Use of Chlorella sorokinina or its extracts in the preparation of drugs for inhibiting SARS-CoV-2 infection

Abstract

Compositions comprising eukaryotic microalgae or extracts thereof can be used to treat viral diseases, such as coronavirus, and as inhibitors of the binding of the receptor binding domain (RBD) of the spike protein to angiotensin-converting enzyme 2 (ACE2). Compounds derived from eukaryotic microalgae can also be used to treat viral diseases. Eukaryotic microalgae, in turn, can be used as antiviral agents.

Description

Technical Field

[0001] This invention relates to compositions comprising eukaryotic microalgae or extracts thereof. More specifically, this invention relates to compositions comprising eukaryotic microalgae or extracts thereof for the treatment of viral diseases, compounds derived from eukaryotic microalgae for the treatment of viral diseases, compositions comprising eukaryotic microalgae or extracts thereof as inhibitors of the binding of the spike protein receptor-binding domain (RBD) to angiotensin-converting enzyme 2 (ACE2), and the use of eukaryotic microalgae or extracts thereof as antiviral agents. Background Technology

[0002] A virus is a small infectious agent that replicates only within the living cells of other organisms. Viruses can infect a wide variety of life forms, from animals and plants to microorganisms, including bacteria and archaea. Viral infections typically cause symptoms of illness in infected individuals, which can range from mild to severe, leading to morbidity and death.

[0003] Some of the most common symptoms associated with viral infections come from human respiratory illnesses. Examples of human respiratory illnesses caused by viruses include influenza, caused by various influenza viruses; severe acute respiratory syndrome (SARS), caused by a coronavirus (SARS-CoV); respiratory illnesses possibly caused by adenoviruses; and bronchitis and childhood pneumonia caused by respiratory syncytial virus (RSV).

[0004] Coronavirus disease 2019, commonly known as COVID-19, is an infectious disease caused by Severe Acute Respiratory Syndrome Coronavirus type 2 (SARS-CoV-2). The COVID-19 pandemic, also called the coronavirus pandemic, is an ongoing outbreak with over 88 million confirmed cases and over 1.89 million deaths globally as of March 2021. The elderly and those with underlying health conditions have the highest mortality rates. While most people experience mild symptoms, some develop severe symptoms such as pneumonia, respiratory failure, shock, or multiple organ dysfunction.

[0005] The high mortality rate among patients admitted to intensive care units due to severe acute SARS-CoV-2 infection is attributed to the combined effects of hyperinflammatory syndrome, coagulation disorders, and pathological activation of the immune pathway leading to cytokine release syndrome.

[0006] There is an urgent need for effective early treatments that have both antiviral and disease-mitigating effects to limit the strain on healthcare infrastructure caused by severe COVID-19 cases. While vaccination is the most effective primary prevention of SARS-CoV-2 infection, intranasal or oral delivery of antiviral agents may be an important additional method for preventing infection and transmission of SARS-CoV-2 and other respiratory viruses.

[0007] Typically, SARS-CoV-2 is primarily transmitted through aerosols expelled by infected individuals when they breathe, cough, sneeze, or talk. Therefore, one of the main entry points for the SARS-CoV-2 virus into the human body is through the mouth, nose, or eyes. Blocking these entry points with medication may help reduce the number of COVID-19 cases. When the virus enters a subject through the mouth, nose, or eyes, it replicates by attaching to the surface of host cells before entering the cells. The spike protein receptor-binding domain (RBD) recognizes and attaches to the angiotensin-converting enzyme 2 (ACE2) receptor found on the surface of type I and II lung cells, endothelial cells, and ciliated bronchial epithelial cells. ACE2 is an exopeptidase that catalyzes the conversion of angiotensin II to angiotensin 1–7 and L-phenylalanine. Angiotensin II is part of the classic renin-angiotensin system (RAS), a hormonal system that regulates fluid homeostasis, blood pressure, and maintains vascular tone. ACE2 has also been shown to be the primary receptor for respiratory viral entry, including human respiratory coronavirus NL63, SARS coronavirus (SARS-CoV), and other SARS-CoV-2 variants such as novel B117. The binding of SARS-CoV and SARS-CoV-2 to ACE2, subsequent membrane fusion, and viral entry into cells are associated with downregulation of the ACE2 receptor. This downregulation of ACE2 also impairs its crucial protective function, the degradation of angiotensin II into angiotensin 1–7. Angiotensin II is associated with a variety of adverse health outcomes, including cardiovascular disease, increased inflammation due to oxidative stress, and a hypercoagulable state. Furthermore, angiotensin II is a macrophage activator, leading to increased secretion of IL-6 and TNFα, as well as other inflammatory responses. The ACE2 receptor counteracts angiotensin II by degrading and thus eliminating it, and by catalyzing its conversion into angiotensin 1–7, thus generating a variety of positive and even counter-regulatory effects against angiotensin II. Although selective ACE2 inhibitors, such as MLN-4760, may inhibit cell attachment of viruses such as SARS-CoV-2, they also block the degradation of angiotensin II into angiotensin 1–7.

[0008] Therefore, a new therapeutic modality is needed to block the virus from entering through the SARS-CoV-2 RBD without inhibiting ACE2. Summary of the Invention

[0009] The present invention provides a composition comprising eukaryotic microalgae or extracts thereof for the treatment of viral diseases.

[0010] As used herein, the term “treatment” can include preventive (preventive) treatment; curative treatment; maintenance treatment; and promotional treatment.

[0011] Surprisingly, compositions containing eukaryotic microalgae or their extracts have been found to have beneficial effects in preventing the spread of viral diseases such as COVID-19.

[0012] Viral illnesses can be respiratory viral illnesses. Viral illnesses may be caused by one or more of the following: influenza virus, respiratory syncytial virus (RSV), parainfluenza virus, or respiratory adenovirus. Viral illnesses can be severe acute respiratory syndrome (SARS). Viral illnesses can be MERS and / or COVID-19. Viral illnesses may be caused by SARS-related viruses. Viral illnesses may be caused by coronaviruses (SARS-CoV-2). In this article, coronaviruses are understood to include the subfamily Orthocoronavirinae within the family Coronaviridae.

[0013] More specifically, compositions comprising eukaryotic microalgae or extracts thereof have been found to inhibit the binding of the viral spike protein receptor-binding domain (RBD) to the angiotensin-converting enzyme 2 (ACE2) receptor. Advantageously, compositions comprising eukaryotic microalgae or extracts thereof do not affect ACE2 activity. Conversely, compositions comprising eukaryotic microalgae or extracts thereof do not impair respiratory function or cause cardiovascular disease, increased inflammation in response to oxidative stress, or a hypercoagulable state. Advantageously, the composition prevents viral diseases from reaching the brain or bronchi and lungs. Therefore, the compositions of the present invention can be used to inhibit or prevent damage to the respiratory system. Furthermore, by preventing viral infection from colonizing olfactory and gingival epithelial cells, which can migrate from olfactory and gingival epithelial cells to the central nervous system to attack the respiratory center and affect neurons, the compositions of the present invention can also prevent anosmia caused by SARS-CoV-2 infection.

[0014] In an alternative embodiment, the invention may relate to prokaryotic microalgae or extracts thereof. The aspects of the invention described with respect to eukaryotic microalgae or extracts thereof are equally applicable to prokaryotic microalgae or extracts thereof. Specifically, the invention may relate to compositions comprising prokaryotic microalgae or extracts thereof for use in the treatment of viral diseases. Prokaryotic microalgae may include cyanobacteria (formerly known as "blue-green algae").

[0015] The term "eukaryotic microalgae" is understood to refer to different groups of aquatic organisms capable of photosynthesis. Eukaryotic microalgae are unicellular species that exist individually, in chains, or in groups. Eukaryotic microalgae can include green algae, brown algae, and red algae. More specifically, eukaryotic microalgae can include the following phyla: Rhodophyta (red algae), Chlorophyta (green algae), and Phaeophyta (brown algae). Eukaryotic microalgae can belong to the genera *Nannochloropsis* or *Tetraselmis*.

[0016] Preferably, the eukaryotic microalgae can be green eukaryotic microalgae, for example, green eukaryotic microalgae capable of photosynthesis. The eukaryotic microalgae can be from the Chlorophyta phylum, preferably *Chlorella*. The term "Chlorella" is understood to refer to a single-celled eukaryotic green alga belonging to the Chlorophyta phylum. Advantageously, *Chlorella* is a FDA-recognized as safe (GRAS) substance.

[0017] Preferably, the eukaryotic microalgae may be selected from one or more of the following: Chlorella autotrophica, Chlorella colonials, Chlorella lewinii, Chlorella minutissima, Chlorella pituita, Chlorella pulchelloides, Chlorella pyrenoidosa, Chlorella rotunda, Chlorella singularis, Chlorella sorokinina, Chlorella variabilis, Chlorella volutis, Chlorella vulgaris, Tetraselmis Chuii, and Nannochloropsis gaditana.

[0018] More preferably, the composition may comprise eukaryotic microalgae or extracts thereof for treating viral diseases caused by coronavirus SARS-CoV-2, wherein the eukaryotic microalgae is Chlorella.

[0019] Surprisingly, compositions containing Chlorella or its extracts have been found to have potential beneficial effects in the treatment of COVID-19. More specifically, Chlorella has been found to inhibit the binding of the viral RBD to the ACE2 receptor without affecting ACE2 activity. Therefore, compositions containing Chlorella or its extracts could be used to treat SARS-CoV-2 infection and COVID-19.

[0020] Furthermore, it has been found that Chlorella prevents viruses from binding to ACE2 receptors present on cells in the mouth, nose, and / or eyes. By preventing viral diseases from binding to these cell surfaces, Chlorella inhibits the ability of viral diseases to enter and replicate within cells, and prevents further infection from viral disease particles released by already infected cells.

[0021] Extracts from eukaryotic microalgae may contain one or more of the following: proteins, lipids, vitamins, minerals, folic acid, iron, fiber, and / or pigments. Vitamins may include vitamins B and D12.

[0022] The protein may be selected from one or more of the following: plastocyanin (A0A2P6TGD1), glycine-rich 2 (A0A2P6TDY2), containing SMAD FHA domain (A0A2P6U3K7), thylakoid lumen kDa chloroplast (A0A2P6TNZ5), peptidyl prolyl isomerase (A0A2P6TGB0), calmodulin (A0A2P6TFR8), 10kDa chaperone protein-like protein (A0A2P6THP7), membrane isoform A (A0A2P6TEF6), L-inducible nipa (A0A2P6TU12), β-Ig-H3 bundler protein (A0A2P6TQQ9).

[0023] The pigment can be chlorophyll. Chlorophyll can be one or more of chlorophyll a, chlorophyll b, chlorophyll c1, chlorophyll c2, chlorophyll d, and chlorophyll f. Preferably, the extract can contain chlorophyll c1 and / or c2, which are ubiquitous in many algae. The extract can contain a chlorophyll precursor, such as a precursor derived from glutamic acid.

[0024] Extracts from eukaryotic microalgae can contain natural or synthetic chlorophyll. Advantageously, synthetic chlorophyll, registered as a food additive coloring agent (E140), is safe to consume.

[0025] Compositions containing eukaryotic microalgae or extracts thereof for the treatment of viral diseases can be applied topically. Preferably, the compositions according to the invention can be applied topically. The composition can be a powder; liquid; aerosol; gel; paste or cream. The composition can be applied to the nasal cavity or oral cavity. Preferably, the compositions according to the invention can be administered as a nasal spray; mouthwash; or oral inhalation formulation. Topical application to the respiratory tract is ideal because it helps maintain a high local concentration of eukaryotic microalgae or extracts thereof at the site of primary infection (e.g., airway respiratory epithelium) while maintaining a lower systemic load compared to systemic application. Advantageously, this provides a dual beneficial effect: firstly, the composition administered as a nasal spray or mouthwash can prevent viral infection in healthy subjects via the nasal cavity and oral cavity by shielding the epithelial cells of the nasal and oral mucosa; and secondly, in the case of newly diagnosed infected subjects, the composition administered as a nasal spray or mouthwash can limit or prevent viral particles released by dying cells from colonizing new cells, such as olfactory and gingival epithelium. By limiting and / or preventing further colonization, the virus can be prevented from spreading to new pathways, such as the cardiovascular system, and / or from infecting more respiratory epithelial cells or lower respiratory system cells.

[0026] The compositions according to the invention may contain at least about 0.001 μg / mL, at least about 0.01 μg / mL, at least about 0.1 μg / mL, at least about 1 μg / mL of eukaryotic microalgae or extracts thereof.

[0027] The compositions according to the present invention may contain not more than about 10,000 μg / mL, not more than about 1,000 μg / mL, not more than about 100 μg / mL, not more than about 10 μg / mL, or not more than about 10 μg / mL of eukaryotic microalgae or extracts thereof.

[0028] The compositions according to the invention may contain eukaryotic microalgae or extracts thereof in amounts of about 0.001 μg / mL to about 10000 μg / mL, about 0.01 μg / mL to about 1000 μg / mL, about 0.1 μg / mL to about 100 μg / mL, about 1 to about 10 μg / mL.

[0029] Compositions containing eukaryotic microalgae or extracts thereof may contain any range of given endpoints. Compositions containing eukaryotic microalgae or extracts thereof may enable effective doses of the extract for, for example, the treatment of viral diseases.

[0030] This invention also provides compounds derived from eukaryotic microalgae for use in the treatment of viral diseases. In this document, the expression "compound derived from eukaryotic microalgae" should be understood to mean that the compound can be isolated or extracted from microalgae, for example, from microalgal cultures. Conventional methods for isolating or extracting compounds from microalgae are available to those skilled in the art, for example, in the form of commercial kits for isolating or extracting compounds from microalgae. The expression "compound derived from eukaryotic microalgae" may also include chemically synthesized compounds that are naturally occurring in eukaryotic microalgae and therefore "derived" from eukaryotic microalgae by conventional methods.

[0031] The present invention also provides a composition for use as an inhibitor of the binding of the spike protein receptor-binding domain (RBD) to angiotensin-converting enzyme 2 (ACE2), comprising eukaryotic microalgae or extracts thereof. Preferably, a composition for use as an inhibitor of RBD-ACE2 binding in vivo comprises eukaryotic microalgae or extracts thereof. For example, a composition for use as an inhibitor of RBD-ACE2 binding in human subjects comprises eukaryotic microalgae or extracts thereof.

[0032] Preferably, the composition comprising eukaryotic microalgae or extracts thereof is used as an inhibitor of RBD binding to ACE2 without affecting ACE2 activity.

[0033] This invention provides compositions comprising eukaryotic microalgae or extracts thereof for the treatment of viral diseases and / or as inhibitors of the binding of the spike protein receptor-binding domain (RBD) to angiotensin-converting enzyme 2 (ACE2), thereby reducing adverse events. Adverse events may include, but are not limited to, events caused by angiotensin II overdose, such as dyspnea; cardiovascular events; inflammatory responses, such as inflammation in response to oxidative stress; blood clots associated with thrombocytopenia; and hypercoagulable states, such as disseminated intravascular coagulation (DIC). Attached Figure Description

[0034] Embodiments of the invention will now be described by way of illustration only with reference to the accompanying drawings, wherein:

[0035] Figure 1 This is an image showing a protein gel of Chlorella extract;

[0036] Figure 2 A and Figure 2 B showed inhibition of ACE2:SARS-CoV-2 spike binding using different concentrations of Chlorella extract [MIX0000036]( Figure 2 A) and EDTA were used as positive controls. Figure 2 B) Measurement by chemiluminescence;

[0037] Figure 3 The activity of ACE2 in the presence of Chlorella extract was demonstrated; and

[0038] Figure 4 The effects of Chlorella extract and Remdesivir on active SARS-CoV-2-infected VeroE6 cell cultures were demonstrated. Detailed Implementation

[0039] Experiment 1 - Proteomics Characterization

[0040] The following experiments were conducted using Chlorella extract obtained from Algosource, Saint-Nazaire, France, which showed the greatest homology with Chlorella soxehii after proteomic analysis.

[0041] The proteome of Chlorella extract was evaluated using microfluidic electrophoresis (LabChip protein characterization system) and liquid chromatography-mass spectrometry (LC-MS / MS) in a data-dependent acquisition mode (proteomic profiling analysis).

[0042] Microfluidic electrophoresis showed that proteins with the highest abundance were found in the low molecular weight range. Figure 1 The image shows a protein gel, confirming that proteins below 20 kDa have the highest abundance.

[0043] These observations were confirmed by total protein abundance measurements performed by LC-MS using a data-dependent acquisition method (Table 1). Table 1 includes a list of the most abundant proteins along with their accession numbers and normalized spectral abundance factor (NSAF) scores. Spectral counting analysis indicated that several proteins involved in photosynthesis were particularly abundant.

[0044] Table 1: Total protein analysis of Chlorella extract

[0045]

[0046] Experiment 2 - Inhibitor Screening Assay

[0047] Following the standard ACE2:SARS-CoV-2 spike inhibitor screening assay protocol (BPSBioscience, San Diego, US), the assay plate was coated with ACE2-His solution (1 μg / mL) at room temperature (RT) for 1 h. The ACE2-His coating solution was washed off, leaving only ACE2-His coated on the assay plate. The assay plate was incubated with blocking buffer for 10 min to provide nonspecific spike binding. After removing the blocking buffer, Chlorella extract at concentrations ranging from 0.001 to 100 μg / mL was incubated for 1 h, followed by initiation of the binding reaction with a SARS-CoV-2 spike (20 ng / well) solution. The mixture was incubated again at room temperature for 1 h, then washed and blocked again for 10 min. Finally, the plate was incubated with a secondary HRP-labeled antibody solution (1:1000) against the SARS-CoV-2 spike for 1 h at room temperature. The HRP-labeled antibody was washed away, and chemiluminescence was initiated. Measurements were then performed using an enhanced chemiluminescent substrate and a FLUOstar Omega plate reader (BMG Labtech).

[0048] To characterize the effect of Chlorella extract on ACE2: a SARS-CoV-2 binding assay, a commercially available recombinant SARS-CoV-2 spike inhibitor screening assay, was used for inhibitor screening and characterization (BPSBioscience, San Diego, US).

[0049] Figure 2 A indicates that the Chlorella extract exhibits a dose-dependent inhibitory effect on the binding of ACE2 to SARS-CoV-2. This, in turn, suggests that the main entry point for the virus into cells is blocked. More specifically, Figure 2 A and Figure 2 B showed inhibition of ACE2:SARS-CoV-2 spike binding, using different concentrations of ( Figure 2 A, MIX0000036, Chlorella extract) and EDTA were used as positive controls. Figure 2 B) Measured by chemiluminescence.

[0050] ACE2:SARS-CoV-2 spike binding was measured in relative optical units (RLU). The half-maximal inhibitory concentration (IC50) of the Chlorella extract was determined, with an IC50 of 16.58 μg / mL. Figure 2B confirms the validity of the assay itself. As a negative control, no SARS-CoV-2 spike protein was applied (negative); as a positive control, 1 ng / μL of SARS-CoV-2 spike protein was applied (positive). ACE2:SARS-CoV-2 spike protein binding was confirmed using EDTA in a test range of 0.45 μM to 1000 μM.

[0051] Experiment 3 - Catalytic pathway without interfering with ACE2

[0052] To characterize the effect of Chlorella extract on ACE2 enzyme activity, a cell assay was used, which monitored the catalytic activity of ACE2 using a fluorescent ACE2 substrate to demonstrate successful inhibition through fluorescence loss.

[0053] The ACE2 enzyme activity assay was designed to measure the exopeptidase activity of ACE2 and screen for inhibitors. Commercially available synthetic fluorescent substrates (R&D systems, Zug) targeting ACE2 and rACE2 were used to measure activity. A standard curve for rACE2 was generated using assay buffer, 75 mM TRIS, 1 M NaCl, and pH 7.5. Natural extracts were screened using 100 ng / mL rACE2 and 15 μM MACE2 fluorescent substrates, along with different concentrations of Chlorella extract. The mixtures were then incubated at 37 °C for 1 h and measured using a Flexstation 3 (Molecular Devices, San Jose, CA, USA) at excitation at 320 nm and emission at 405 nm.

[0054] Chlorella extracts in the range of 4.5 μg / mL to 10000 μg / mL did not show any effect on ACE2 enzyme activity, indicating that the main function of ACE2 in degrading angiotensin II to angiotensin I-7 was maintained. Figure 3 As a positive control, a known ACE2 inhibitor (MLN-4760) in the range of 0.045 nM to 11100 nM was used. More specifically, Figure 3 The results show the different concentrations of Chlorella extract (MIX0000036). Figure 3 A) and MLN-4760 were used as positive controls. Figure 3 The activity of the ACE2 catalytic pathway was measured by fluorescence in the presence of B), where RFU is the relative fluorescence unit. The half-maximal inhibitory concentration (IC50) of MLN-4760 was 0.19 nM.

[0055] Experiment 4 - Chlorella extract protects cells from infection by active SARS-CoV-2.

[0056] To characterize the protective effect of Chlorella extract against SARS-CoV-2 infection in Vero E6 cells, a virus neutralization test was performed to monitor cell survival.

[0057] A viral suspension containing 200 TCID50 of SARS-CoV-2 was prepared for use with different concentrations of Chlorella and Remdesivir in a final volume of 200 μl. The resulting viral suspension was incubated at 37 °C for 1 h in a transport chamber containing a CO2 bag and H2O.

[0058] According to the manufacturer's protocol, Vero E6 cells were grown in MEM (M3303), 1.25% L-glutamine, 1% P / S, 1% NEAA, bicarbonate, and 10% FCS (all from Bioswisstec, Switzerland) until confluence. Cells were then seeded at 200,000 cells / well in 96-well plates. Infection of Vero E6 cells was performed by removing the cell culture medium from the cell suspension and adding 200 μl of a prepared viral suspension. Cell suspensions containing Vero E6 cells and SARS-CoV-2 / Chlorella / Remdesivir were incubated at 37°C for 4 days in a transport chamber containing a CO2 bag and H2O. After incubation, Vero E6 cell viability was measured using a Celltiter Glo (Promega) and a GloMax plate reader (Promega) according to the manufacturer's protocol.

[0059] Figure 4 The antiviral activity of Chlorella extract (MIX0000036, white circle) in active SARS-CoV-2 (French isolate) and VeroE6 cells is shown. Viral fusion in VeroE6 cells mainly occurs after ACE2 binding, followed by endocytosis. Pretreatment with Chlorella extract at test concentrations ranging from 63 μg / mL to 2000 μg / mL, followed by SARS-CoV-2 infection, inhibited SARS-CoV-2 viral entry in VeroE6 cells for 72 hours, with an IC50 of approximately 140 μg / mL. The percentage of VeroE6 cells surviving SARS-CoV-2 infection peaked at 250 μg / mL in the presence of Chlorella extract.

[0060] As a positive control, the broad-spectrum antiviral drug remdesivir was used. Figure 4 (black circle), which blocks SARS-CoV-2 replication in a test range of 0.04 μg / mL to 10 μg / mL by inhibiting viral RNA synthesis.

Claims

1. Chlorella sogii ( Chlorella sorokiniana The use of (or its extracts) in the preparation of drugs for inhibiting SARS-CoV-2 infection.

2. The use according to claim 1, wherein the extract may contain one or more of the following: proteins, lipids, vitamins, minerals, folic acid, iron, fiber and / or pigments.

3. The use according to claim 2, wherein the protein is selected from one or more of the following: plastocyanin (A0A2P6TGD1), glycine-rich 2 (A0A2P6TDY2), containing SMAD FHA domain (A0A2P6U3K7), thylakoid lumen kDa chloroplast (A0A2P6TNZ5), peptidyl prolyl isomerase (A0A2P6TGB0), calmodulin (A0A2P6TFR8), 10kDa chaperone protein-like protein (A0A2P6THP7), membrane isoform A (A0A2P6TEF6), L-inducible nipa (A0A2P6TU12), β-Ig-H3 bundler protein (A0A2P6TQQ9).

4. The use according to claim 1, wherein Chlorella vulgaris or its extract is applied topically.

5. The use according to claim 1, wherein Chlorella vulgaris or its extracts inhibit the binding of the spike protein receptor-binding domain to angiotensin-converting enzyme 2.

6. The use according to claim 5, wherein Chlorella vulgaris or its extracts do not affect angiotensin-converting enzyme 2 activity.

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