was confirmed that the color gradually changed to black as the reaction progressed. For purification, the prepared bMNS was collected by centrifugation at 17,000 rpm for 20 min and washed three times with deionized water. Subsequently, to prepare DNase-I-coated melanin nanoparticles, DNase-I was deposited on the surface of bMNS by performing a one-pot process, referring to [ Science 2018 , 361, eaao4227] and [ Sci Transl Med 2016 , 8, 361ra138]. was coated (Figure 2). The obtained bMNS (10 mg) was incubated with Tris buffer (5 mL, 10 mg) containing DNase-I (2, 5, 10, or 20 mg) and polyethylene glycol (10 mg, 4 arm-amine terminal, HCL salt). mM, pH 8.5) and stirred at 4°C for 3 hours to prepare polyethylene glycol-coated melanin nanoparticles with DNase-I bound to the surface (DNase-I pMNS). The prepared DNase-I pMNS was purified by the same method as described above and then washed several times with deionized water. As a control, PEG-coated MNS (pMNS), in which DNase-I was not bound to the surface, was prepared in the same manner as DNase-I pMNS, but the addition of DNase-I was omitted.

3-2. Characterization of DNase-I pMNS

The particle size and surface charge of the nanoparticles (nanospheres) according to the present invention were measured using dynamic light scattering (DLS; Malvern Instruments, MA, USA). As shown in Table 3 and Figure 3 below, the particle sizes (diameters) of bMNS, pMNS, and DNase-I pMNS were all similar at 170 nm. The surface charge measurement results are shown in Figure 4. The surface charge of bMNS was measured to be -12.4 mV, and when pMNS was prepared by coating PEG, the surface charge was neutralized to -0.15 mV. However, in the case of DNase-I pMNS, the surface charge was found to be -10.9 mV as it was coated with DNase-I, which has a negative charge.

division bMNS pMNS DNase-I pMNS Size (nm) 170 172 170 PDI 0.090 0.123 0.091

The morphology of nanoparticles was observed using field emission scanning electron microscopy (FE-SEM) (JEM-7500F; Akishima, Japan). As a result, as shown in Figure 5, the nanoparticles according to the present invention were found to be spherical and have a relatively monodispersed size.

Next, the enzymatic activity of DNase-I pMNS, i.e., DNA degrading ability, was confirmed by measuring the degree to which polymerized salmon sperm DNA (Sigma-Aldrich; USA, PA) was degraded using gel electrophoresis. For this purpose, bMNS, pMNS, or DNase-I Pmns were incubated with 1 μg of salmon sperm DNA at 37°C for 10 minutes and then electrophoresed. As shown in Figure 6, free DNase-I (1U) completely degraded 1 μg of DNA, while pMNS and bMNS did not degrade DNA because there was no DNase-I on the surface. However, DNase-I pMNS coated on the surface of DNase-I degraded DNA at a nanosphere concentration of 1.0 μg or more, showing that the DNA decomposition effect of the nanoparticles was due to DNase-I coated on the particle surface.

Furthermore, to confirm the amount of DNase-I that can bind to pMNS, nanoparticles were prepared with various ratios of pMNS and DNase-I. The binding amount and binding efficiency of DNase-I on pMNS were evaluated by BCA analysis, and the results are shown in Table 4 below.

Sample Feed (mg) DNase-I pMNSs ^a DNase-I ^b Binding amount
(Contents)
(wt%) ^c Binding efficiency
Efficiency)
(%) ^d DNase-I pMNSs1 10 2 13.8 80.1 DNase-I pMNSs2 10 5 29.4 83.1 DNase-I pMNSs3 10 10 45.4 83.1 DNase-I pMNSs4 10 20 61.1 78.5 ^a Weight of feed pMNSs
^b Weight of feed DNase-I
^c Binding amount = (actual mass of DNase-I/actual total weight of DNase-I pMNSs) × 100, calculated by measurement by BCA analysis.
^d Binding efficiency = (actual weight of DNase-I/processed weight (feed mass) of DNase-I) × 100, calculated by measurement by BCA analysis.

As shown in Table 4, as the feed amount of DNase-I increased, the actual amount of binding of DNase-I to nanoparticles increased, but the binding efficiency of DNase-I was 83%. was limited to that extent. However, in the case of DNase-I pMNSs3, which contains ~45% DNase-I-, it was confirmed that stability and DNA decomposition ability were very excellent. Therefore, DNase-I pMNSs3 was used in subsequent experiments.

Finally, the binding stability of nanoparticles and DNase-I over time was evaluated in PBS containing 10% FBS or simple PBS. As a result of measuring DNase activity after incubating long-acting DNase-I in PBS containing 10% FBS, the activity of DNase-I pMNS was found to persist for up to 36 hours and then decrease after 48 hours ( Figure 7a). However, in PBS, the activity of DNase-I pMNS appeared to be maintained for up to 72 hours (Figure 7b).

Example 4. Confirmation of the inhibitory effect of exogenous DNase-I pMNS on NETosis and neutrophils in the plasma of severe COVID-19 patients

The present inventors confirmed whether DNase-I-coated nanoparticles, DNase-I pMNS, prepared in Example 3 above inhibit NETosis of neutrophils. To verify the DNA degradation effect of DNase-I, plasma of severe COVID-19 patients was treated with free DNase-I or DNase-I pMNS. The results showed that both forms of DNase-I significantly reduced eDNA levels, and exposure of DNase-I to the plasma of severe COVID-19 patients appeared to increase the activity of DNase-I. , the eDNA degradation effect and increase in DNase activity by DNase-I pMNS were found to be higher (Figure 8). Additionally, when both forms (free DNase-I or DNase-I pMNS) were treated with the plasma of severe COVID-19 patients, the NET level, MPO activity, and NE level in neutrophils were significantly compared to the control group (PBS treatment). It was confirmed that there was a decrease, and in particular, a more significant decrease was confirmed by DNase-I pMNS (FIG. 9).

As shown in Table 1 above, more than 50% of severe COVID-19 patients were diagnosed with ARDS and sepsis at the time of hospitalization. Typically, sepsis is accompanied by a fatal systemic inflammatory response, a cytokine storm, which is caused by an explosive increase in the production of pro-inflammatory cytokines by immune effector cells. Therefore, it was confirmed whether DNase-I pMNS according to the present invention can inhibit NF-κB activation and cytokine production by neutrophils. As a result, when treated with free-DNase-I, the activity of NF-κB was decreased, the secretion of cytokines such as IL-1β, IL-6, IFN-γ, and TNF-α was decreased, and such inflammation The reaction inhibition effect was found to be more excellent when treated with DNase-I pMNS (FIG. 10).

The above results show that the nanoparticles according to the present invention can effectively suppress the increase in NETosis and inflammatory factors by complementing the decrease in DNase-I in severe COVID-19 patients, and thus, sepsis, cytokine storm phenomenon, or systemic inflammation caused by COVID-19. It shows that inflammatory reactions, etc. can be improved.

Example 5. Confirmation of NETosis alleviation effect in sepsis mouse model by DNase-I pMNS

In Example 4, the inhibitory effect of DNase-I pMNS on the number of neutrophils and neutrophil-related NETosis factors in severe COVID-19 patients was confirmed through in vitro experiments. As a next step, the effect of DNase-I pMNS was tested in vivo. Confirmed. Considering that neutrophil activity can be a therapeutic target to prevent or suppress sepsis, a representative COVID-19 related disease, and improve survival rate, we used a cecal ligation and perforation (CLP)-performed sepsis mouse model. Changes following administration of DNase-I pMNS were confirmed using this method. The CLP mouse model is the most frequently used model to identify the complex molecular mechanisms of sepsis. Sepsis is characterized by microbial infection within the abdominal cavity followed by microorganisms moving through the blood and causing a systemic inflammatory response. Similar phenotypes are observed in the CLP model and severe COVID-19 patients. In particular, NETosis, cytokine release syndrome (CRS), and multiple organ failure (MOF) syndrome can also be induced in the CLP model. Therefore, the present inventors used the CLP model as an in vivo model of severe COVID-19 patients caused by SARS-CoV-2.

To confirm the effect of DNase-I nanoparticles, phosphate-buffered saline (PBS; control), PEG-Nano (pMNS), and free DNase-I ( 100 units), or DNase-I pMNS (100 units) was injected intravenously. DNase-I activity and NET levels were measured at a certain time after drug administration. To suppress the rapid increase in NETs, the drug was administered 12 and 24 hours after CLP surgery, and the effects of DNase-I pMNS, etc. were confirmed 24 hours after drug administration.

First, as a result of checking the survival rate of the mouse model according to each drug administration, among the CLP-treated mice, all mice administered PBS, PEG-Nano, or free-DNase-I died within 90 hours after CLP treatment (Figure 11 ). The above results mean that sepsis caused by CLP is very fatal. In particular, the high mortality rate in mice administered free DNase-I is believed to be because the half-life of recombinant human DNase-I in serum is very short ( Immunol 1998 , 113, 289). On the other hand, it was confirmed that mice administered DNase-I pMNS showed a survival rate of more than 40% even more than 132 hours after CLP, leading to complete recovery (Figure 11). The above results show that in the case of DNase-I pMNS according to the present invention, the half-life in the plasma of DNase-I coated on nanoparticles increases and its activity can be stably maintained, thereby treating sepsis symptoms. In addition, as a result of analyzing the lung tissue of a mouse model, morphological changes such as pulmonary edema, hemorrhage, alveolar collapse, and inflammatory cell infiltration induced by CLP were significantly observed by DNase-I pMNS. It was confirmed that there was a significant decrease (Figure 12). The above results show that DNase-I pMNS nanoparticles according to the present invention can improve lung tissue damage caused by sepsis.

Changes in intraperitoneal neutrophils, NET, eDNA, DNase activity, NE activity, and MPO activity in mice following administration of DNase-I pMNS nanoparticles are shown in Figure 13. Similar to in vitro experiments using plasma and neutrophils from severe COVID-19 patients, CLP mice administered DNase-I pMNS showed a dramatic decrease in the levels of peritoneal neutrophils, NET, eDNA, NE activity, and MPO activity, compared with PBS and pMNS. Of course, it was found that the rate was slowed down to a greater extent compared to when free DNase-I was administered. Additionally, DNase activity also increased most dramatically in CLP mice administered DNase-I pMNS. The above results show that the DNase-I pMNS nanoparticles according to the present invention have an excellent NETosis inhibition effect and DNase activity promotion effect even in vivo .

Next, we confirmed the effect of DNase-I pMNS on controlling inflammatory responses in a mouse model of sepsis caused by CLP. As described above, the systemic inflammatory response associated with sepsis can cause multiple organ failure syndrome, and it is known that multiple organ failure occurs especially in the kidneys and liver. In fact, mice that underwent CLP surgery had liver damage markers such as alanine transferase (AST) and aspartate transaminase (AST) in their serum; blood urea nitrogen (BUN) and creatine, markers of kidney damage; lactate dehydrogenase (LDH), a tissue damage marker; And the level of C-reactive protein (CRP), a marker for pneumonia and sepsis, rapidly increased. However, in mice administered DNase-I pMNS nanoparticles according to the present invention, the levels of all of the above markers were significantly reduced, especially compared to mice administered free DNase-I (FIG. 14). The above results show that DNase-I nanoparticles according to the present invention can suppress systemic inflammatory responses and effectively improve multiple organ failure.

Next, we evaluated the absolute neutrophil count, absolute white blood cell count, and total white blood cell count in the blood of a mouse model of sepsis caused by CLP. Similar to the changes in the abdominal cavity seen previously, DNase-I pMNS dramatically reduced the absolute neutrophil count and total leukocyte count in the blood compared to the control and free DNase-I (Figure 15). However, in the case of absolute white blood cell count, there was no significant change even after treatment with DNase-I pMNS. The results show that DNase-I pMNS nanoparticles alleviate the inflammatory response by inhibiting excessive proliferation of inflammatory cells.

Furthermore, the anti-NETosis effect of DNase-I pMNS in a sepsis mouse model was evaluated through cytokine array analysis of mouse lung tissue. As shown in Figure 16, the mouse model that underwent CLP surgery had significantly increased levels of inflammatory cytokines such as IL-6, IL-8, IL-1β, and CCL2. The increase in cytokines in lung tissue in the CLP mouse model is similar to the rapid rise in systemic inflammatory responses, including a cytokine storm, following SARS-CoV-2 infection. However, mice administered DNase-I pMNS showed significantly reduced levels of cytokines such as IL-6, IL-8, IL-1β, and CCL2. The above results suggest that DNase-I pMNS nanoparticles suppress inflammatory responses in the body by reducing the levels of various inflammatory factors.

Next, since SARS-CoV-2 damages vascular endothelial cells and causes severe lung damage, transendothelial permeability was observed in a sepsis mouse model to confirm that the DNase-I nanoparticles according to the present invention protect the vascular barrier. It was confirmed whether it was effective. As a result, free DNase-I was observed to have an effect of suppressing the increase in vascular permeability caused by CLP at the beginning of administration, but the effect rapidly decreased more than 72 hours after administration. On the other hand, the group administered DNase-I pMNS showed a high vascular barrier protection effect maintained even 72 hours after administration (Figure 17). The above results show that the DNase-I nanoparticles according to the present invention have higher stability in the blood compared to free DNase-I and can effectively inhibit vascular endothelial cell damage caused by SARS-CoV-2.

Finally, to determine whether the sepsis treatment effect of DNase-I pMNS was DNase-I pMNS-concentration dependent, various concentrations of DNase-I pMNS were administered to mice after CLP was performed and the survival rate over time was checked (i.e. , nanoparticles are constant and only vary in DNase-I; 25, 50, and 100 units). As a result, as shown in Figure 18, the survival rate of CLP mice increased as the treatment concentration of DNase-I pMNS increased, confirming that the sepsis treatment effect of DNase-I nanoparticles according to the present invention was concentration dependent.

As seen above, the present inventors found that when DNase-I was coated on melanin nanoparticles and administered (DNase-I pMNS), the levels of NETosis, inflammatory cells, and various inflammation-related factors in COVID-19 patients were reduced, It was confirmed that the increased activity of DNase-I not only significantly improved the symptoms of sepsis caused by SARS-CoV-2 infection but also increased the survival rate of the sepsis mouse model. The therapeutic effect of DNase-I pMNS was confirmed by using DNase-I. It was confirmed that it was superior to administering it alone. In other words, the DNase-I-coated melanin nanoparticles according to the present invention effectively inhibit NETosis, which is an exacerbating factor in COVID-19 patients, and alleviate systemic inflammatory responses, thereby preventing the progression of COVID-19 patients to acute respiratory syndrome, pneumonia, and sepsis. It is expected to be used as a new coronavirus treatment strategy as it can prevent and potentially treat COVID-19 symptoms.

The description of the present invention described above is for illustrative purposes, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical idea or essential features of the present invention. will be. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

<110> Research & Business Foundation SUNGKYUNKWAN UNIVERSITY <120> Pharmaceutical compositions for preventing or treating SARS-CoV-2 infection <130> MP20-179P1 <150> KR 10-2020-0117435 <151> 2020-09-14 <160> 5 <170> KoPatentIn 3.0 <210> 1 <211> 282 <212> PRT <213> Artificial Sequence <220> <223> DNase-1 isoform 1_amino acid seq_full length <400> 1 Met Arg Gly Met Lys Leu Leu Gly Ala Leu Leu Ala Leu Ala Ala Leu 1 5 10 15 Leu Gln Gly Ala Val Ser Leu Lys Ile Ala Ala Phe Asn Ile Gln Thr 20 25 30 Phe Gly Glu Thr Lys Met Ser Asn Ala Thr Leu Val Ser Tyr Ile Val 35 40 45 Gln Ile Leu Ser Arg Tyr Asp Ile Ala Leu Val Gln Glu Val Arg Asp 50 55 60 Ser His Leu Thr Ala Val Gly Lys Leu Leu Asp Asn Leu Asn Gln Asp 65 70 75 80 Ala Pro Asp Thr Tyr His Tyr Val Val Ser Glu Pro Leu Gly Arg Asn 85 90 95 Ser Tyr Lys Glu Arg Tyr Leu Phe Val Tyr Arg Pro Asp Gln Val Ser 100 105 110 Ala Val Asp Ser Tyr Tyr Tyr Asp Asp Gly Cys Glu Pro Cys Gly Asn 115 120 125 Asp Thr Phe Asn Arg Glu Pro Ala Ile Val Arg Phe Phe Ser Arg Phe 130 135 140 Thr Glu Val Arg Glu Phe Ala Ile Val Pro Leu His Ala Ala Pro Gly 145 150 155 160 Asp Ala Val Ala Glu Ile Asp Ala Leu Tyr Asp Val Tyr Leu Asp Val 165 170 175 Gln Glu Lys Trp Gly Leu Glu Asp Val Met Leu Met Gly Asp Phe Asn 180 185 190 Ala Gly Cys Ser Tyr Val Arg Pro Ser Gln Trp Ser Ser Ile Arg Leu 195 200 205 Trp Thr Ser Pro Thr Phe Gln Trp Leu Ile Pro Asp Ser Ala Asp Thr 210 215 220 Thr Ala Thr Pro Thr His Cys Ala Tyr Asp Arg Ile Val Val Ala Gly 225 230 235 240 Met Leu Leu Arg Gly Ala Val Val Pro Asp Ser Ala Leu Pro Phe Asn 245 250 255 Phe Gln Ala Ala Tyr Gly Leu Ser Asp Gln Leu Ala Gln Ala Ile Ser 260 265 270 Asp His Tyr Pro Val Glu Val Met Leu Lys 275 280 <210> 2 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> DNase-1 isoform 1_amino acid seq_active site <400> 2 Ile Val Pro Leu His Ala Ala Pro Gly Asp Ala Val Ala Glu Ile Asp 1 5 10 15 Ala Leu Tyr Asp Val 20 <210> 3 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> DNase-1 isoform 1_amino acid seq_conserved site <400> 3 Gly Asp Phe Asn Ala Gly Cys Ser 1 5 <210> 4 <211> 228 <212> PRT <213> Artificial Sequence <220> <223> DNase-1 isoform 1_amino acid seq_domain <400> 4 Phe Asn Ile Gln Thr Phe Gly Glu Thr Lys Met Ser Asn Ala Thr Leu 1 5 10 15 Val Ser Tyr Ile Val Gln Ile Leu Ser Arg Tyr Asp Ile Ala Leu Val 20 25 30 Gln Glu Val Arg Asp Ser His Leu Thr Ala Val Gly Lys Leu Leu Asp 35 40 45 Asn Leu Asn Gln Asp Ala Pro Asp Thr Tyr His Tyr Val Val Ser Glu 50 55 60 Pro Leu Gly Arg Asn Ser Tyr Lys Glu Arg Tyr Leu Phe Val Tyr Arg 65 70 75 80 Pro Asp Gln Val Ser Ala Val Asp Ser Tyr Tyr Tyr Asp Asp Gly Cys 85 90 95 Glu Pro Cys Gly Asn Asp Thr Phe Asn Arg Glu Pro Ala Ile Val Arg 100 105 110 Phe Phe Ser Arg Phe Thr Glu Val Arg Glu Phe Ala Ile Val Pro Leu 115 120 125 His Ala Ala Pro Gly Asp Ala Val Ala Glu Ile Asp Ala Leu Tyr Asp 130 135 140 Val Tyr Leu Asp Val Gln Glu Lys Trp Gly Leu Glu Asp Val Met Leu 145 150 155 160 Met Gly Asp Phe Asn Ala Gly Cys Ser Tyr Val Arg Pro Ser Gln Trp 165 170 175 Ser Ser Ile Arg Leu Trp Thr Ser Pro Thr Phe Gln Trp Leu Ile Pro 180 185 190 Asp Ser Ala Asp Thr Thr Ala Thr Pro Thr His Cys Ala Tyr Asp Arg 195 200 205 Ile Val Val Ala Gly Met Leu Leu Arg Gly Ala Val Val Pro Asp Ser 210 215 220 Ala Leu Pro Phe 225 <210> 5 <211> 187 <212> PRT <213> Artificial Sequence <220> <223> DNase-1 isoform 2_amino acid seq_full length <400> 5 Met His Gln Thr Pro Ile Thr Thr Trp Ser Val Ser His Trp Asp Gly 1 5 10 15 Thr Ala Ile Arg Ser Ala Thr Cys Ser Cys Thr Gly Leu Thr Arg Cys 20 25 30 Leu Arg Trp Thr Ala Thr Thr Thr Met Met Ala Ala Ser Pro Ala Gly 35 40 45 Thr Thr Pro Ser Thr Glu Ser Gln Pro Leu Ser Gly Ser Ser Pro Gly 50 55 60 Ser Gln Asp Val Met Leu Met Gly Asp Phe Asn Ala Gly Cys Ser Tyr 65 70 75 80 Val Arg Pro Ser Gln Trp Ser Ser Ile Arg Leu Trp Thr Ser Pro Thr 85 90 95 Phe Gln Trp Leu Ile Pro Asp Ser Ala Asp Thr Thr Ala Thr Pro Thr 100 105 110 His Cys Ala Tyr Asp Arg Ile Val Val Ala Gly Met Leu Leu Arg Gly 115 120 125 Ala Val Val Pro Asp Ser Ala Leu Pro Phe Asn Phe Gln Ala Ala Tyr 130 135 140 Gly Leu Ser Asp Gln Leu Phe Ser Val His Thr Cys Ser Gly Ala Gly 145 150 155 160 Leu Gly Glu Arg His Gly Leu Pro Ala Ser Ala Ala Leu Pro Ser Asn 165 170 175 Thr Cys Arg Ala Gly Thr His Arg Val Ser Thr 180 185

Claims (17)

Contains biopolymer nanoparticles with DNase-I protein bound to the surface as an active ingredient,
A pharmaceutical composition for preventing or treating SARS-CoV-2 virus infection, wherein the binding ratio of DNase-I protein to the biopolymer nanoparticle is 10 to 70% by weight.
According to paragraph 1,
The biopolymers include melanin, chitosan, alginate, heparin, cellulose, gelatin, polyvinyl alcohol, polyvinylpyrrolidione, In the group consisting of collagen, gelatin, polycaprolactone, poly(lactic-co-glycolic acid), polylactide, and derivatives thereof A pharmaceutical composition, characterized in that it is one or more selected.
According to paragraph 1,
A pharmaceutical composition, wherein the biopolymer nanoparticles have a surface coated with polyethylene glycol.
According to paragraph 1,
A pharmaceutical composition characterized in that the biopolymer nanoparticle having the DNase-I protein bound to its surface satisfies one or more of the following characteristics:
(a) the average diameter of the nanoparticles is 100 to 300 nm;
(b) the surface charge of the nanoparticles is -20 mV to -0.1 mV;
(c) the polydispersity of the nanoparticles is 0.02 to 0.15; or
(d) The nanoparticles are spherical.
delete According to paragraph 1,
The SARS-CoV-2 virus infection is selected from the group consisting of coronavirus infection-19, sepsis, acute respiratory syndrome, pneumonia, cytokine storm, cytokine release syndrome, systemic inflammatory response syndrome, and multiple organ failure. A pharmaceutical composition, characterized in that it contains one or more.
According to paragraph 1,
The pharmaceutical composition is for the treatment of an individual with an increase in one or more levels selected from the group consisting of extracellular DNA and citrullinated histone H3 levels in the blood compared to normal people.
According to paragraph 1,
The pharmaceutical composition is for the treatment of individuals with reduced activity or level of DNase-I compared to normal people.
According to paragraph 1,
The pharmaceutical composition is characterized in that it increases the activity or level of DNase-I in the blood.
According to paragraph 1,
The pharmaceutical composition inhibits one or more selected from the group consisting of neutrophil proliferation, myeloid peroxidase activity, neutrophil elastase activity, neutrophil extracellular trap formation (NETosis), and neutrophil NF-κB activity. A pharmaceutical composition, characterized in that.
According to paragraph 1,
The pharmaceutical composition is characterized in that it inhibits the production of inflammatory cytokines.
According to clause 11,
A pharmaceutical composition, wherein the inflammatory cytokine is one or more selected from the group consisting of IL-1β, IL-6, IL-8, CCL2, IFN-γ, and TNF-α.
A kit for preventing or treating SARS-CoV-2 virus infection, comprising the pharmaceutical composition of any one of claims 1 to 4 and 6 to 12.
A quasi-drug composition for preventing or improving SARS-CoV-2 virus infection containing biopolymer nanoparticles with DNase-I protein bound to the surface as an active ingredient,
A quasi-drug composition for preventing or improving SARS-CoV-2 virus infection, wherein the binding ratio of DNase-I protein to the biopolymer nanoparticle is 10 to 70% by weight.
A method for producing biopolymer nanoparticles with DNase-I protein bound to the surface, comprising the following steps:
Method for producing biopolymer nanoparticles with DNase-I protein bound to the surface, wherein the binding ratio of DNase-I protein to the biopolymer nanoparticle is 10 to 70% by weight:
(S1) preparing nanoparticles by adding an alkaline solution to the biopolymer solution; and
(S2) Adding the nanoparticles obtained in step (S1) to a buffer containing DNase-I.
According to clause 15,
A preparation method, wherein the buffer solution containing DNase-I in step (S2) further contains polyethylene glycol.
According to clause 15,
In the step (S2), the nanoparticles and DNase-I are mixed at a weight ratio of 1:0.2 to 2.