Methods for maximizing coronavirus-killing activity of high density lipoproteins and pharmaceutical compositions for preventing or treating COVID-19

Abstract

The present invention relates to a method for maximizing the coronavirus-killing activity of high-density lipoprotein and a pharmaceutical composition for preventing or treating COVID-19. As confirmed by the present inventors, non-glycated normal high-density lipoprotein (HDL) exhibits superior killing activity against coronaviruses (SARS-CoV-2) compared with glycated HDL. Therefore, a pharmaceutical composition for preventing and treating COVID-19 containing non-glycated normal HDL as an active ingredient is provided. Furthermore, based on the inventors' confirmation, the present invention is useful because it can provide a method for maximizing coronavirus-killing activity using an HDL glycation inhibitor and a method for screening pharmaceutical compositions for preventing and treating COVID-19 by evaluating the degree of HDL glycation inhibition of candidate drugs.

Description

[Technical field]

[0001] 1. Field of the Invention The present invention relates to a method for maximizing the coronavirus-killing activity of high density lipoproteins and a pharmaceutical composition for preventing or treating COVID-19. [Background technology]

[0002] 2. Description of Related Art COVID-19, which broke out in Wuhan, Hubei Province, China in December 2019, had spread to approximately 114 countries by March 2020. As a result, the World Health Organization (WHO) declared COVID-19 to be in a global pandemic state. Since then, by September 2020, approximately 35 million people worldwide have been infected, of which approximately 1 million have died, and the number of infected people and deaths continues to increase.

[0003] In addition to fever, COVID-19 can cause respiratory symptoms such as dry cough, phlegm, and difficulty breathing, and can lead to complications such as acute respiratory distress syndrome, heart failure, and arrhythmia. In this case, conservative treatment is performed using oxygen therapy and the administration of antiviral drugs and antibiotics, but since this is not a treatment for coronavirus, there is a problem that it is not sufficiently effective.

[0004] COVID-19 is primarily transmitted by droplets produced by coughing and sneezing, and is known to be more contagious than influenza, with a basic reproduction number (the average number of people an infected person infects during the infectious period) of 2 to 2.5. Furthermore, in the case of COVID-19, even asymptomatic infected individuals can spread the virus, making it difficult to block it effectively.

[0005] Coronavirus (CoV) refers to viruses that phylogenetically belong to the Coronaviridae family, and the subgroup Ortho Coronaviridae is classified into four genera: AlphaCoV, BetaCoV, DeltaCoV, and GammaCoV. Of these, only AlphaCoV and BetaCoV infect mammals, while DeltaCoV and GammaCoV infect birds and some mammals.

[0006] So far, there are seven types of coronaviruses (HCoVs) that can infect humans: the alpha-CoVs HCoV-229E and HCoV-NL63, the beta-CoVs HCoV-OC43, HCoV-HKU1, SARS-CoV and MERS-CoV, and SARS-CoV-2, the causative virus of coronavirus disease 2019 (COVID-19). HCoV-229E, HCoV-NL63, HCoV-OC43, and HCoV-HKU1 cause cold or gastrointestinal illness in humans, while SARS-CoV and MERS-CoV, together with SARS-CoV-2, cause severe acute respiratory infections.

[0007] SARS-CoV-2 belongs to the BetaCoV family in the phylogenetic tree, together with SARS-CoV and MERS-CoV, but is clearly distinct from SARS-CoV in terms of molecular phylogeny and evolved long ago from MERS-CoV; their nucleotide sequence similarities are about half as similar, as shown in the evolutionary analysis of SARS-CoV, MERS-CoV and SARS-CoV-2 using maximum likelihood (Evolutionary analysis of SARS-CoV, MERS-CoV-2 and SARS-CoV-2, Maximum Likelihood).

[0008] Furthermore, although SARS-CoV-2 shares some similarities with SARS-CoV in terms of structure and pathogenicity, there are clear structural differences in the protein structure of greatest importance for vaccine development, namely the spike protein (S). The presence of a furin-like cleavage site (SLLR-ST) in SARS-CoV-2 promotes priming of the S protein, further enhancing the infectivity of SARS-CoV-2 compared to SARS-CoV (Le Infezioni in Medicina, n. 2, 174-184, 2020, SARS-CoV-2, SARS-CoV, and MERS-CoV: a comparative overview).

[0009] More specifically, cleavage of the MERS-CoV S protein by RSVR↓SV is mediated by furin during virus release, whereas the SARS-CoV S protein is not completely cleaved due to the lack of a furin-like cleavage site (SLLR-ST) in SARS-CoV. In MERS-CoV, cleavage of the S protein occurs at the conserved sequence AYT↓M by proteases expressed by target cells (elastase, cathepsin L or TMPRS). On the other hand, the S protein of SARS-CoV-2 has 12 additional nucleotides upstream of the single Arg↓ cleavage site1 to form a PRRAR↓SV sequence, which corresponds to the furin-like cleavage site (SLLR-ST). As mentioned above, the presence of the furin-like cleavage site (SLLR-ST) in SARS-CoV-2 facilitates the priming of the S protein and, in turn, enhances the infectivity of SARS-CoV-2 compared to SARS-CoV. Thus, there are clear structural differences between SARS-CoV-2 and SARS-CoV.

[0010] Furthermore, the RNA nucleotide sequence of SARS-CoV-2 has clear differences compared to the RNA nucleotide sequences of existing SARS-CoV and MERS-CoV. For example, the RNA nucleotide sequence of SARS-CoV-2 is 17.7% different from the RNA nucleotide sequence of existing SARS-CoV. This difference is obviously so large that existing SARS-CoV treatments cannot be used as is to prevent and treat SARS-CoV-2, and the development of a new vaccine for SARS-CoV-2 is absolutely necessary.

[0011] On the other hand, it has been recently reported that the levels of total cholesterol, HDL cholesterol, and LDL cholesterol in the blood of COVID-19 patients are generally lower than those of normal people, and patients with lower levels of HDL cholesterol have higher severity of COVID-19 disease (Wang et al. Lipids in Health and Disease (2020) 19:204). However, another report suggested that when coronavirus (SARS-Co-V-2) enters cells, it binds to HDL cholesterol and enters cells through SR-B-1, a receptor for HDL (Nature metabolism 2020.11.26., https: / / www.nature.com / articles / s42255-020-00324-0). Thus, so far, the function and role of HDL in COVID-19 infection is unclear. Furthermore, the above reports are only early-stage reports comparing COVID-19 patients with normal people based on various blood indicators, and their direct relationship or function with COVID-19 disease has not yet been clarified, requiring further research and development in various ways. Summary of the Invention

[0012] In the above context, the present inventors first confirmed the ability of native HDL (high density lipoprotein) isolated from human serum by ultracentrifugation to kill coronavirus (SARS-Co-V-2). Furthermore, the present inventors prepared glycated HDL from native HDL by fructose treatment and investigated the changes in HDL structure and function due to glycation. Observation with an electron microscope confirmed that the structure and shape of native HDL were changed by glycation and that paraoxonase activity was inhibited by glycation. The present inventors also confirmed for the first time that when the structure and function of HDL were impaired by glycation of HDL, its ability to kill coronavirus was also reduced. As a result, based on the facts disclosed by the inventors, the inventors have confirmed that it is possible to provide a pharmaceutical composition for preventing and treating COVID-19 infection, which contains HDL (high density lipoprotein) as an active ingredient, a method for maximizing the coronavirus-killing activity of HDL (high density lipoprotein), and a screening method for developing drugs for preventing and treating COVID-19 infection, thereby completing the present invention.

[0013] Summary of the Invention The object of the present invention is to provide a pharmaceutical composition for preventing and treating Corona 19 infection (COVID-19) capable of killing coronavirus (SARS-Co-V-2), a method for maximizing the coronavirus-killing activity of HDL (high-density lipoprotein), and a screening method for developing drugs for preventing and treating Corona 19 infection (COVID-19).

[0014] To achieve the above objective, the present invention provides a pharmaceutical composition for the prevention and treatment of Corona 19 infection (COVID-19), comprising HDL (high density lipoprotein) as an active ingredient.

[0015] The present invention also provides a method of maximizing the coronavirus-killing activity of HDL (high density lipoprotein) using an inhibitor of HDL glycation.

[0016] The present invention also provides an HDL (high density lipoprotein) glycation induction model prepared by treating HDL (high density lipoprotein) with any one or more sugar compounds selected from the group consisting of fructose, glucose and galactose.

[0017] The present invention also relates to a method for screening a pharmaceutical composition for the prevention and treatment of Corona 19 infection (COVID-19), comprising the following steps: Using the HDL glycation-induced model as a control group and the HDL glycation-induced model treated with the candidate drug as an experimental group, the degree of glycation is compared and evaluated; and determining that the candidate drug is effective against COVID-19 if the degree of glycation is lower in the experimental group treated with the candidate drug compared to the control group; The present invention provides a method comprising the steps of:

[0018] Furthermore, the present invention relates to a method for providing information for evaluating the prognosis of COVID-19, comprising the following steps: Using the HDL glycation induction model as a control group and HDL isolated from the subject's blood sample as an experimental group, the degree of glycation is comparatively evaluated; and determining that the prognosis of COVID-19 is good if the degree of glycation of the experimental HDL isolated from the blood sample is lower than that of the control group; The present invention provides a method comprising the steps of:

[0019] Beneficial Effects As confirmed by the present inventors, non-glycated (non-glycated) normal high density lipoprotein (HDL) exhibits superior killing activity against coronavirus (SARS-Cov-2) to glycated HDL, and therefore a pharmaceutical composition for preventing and treating COVID-19, which contains non-glycated natural HDL as an active ingredient, is provided. Furthermore, based on the confirmation by the present inventors, the present invention is useful because it can provide a method for maximizing coronavirus killing activity by using an inhibitor of HDL glycation, and can provide a method for screening pharmaceutical compositions for preventing and treating COVID-19 by evaluating the degree of HDL glycation inhibition of candidate drugs. [Brief description of the drawings]

[0020] [Figure 1] FIG. 1 is a photograph showing the electrophoretic patterns of native HDL and glycated HDL according to the present invention. [Diagram 2] FIG. 2 is a graph showing the results of fluorescence analysis of native HDL and glycated HDL. [Diagram 3] FIG. 3 is a set of electron micrographs showing the structures of native and glycated HDL. [Figure 4] FIG. 4 is a graph showing a comparison of paraoxonase activity between native HDL and glycated HDL. [Figure 5A] FIG. 5A is a photograph showing normal macrophages. [Figure 5B] FIG. 5B is a photograph showing macrophage phagocytosis of acetylated LDL. [Figure 5C] FIG. 5C is a photograph showing that macrophage phagocytosis of acetylated LDL was suppressed by co-treatment with native HDL. [Figure 5D] FIG. 5D is a photograph showing that macrophage phagocytosis of acetylated LDL was suppressed by co-treatment with glycated HDL. [Figure 6]FIG. 6 is a graph showing the amount of oxidized species detected in normal control cell culture medium, cell culture medium treated with acetylated LDL, cell culture configurations co-treated with acetylated LDL and native HDL, and cell culture medium co-treated with acetylated LDL and glycated HDL, expressed as the amount of MDA (malondialdehyde). [Figure 7] FIG. 7 is a graph showing the effect of native HDL and glycated HDL on the viability of African green monkey kidney cells. [Figure 8] FIG. 8 is a graph showing the coronavirus-killing effect of native HDL according to the present invention, in comparison with that of glycated HDL. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] Description of the Preferred Embodiments The present invention will be described in detail below.

[0022] The present invention provides a pharmaceutical composition for the prevention and treatment of Corona 19 infection (COVID-19), comprising HDL (high density lipoprotein) as an active ingredient.

[0023] HDL is non-glycosylated HDL and has the ability to kill coronavirus (SARS-Co-V-2). HDL is non-glycosylated HDL and has the ability to kill coronavirus (SARS-Co-V-2) due to the normal function of HDL; for example, HDL has the ability to kill coronavirus (SARS-Co-V-2) due to the paraoxonase activity of HDL or the activity of preventing macrophage phagocytosis of acetylated LDL.

[0024] The present inventors compared and evaluated the structure, function, and coronavirus (SARS-CoV-2) killing activity of natural HDL and an HDL glycation-induced model. As a result, it was confirmed that the normal structure and function of HDL were impaired in the HDL glycation-induced model. In particular, natural HDL showed excellent killing ability against coronavirus (SARS-CoV-2), and it was confirmed that a pharmaceutical composition for preventing and treating coronavirus 19 infection (COVID-19) containing natural HDL as an active ingredient can be provided.

[0025] In one embodiment of the present invention, natural HDL (high density lipoprotein) was prepared by separating it from human serum by ultracentrifugation, and the HDL was treated with fructose to prepare an HDL glycation-induced model; the natural HDL and the HDL glycation-induced model were analyzed by electron microscopy. As a result, it was confirmed that the natural HDL had a large particle size of 18 to 21 nm in diameter and had a clear structure and outline, whereas the glycated HDL had a particle structure that had changed due to glycation modification by fructose and oxidative stress, had a small particle size of 13 to 16 nm in diameter, and had a blurred structure and outline (see FIG. 3).

[0026] In one embodiment of the present invention, the paraoxonase activity of native HDL was compared with that of an HDL glycation-induced model and evaluated. As a result, it was shown that the paraoxonase activity of glycated HDL was less than half that of native HDL, confirming that the function of HDL was impaired by glycation (see FIG. 4).

[0027] In one embodiment of the present invention, the preventive activity of natural HDL and HDL glycation induction model against macrophage phagocytosis of acetylated LDL, which is an early reaction of atherosclerosis, was compared and evaluated. As a result, when natural HDL was co-treated with acetylated LDL, the phagocytosis of LDL was reduced by more than 60% compared to when acetylated LDL was treated alone, whereas when glycated HDL was co-treated with acetylated LDL, the phagocytosis was increased by 1.3 times compared to when natural HDL was treated, confirming that the function of HDL was impaired due to glycation (see FIG. 5).

[0028] In one embodiment of the present invention, the amount of oxidants produced in the cell culture medium was compared between native HDL and HDL glycation-induced model by thiobarbituric acid reaction assay. As a result, the highest MDA (malondialdehyde) of 3.8 nM was detected when acetylated LDL was treated, whereas the lowest MDA of 1.3 nM was detected when native HDL was co-treated, and 2.8 nM was detected when glycated HDL was co-treated, confirming that HDL function was impaired by glycation (see FIG. 6).

[0029] In one embodiment of the present invention, the cytotoxicity of native HDL and an HDL glycation-induced model was compared and evaluated. As a result, native HDL showed a cell viability of 68-73%, whereas glycated HDL showed a cell viability of 40-49%, confirming that the cytotoxicity of HDL is increased by glycation (see FIG. 7).

[0030] In one embodiment of the present invention, the ability of natural HDL and HDL glycation-induced model to kill coronavirus (SARS-Co-V-2) was compared and evaluated. As a result, natural HDL showed 62% killing ability, while glycated HDL showed 17% killing ability, confirming that HDL's coronavirus (SARS-Co-V-2) killing ability was impaired by glycation (see FIG. 8). Furthermore, it was confirmed that natural HDL has 3.6 times higher coronavirus (SARS-Co-V-2) killing ability than glycated HDL (see FIG. 8).

[0031] Therefore, non-glycosylated native HDL (high density lipoprotein) showed no damage in terms of HDL structure and function and exhibited excellent killing ability against coronavirus (SARS-CoV-2); therefore, a pharmaceutical composition for the prevention and treatment of coronavirus 19 infection (COVID-19) containing native HDL as an active ingredient can be provided.

[0032] The present invention also provides a method of maximizing the coronavirus-killing activity of HDL (high density lipoprotein) using an inhibitor of HDL glycation.

[0033] By inhibiting HDL glycation, HDL glycation inhibitors can prevent the reduction in HDL's coronavirus (SARS-CoV-2)-killing ability due to structural and functional damage to HDL, and can also maximize the coronavirus (SARS-CoV-2)-killing ability of HDL by inhibiting HDL glycation.

[0034] The present invention also provides an HDL (high density lipoprotein) glycation induction model prepared by treating HDL (high density lipoprotein) with any one or more sugar compounds selected from the group consisting of fructose, glucose and galactose.

[0035] The sugar compound can be used at a concentration equal to or greater than the concentration that maximally saturates HDL glycation.Furthermore, the sugar compound treatment is preferably a fructose treatment.

[0036] In one embodiment of the present invention, HDL glycation-induced models were prepared by separating HDL from human serum by ultracentrifugation and treating the separated HDL with excess fructose (5 mM to 250 mM per 1 to 10 mg / ml HDL) for 72 hours. Electrophoresis confirmed that the HDL glycation-induced models were generated and that apoA-I with increased molecular weight was observed in the glycated HDL; it was also confirmed that multimerized HDL such as dimers to tetramers was generated (see FIG. 1). Furthermore, it was confirmed that the HDL glycation-induced models had a 7-fold increase in fluorescence intensity compared to native HDL, confirming that the HDL glycation-induced models were successfully generated (see FIG. 2).

[0037] The present invention also relates to a method for screening a pharmaceutical composition for preventing or treating coronavirus 19 infection (COVID-19), comprising the steps of: Using the HDL glycation-induced model as a control group and the HDL glycation-induced model treated with the candidate drug as an experimental group, the degree of glycation is compared and evaluated; and determining that the candidate drug is effective against COVID-19 if the degree of glycation is lower in the experimental group treated with the candidate drug compared to the control group; The present invention provides a method comprising the steps of:

[0038] The step of comparatively assessing the degree of glycation may involve detecting and quantifying a fluorescent signal generated by glycation, thereby comparatively assessing the degree of glycation.

[0039] In one embodiment of the present invention, the degree of glycation in the HDL glycation-induced model compared to native HDL was quantitatively measured and assessed by confirming that the fluorescence intensity was increased 7-fold in the HDL glycation-induced model compared to native HDL (see FIG. 2 ).

[0040] According to the determination that a candidate drug is effective against COVID-19 if the degree of glycation in the experimental group treated with the candidate drug is lower than that in the control group, a drug that inhibits the glycation of natural HDL can be understood as a drug that prevents damage to the coronavirus (SARS-CoV-2)-killing ability of HDL.

[0041] In one embodiment of the present invention, the killing ability of natural HDL and HDL glycation-induced model against coronavirus (SARS-CoV-2) was compared and evaluated. As a result, natural HDL showed 62% killing ability, while glycated HDL showed 17% killing ability, confirming that the killing ability of HDL against coronavirus (SARS-CoV-2) was impaired by glycation.

[0042] Therefore, HDL glycation inhibitors that prevent glycation-induced damage to HDL's killing ability against coronavirus (SARS-CoV-2) and maximize the killing ability can be screened as active ingredients for preventing or treating coronavirus 19 infection (COVID-19).

[0043] The step of comparatively assessing the degree of glycation of HDL may further include a step of comparatively assessing at least one activity of HDL paraoxonase activity and an activity of HDL that prevents macrophage phagocytosis of acetylated LDL.

[0044] If at least one of the HDL paraoxonase activity and the HDL activity that prevents macrophage phagocytosis of acetylated LDL is higher in the experimental group treated with the candidate drug compared to the HDL glycation induction model control group, the candidate drug can be determined to be effective against coronavirus 19 infection (COVID-19).

[0045] In one embodiment of the present invention, the paraoxonase activity of native HDL was compared with that of an HDL glycation-induced model, and was evaluated. As a result, it was shown that the paraoxonase activity of glycated HDL was less than half that of native HDL, confirming that the function of HDL was impaired by glycation (see FIG. 4).

[0046] In one embodiment of the present invention, the activity of natural HDL to prevent macrophage phagocytosis of acetylated LDL was compared and evaluated in a HDL glycation induction model. As a result, the phagocytosis of LDL was reduced by 60% or more when natural HDL was co-treated with acetylated LDL compared to when it was treated alone, whereas the phagocytosis of LDL was increased by 1.3 times when glycated HDL was co-treated compared to when it was treated with natural HDL, confirming that the function of HDL was impaired by glycation (see FIG. 5).

[0047] Therefore, HDL glycation inhibitors that prevent glycation-induced damage to HDL paraoxonase activity and HDL activity preventing macrophage phagocytosis of acetylated LDL, and maximize the killing ability of HDL against coronavirus (SARS-CoV-2), may be screened as active ingredients for preventing or treating coronavirus 19 infection (COVID-19).

[0048] Furthermore, the present invention relates to a method for providing information for evaluating the prognosis of COVID-19, comprising the following steps: Using the HDL glycation induction model as a control group and HDL isolated from the subject's blood sample as an experimental group, the degree of glycation is comparatively evaluated; and determining that the prognosis for COVID-19 is good if the degree of glycation of the experimental HDL isolated from the blood sample is lower than that of the control group; The present invention provides a method comprising the steps of:

[0049] The step of comparatively assessing the degree of glycation may involve detecting and quantifying a fluorescent signal generated by glycation, thereby comparatively assessing the degree of glycation.

[0050] In one embodiment of the present invention, the degree of glycation in the HDL glycation-induced model compared to native HDL was quantitatively measured and assessed by confirming that the fluorescence intensity was increased 7-fold in the HDL glycation-induced model compared to native HDL (see FIG. 2 ).

[0051] The prognosis of COVID-19 is judged to be good if the degree of glycation of the experimental HDL isolated from blood samples is lower than that of the HDL glycation induction model control group, based on the judgment that the coronavirus (SARS-CoV-2)-killing ability of HDL is not damaged by glycation and the coronavirus (SARS-CoV-2)-killing ability of natural HDL is maintained.

[0052] In one embodiment of the present invention, the killing ability of natural HDL and HDL glycation-induced model against coronavirus (SARS-CoV-2) was compared and evaluated. As a result, natural HDL showed 62% killing ability, while glycated HDL showed 17% killing ability, confirming that the killing ability of HDL against coronavirus (SARS-CoV-2) was impaired by glycation.

[0053] Therefore, by confirming that HDL glycation is absent or reduced compared to the control group, it can be confirmed that there is no or reduced damage caused by glycation to HDL's coronavirus (SARS-CoV-2)-killing ability, and thus the prognosis for COVID-19 can be determined to be good.

[0054] The step of comparatively assessing the degree of glycation of HDL may further include a step of comparatively assessing at least one activity of HDL paraoxonase activity and an activity of HDL that prevents macrophage phagocytosis of acetylated LDL.

[0055] If at least one of the HDL paraoxonase activity and the HDL activity that prevents macrophage phagocytosis of acetylated LDL is higher in the experimental HDL isolated from blood samples compared to the HDL glycation induction model control group, the prognosis for COVID-19 can be determined to be good.

[0056] In one embodiment of the present invention, the paraoxonase activity of native HDL was compared with that of an HDL glycation-induced model and evaluated. As a result, it was shown that the paraoxonase activity of glycated HDL was less than half that of native HDL, confirming that the function of HDL was impaired by glycation (see FIG. 4).

[0057] In one embodiment of the present invention, the activity of natural HDL to prevent macrophage phagocytosis of acetylated LDL was compared and evaluated in a HDL glycation induction model. As a result, the phagocytosis of LDL was reduced by more than 60% when co-treated with natural HDL compared to when acetylated LDL was treated alone, whereas the phagocytosis was increased by 1.3 times when co-treated with glycated HDL compared to when treated with natural HDL, confirming that the function of HDL was impaired by glycation (see FIG. 5).

[0058] Therefore, by confirming that the paraoxonase activity of HDL and the activity of HDL to prevent macrophage phagocytosis of acetylated LDL are intact or not reduced compared to the control group, it can be confirmed that there is no or little damage caused by glycation to the coronavirus (SARS-CoV-2)-killing ability of HDL, and thus the prognosis for COVID-19 can be determined to be good. EXAMPLES

[0059] The present invention will now be described in detail with reference to the following examples and experimental examples.

[0060] However, the following examples and experimental examples are for the purpose of illustrating the present invention and are not intended to limit the scope of the present invention.

[0061] Example 1: Preparation of native HDL Native HDL was isolated from human plasma by ultracentrifugation (100,000 g) for 48–72 h according to the method proposed by the inventors in our previous paper (Cho, K.-H.; Shin, D.-G.; Baek, S.-H.; Kim, J.-R. Myocardial infarction patients show altered lipoprotein properties and functions when compared with stable angina pectoris patients. Exp Mol Med 2009, 41, 67-76, doi:10.3858 / emm.2009.41.2.009.).

[0062] Example 2: Preparation of HDL glycation induction model The HDL glycation induction model was prepared by treating a portion of the human serum-derived HDL prepared in Example 1 with excess fructose.

[0063] Specifically, purified HDL (1-10 mg / mL) was reacted with 5 mM-250 mM D-fructose [in 200 mM potassium phosphate / 0.02% sodium azide buffer (pH 7.4)] at 37°C in air containing 5% CO2 for 24-72 hours.

[0064] After the glycation reaction, HDL was compared and confirmed by 12% SDS-PAGE, which separates proteins according to their molecular weight. As a result, as shown in Figure 1, apoA-I with increased molecular weight was observed in glycated HDL, and several polymerized HDLs such as dimers and tetramers were confirmed.

[0065] On the other hand, the degree of glycation in the HDL glycation induction model was determined by measuring the fluorescence intensity of the generated fluorescent substance at 370 nm (excitation) and 440 nm (emission); the results are shown in FIG. 2.

[0066] As shown in Figure 2, after treatment with fructose for 72 hours, the fluorescence intensity of the HDL glycation induction model increased by approximately 7-fold compared to native HDL, confirming that the HDL glycation induction model was successfully constructed.

[0067] Experimental Example 1: Evaluation of structural changes in HDL due to glycation The structural changes of HDL caused by glycation were evaluated by analyzing the structural differences between native HDL and the HDL glycation-induced model.

[0068] Specifically, the protein concentrations of native HDL and HDL glycation-induced model were adjusted to 0.3 mg / mL, stained with 1% sodium phosphotungstate, and observed under a high voltage of 80 kV using a transmission electron microscope (Hitachi HT-7800, 40,000X); the photographs are shown in Figure 3.

[0069] As shown in Figure 3, native HDL had a large particle size of 18-21 nm in diameter and a clear structure and shape, whereas glycated HDL had a small particle size of 13-16 nm in diameter and a blurred structure and shape. These results indicate that glycation modification and oxidative stress caused by fructose treatment caused the cleavage and aggregation of apoA-I, altering the particle structure of HDL.

[0070] Experimental Example 2: Evaluation of changes in HDL function due to glycation The functional changes of HDL due to glycation were evaluated by analyzing the functional differences between the native HDL prepared in Example 1 and the HDL glycation-induced model prepared in Example 2.

[0071] <2-1> Analysis of paraoxonase activity The functional changes of HDL due to glycation were evaluated by analyzing the difference in paraoxonase activity between native HDL and the HDL glycation-induced model.

[0072] The activity of paraoxonase, a key enzyme involved in the antioxidant function of HDL, was measured as follows: paraoxonase activity was assessed by measuring the absorbance at 415 nm of p-nitrophenol produced by reacting native HDL and HDL glycation-induced model, respectively, with paraoxon-ethyl as a substrate in an enzyme solution (90 mM Tris-HCl / 3.6 mM NaCl / 2 mM CaCl2 [pH 8.5]); the results are shown in Figure 4.

[0073] As shown in FIG. 4, it was confirmed that the paraoxonase activity in glycated HDL was less than half that of native HDL, indicating that the functional damage of HDL was caused by glycation.

[0074] <2-2>Analysis of the early reaction of atherosclerosis (macrophage phagocytosis of acetylated LDL) We evaluated the functional changes of HDL due to glycation by analyzing the difference in the preventive activity between native HDL and an HDL glycation-induced model against macrophage phagocytosis of acetylated LDL, an early reaction of atherosclerosis.

[0075] THP-1 cells, a human mononuclear cell line, were purchased from the American Type Culture Collection (ATCC #TIB-202™) and maintained in RPMI 1640 (Hyclone) supplemented with 10% fetal bovine serum (FBS). Cells cultured in 24-well plates and at less than 20 passages were reacted with phorbol 12-myristate 13-acetate (PMA; final concentration 150 nM) for 48 hours at 37°C in a humidified incubator (5% CO2, 95% air) to induce differentiation into macrophages.

[0076] We treated differentiated macrophages with acetylated LDL to induce phagocytosis while culturing, and simultaneously treated them with native HDL and glycated HDL to compare the degree of preventive activity of macrophages against phagocytosis. After fixing the cells, the degree of phagocytosis was compared by Oil Red O staining; the results are shown in Figure 5.

[0077] As shown in Figure 5, when acetylated LDL was co-treated with native HDL, the phagocytosis of LDL was reduced by more than 60% compared to when acetylated LDL was treated alone, whereas when glycated HDL was co-treated with acetylated LDL, the phagocytosis was increased by 1.3-fold compared to when native HDL was treated.

[0078] Meanwhile, the amount of oxidants produced in the cell culture medium was compared by thiobarbituric acid reaction assay. As a result, as shown in Figure 6, the highest MDA (malondialdehyde) of 3.8 nM was detected when acetylated LDL was treated alone, whereas the lowest MDA of 1.3 nM was detected when native HDL was co-treated, and 2.8 nM MDA was detected when glycated HDL was co-treated.

[0079] As shown by the above results, glycated HDL was found to have significantly reduced activity in preventing the phagocytosis of acetylated LDL, which is an early reaction in atherosclerosis, confirming that the function of HDL was impaired by glycation.

[0080] Experimental Example 3: Comparative evaluation of cytotoxicity The effect of glycation on HDL cytotoxicity was evaluated by assessing the cytotoxicity of native HDL and an induced HDL glycation model.

[0081] Specifically, the cytotoxicity of native HDL and HDL glycation-induced model was evaluated by comparing them using MTT (3-(4,5-dimethylthiazol-2-yl)-3,5-diphenyltetrazolium bromide) assay. African green monkey kidney cells (ATCC CRL-1586) were cultured in 96-well cell culture plates at 5 × 10 4 The cells were plated at a density of 100 μL / well and cultured in a 5% CO2 incubator at 37°C for 48 hours to obtain a cell monolayer, which was washed twice with saline. The plate was treated with each lipoprotein (100 μL / well) at each test concentration, and then cultured in a 5% CO2 incubator at 37°C for 72 hours. After adding MTT solution (10 μL / well), the plate was placed in a 5% CO2 incubator at 37°C for 4 hours, and after 4 hours of reaction, the formazan crystals were fully dissolved, and then the absorbance was measured at 570 nm. The ratio of cytotoxicity was calculated according to the following formula as the ratio of normal cells to the experimental group treated with lipoproteins; the results are shown in Figure 7.

[0082] Cell viability (%) = test OD / control OD x 100%

[0083] As shown in Figure 7, native HDL showed a cell viability of 68 to 73%, whereas glycated HDL showed a cell viability of 40 to 49%, confirming that the cytotoxicity of HDL was increased by glycation.

[0084] Experimental Example 4: Evaluation of coronavirus killing activity The killing ability of native HDL and HDL glycation-induced model against coronavirus (SARS-CoV-2) was evaluated.

[0085] African green monkey kidney cells (ATCC CRL-1586) were cultured in a 96-well plate at 5 × 10 4 The cells were seeded at a density of 100 μL / well and cultured in a 5% CO2 incubator at 37 °C for 48 hours to obtain a cell monolayer, which was then washed twice with saline. After obtaining a cell monolayer, the cells were washed twice with saline and the number of cells was counted. Then, coronavirus (SARS CoV-2) was dispensed into the plate (100 μL / well) using DMEM (FBS-free, 1% antibiotic-antimycotic) to infect the plate at an MOI of 0.001, and then the plate was left to stand in a 5% CO2 incubator at 37 °C for 1 hour. After infection for 1 hour, the virus was removed, and culture medium containing samples of native HDL and HDL glycation induction model prepared at test concentrations was dispensed into the plate (100 μL / well), followed by culturing for 72 hours in a 5% CO2 incubator at 37 °C. After checking the state of the cells, MTT solution was added to the plate (10 μL / well) and the plate was left to stand in a 5% CO2 incubator at 37 °C for 4 hours. After 4 hours of reaction, MTT solution was added to the plate (100 μL / well), the formazan crystals were thoroughly dissolved using a pipette, and the absorbance was measured at 570 nm using a plate reader. The cytotoxicity ratio was calculated as the ratio of normal cells to the experimental group treated with lipoprotein according to the following formula; the results are shown in Figure 8.

[0086] Viral inhibition rate (%) = (Test OD - Viral OD) / (Control OD - Viral OD) x 100%

[0087] As a result, when treated at 60 μg / mL, native HDL showed 62% of the SARS CoV-2 killing ability, whereas glycated HDL showed 17% of the SARS CoV-2 killing ability under the same conditions. These results indicate that native HDL has 3.6-fold higher SARS CoV-2 killing ability than glycated HDL.

[0088] As shown in Figures 7 and 8, when treated with native HDL, cytotoxicity was low and CC 50 The EC 50 The viral killing ability of natural HDL against coronavirus (SARS-CO-V-2) was confirmed.

[0089] From the above results, it was confirmed that non-glycated (non-glycated) native high density lipoprotein (HDL) can be provided as a pharmaceutical composition for the prevention and treatment of coronavirus 19 infection (COVID-19). Furthermore, as confirmed by the present inventors, it can be used to explain why the risk and mortality of COVID-19 is higher in diabetic and hypertensive patients, in whom HDL structure and function are concerned to be impaired, compared to normal people. This can be used for a method of providing information on the prognosis of COVID-19, and also as a method of screening pharmaceutical compositions for the prevention or treatment of COVID-19.

[0090] Industrial Applicability As confirmed by the present inventors, non-glycated (non-glycated) normal high density lipoprotein (HDL) exhibits superior killing activity against coronavirus (SARS-Cov-2) to glycated HDL, and therefore a pharmaceutical composition for preventing and treating COVID-19, comprising non-glycated natural HDL as an active ingredient, is provided. Furthermore, based on the confirmation by the present inventors, the present invention is useful because it can provide a method for maximizing coronavirus killing activity using an HDL glycation inhibitor, and can provide a method for screening pharmaceutical compositions for preventing and treating COVID-19 by evaluating the degree of HDL glycation inhibition of candidate drugs. The present invention includes, for example, the following embodiments: [1] A pharmaceutical composition for the prevention and treatment of Corona 19 infection (COVID-19), containing HDL (high density lipoprotein) as an active ingredient. [2] The pharmaceutical composition for preventing and treating coronavirus 19 infection (COVID-19) described in [1], wherein the HDL is non-glycosylated HDL. [3] A pharmaceutical composition for the prevention and treatment of coronavirus 19 infection (COVID-19) described in [1], wherein HDL is isolated from human serum. [4] A pharmaceutical composition for the prevention and treatment of coronavirus 19 infection (COVID-19) described in [1], wherein HDL has the ability to kill coronavirus (SARS-CoV-2). [5] A pharmaceutical composition for the prevention and treatment of coronavirus 19 infection (COVID-19) described in [4], wherein HDL has the ability to kill coronavirus (SARS-CoV-2) through paraoxonase activity or activity to prevent macrophage phagocytosis of acetylated LDL. [6] A pharmaceutical composition for preventing and treating coronavirus 19 infection (COVID-19) described in [1], wherein the pharmaceutical composition further contains an HDL glycation inhibitor. [7] A pharmaceutical composition for preventing and treating coronavirus 19 infection (COVID-19) described in [6], in which the HDL glycation inhibitor prevents the decrease in HDL's coronavirus (SARS-CoV-2)-killing ability caused by glycation. [8] A method of maximizing the coronavirus-killing activity of HDL (high-density lipoprotein) using an HDL (high-density lipoprotein) glycation inhibitor. [9] A method of maximizing the coronavirus-killing activity of HDL described in [8], in which an HDL glycation inhibitor prevents the decrease in HDL's coronavirus (SARS-CoV-2)-killing ability caused by glycation.

[10] A HDL (high density lipoprotein) glycation induction model prepared by treating HDL (high density lipoprotein) with any one or more sugar compounds selected from the group consisting of fructose, glucose and galactose.

[11] A method for screening a pharmaceutical composition for preventing or treating coronavirus 19 infection (COVID-19), comprising the steps of: Using the HDL glycation induction model described in

[10] as a control group and the HDL glycation induction model treated with the candidate drug as an experimental group, the degree of glycation is compared and evaluated; and determining that the candidate drug is effective against COVID-19 if the degree of glycation is lower in the experimental group treated with the candidate drug compared to the HDL glycation induction model control group; The method comprising:

[12] The method for screening a pharmaceutical composition for preventing or treating coronavirus 19 infection (COVID-19) described in

[11] , wherein the step of comparatively evaluating the degree of HDL glycation further includes a step of comparatively evaluating at least one activity of paraoxonase activity and activity preventing macrophage phagocytosis of acetylated LDL.

[13] The method for screening a pharmaceutical composition for preventing or treating COVID-19 infection described in

[12] , wherein if at least one of paraoxonase activity and activity preventing macrophage phagocytosis of acetylated LDL is higher in an experimental group treated with the candidate drug than in a control group of an HDL glycation induction model, the candidate drug is determined to be effective against COVID-19 infection.

[14] A method for providing information to evaluate the prognosis of coronavirus disease 2019 (COVID-19), comprising the following steps: Using the HDL glycation induction model described in

[10] as a control group and HDL isolated from the subject's blood sample as an experimental group, the degree of glycation is compared and evaluated; and determining that the prognosis of COVID-19 is good when the degree of glycation of the experimental HDL isolated from the blood sample is lower than that of the HDL glycation induction model control group; The method comprising:

[15] The information providing method for evaluating the prognosis of coronavirus 19 infection (COVID-19) described in

[14] , wherein the step of comparatively evaluating the degree of HDL glycation further includes a step of comparatively evaluating at least one of paraoxonase activity and activity preventing macrophage phagocytosis of acetylated LDL.

[16] An information providing method for evaluating the prognosis of coronavirus 19 infection (COVID-19) described in

[15] , in which the prognosis of COVID-19 is determined to be good if at least one of paraoxonase activity and activity preventing macrophage phagocytosis of acetylated LDL is higher in experimental HDL isolated from a blood sample compared to the HDL glycation induction model control group.

Claims

1. A pharmaceutical composition for the prevention and treatment of Corona 19 infection (COVID-19), comprising non-glycated HDL (high density lipoprotein) as an active ingredient, the non-glycated HDL having the ability to kill coronavirus (SARS-CoV-2).

2. The pharmaceutical composition for the prevention and treatment of coronavirus 19 infection (COVID-19) according to claim 1, wherein the non-glycated HDL is isolated from human serum.

3. The pharmaceutical composition for preventing and treating coronavirus 19 infection (COVID-19) according to claim 1, wherein non-glycated HDL has the ability to kill coronavirus (SARS-CoV-2) through paraoxonase activity or the activity of preventing macrophage phagocytosis of acetylated LDL.

4. A method for screening a pharmaceutical composition for preventing or treating COVID-19, comprising the steps of: Comparing the degree of HDL glycation in an HDL glycation induction model not treated with a candidate drug as a control group with the degree of HDL glycation in an HDL glycation induction model treated in vitro with a candidate drug as an experimental group; and determining that the candidate drug is effective against COVID-19 if the degree of HDL glycation is lower in the experimental group treated with the candidate drug in vitro compared to the degree of HDL glycation in the control group; The method comprising:

5. The method for screening a pharmaceutical composition for preventing or treating coronavirus 19 infection (COVID-19) according to claim 4, wherein the step of comparing and evaluating the degree of HDL glycation further comprises a step of comparing at least one activity of paraoxonase activity and activity preventing macrophage phagocytosis of acetylated LDL.

6. The method for screening a pharmaceutical composition for preventing or treating COVID-19 infection according to claim 5, wherein the candidate drug is determined to be effective against COVID-19 infection when at least one of paraoxonase activity and activity preventing macrophage phagocytosis of acetylated LDL is higher in an experimental group treated with the candidate drug compared to the at least one activity in a control group.

7. A method for predicting the prognosis of a coronavirus disease 2019 (COVID-19), comprising the steps of: Using the HDL glycation induction model as a control group and HDL isolated from the subject's blood sample as an experimental group, comparatively evaluating the degree of glycation; and determining that the prognosis of COVID-19 is good when the degree of glycation of the experimental HDL isolated from the blood sample is lower than that of the HDL glycation induction model control group; The method comprising:

8. The method for predicting the prognosis of coronavirus 19 infection (COVID-19) according to claim 7, wherein the step of comparatively evaluating the degree of HDL glycation further comprises a step of comparatively evaluating at least one activity of paraoxonase activity and activity preventing macrophage phagocytosis of acetylated LDL.

9. The method for predicting the prognosis of coronavirus 19 infection (COVID-19) according to claim 8, wherein the prognosis of COVID-19 is determined to be good when at least one of paraoxonase activity and activity preventing macrophage phagocytosis of acetylated LDL is higher in the experimental group HDL isolated from a blood sample compared to the HDL glycation induction model control group.

10. The method for screening a pharmaceutical composition for preventing or treating Corona 19 infection (COVID-19) according to claim 4, wherein the HDL glycation induction model is prepared by treating HDL in vitro with any one or more sugar compounds selected from the group consisting of fructose, glucose and galactose.

11. The method for predicting the prognosis of coronavirus 19 infection (COVID-19) according to claim 7, wherein the HDL glycation induction model is prepared by treating HDL in vitro with any one or more sugar compounds selected from the group consisting of fructose, glucose and galactose.

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