CROSS-REFERENCE TO RELATED APPLICATION

[0001] The present application claims priority to and the benefit of U.S. Provisional Patent

Application No. 63/196,420 filed on June 3, 2021, the entire contents of which are incorporated herein by reference.

TECHNICAL FIELD

[0002] The present disclosure relates to a method for inhibiting the spread of microorganisms using spike proteins, such as COVID-19, including an aerosol to inhibit the spread of such microorganisms, such as COVID-19.

BACKGROUND OF THE DISCLOSURE

[0003] Human coronavimses (CoV) have been known for years and have been responsible for upper respiratory and digestive tract infections. Recently, a new strain of coronavims, commonly referred to as COVID-19, has brought such CoV to the forefront of everyone’s lives. The emergence and rapid spread of COVID-19 to numerous areas throughout the world has prompted the World Health Organization (WHO) to declare a Public Health Emergency of International Concern. Since its emergence, many treatments and vaccines have been studied to stop the spread of COVID-19.

[0004] For example, a ketone -based treatment has been studied and developed to inhibit the spread of COVID-19. However, using a ketone is expensive and such treatments have low efficacy. Therefore, there is still a need to have a more economical and easily accessible way to inhibit COVID-19.

SUMMARY OF THE PRESENT DISCLSOURE

[0005] A method for inhibiting the spread of microorganisms using spike proteins, such as COVID-

19, includes applying a mixture into nasal passageways or throat, wherein the mixture includes HTRE and BMC concentrate. The method may also include a step of applying the mixture inhibits attachment at a Furin level of a COVID-19 vims. The mixture may include HTRE in an amount from about 0.5% to about 5.0% of a total weight of the mixture and BMC concentrate in an amount from about 95% to about 99.5% of a total weight of the mixture. The mixture may be applied into the nasal passageways by spraying the mixture into each nostril of the nose. The method may include applying the mixture into the nasal passageways every 4 hours, and no more than four times within a 24 hour period.

[0006] A method for preparing a mixture to inhibit the spread of microorganisms using spike proteins, such as COVID-19, including adding BMC concentrate to a mixer and adding HTRE to the mixer and mixing for a time period of 5 minutes to 15 minutes. The HTRE may be included in an amount from about 0.5% to about 5.0% of a total weight of the mixture and the BMC concentrate may be included in an amount from about 95% to about 99.5% of a total weight of the mixture. The mixture is mixed at a temperature from 15°C to 30°C and under vacuum during the first time and the second time period.

[0007] A composition for inhibiting the spread of microorganisms using spike proteins, such as

COVID-19, includes BMC concentrate, HTRE, and water. The composition may be an aerosol. The HTRE may be included in an amount from 0.5% to 5.0% of a total weight of the composition. The BMC concentrate may be included in an amount from about 95% to about 99.5% of a total weight of the composition. DETAILED DESCRIPTION

[0008] Aerosols for inhibiting the infection of microorganisms using spike proteins, such as COVID-

19, and methods for applying such aerosols are provided. While compositions, methods, and uses discussed herein are presented as specific to COVID-19, they may be applicable to other microorganisms using spike proteins.

[0009] Generally, the aerosol of the present disclosure is a composition that inhibits the infection of

COVID-19 by blocking the COVID-19 vims from interacting with enzymes in the human body. Without being limited to a particular theory, the inventors have found that COVID-19 is spread by interacting with various enzymes in the human body. In particular, the inventors found that furin and angiotensin 2 (ACE2) are among such enzymes. ACE2 is an exopeptidase that catalyzes the conversion of angiotensin II to angiotensin 1-7 and L-phenyl alanine. Angiotensin II is part of the Renin Angiotensin Aldosterone system (RAAS), a hormonal system that regulates fluid balance, blood pressure and maintains vascular tonicity. [0010] Furin is an enzyme within the human body that cleaves and activates a variety of mammalian, viral and bacterial substrates. Some proteins are inactive within the body, such that some sections need to be removed from the protein in order to become active. Furin acts by cleaving these sections and activating the proteins. Furin has been studied for years for its role in health and diseases, including cancer, inflammatory and infectious diseases. From these studies, various protein and peptide based inhibitors have been developed to control furin activity to treat these diseases.

[0011] In the present disclosure, furin and its role with the COVID-19 virus has been studied. Furin has been found to catch part of the COVID-19 vims in the body, activating the spike protein. The spike protein may then bind to ACE2, which in turn introduces the protein into the host cell. The inventors have found that to inhibit being infected with COVID-19, it is preferable to block the attachment of the vims at the furin stage, to prevent forming a spike protein that could possibly bind with ACE2. The normal role of ACE2 is to convert the hormone angiotensin II into angiotensin. This conversion of angiotensin II helps reduce constricted blood vessels and causes the kidneys to extract sodium from the blood to lower blood pressure. Therefore, ACE2 plays a protective role for the cardiovascular system and the kidneys. ACE2 has other protective qualities as well, including its anti-inflammatory effects and, presumably, the ability to reduce the risk of excessive blood clots. The inventors found that if there is a high amount of ACE2 inhibition, there are more extreme side effects because of ACE2’s ability to regulate the cardiovascular system and blood pressure system.

Therefore, the inventors have found that it is desirable to inhibit the attachment of the vims at the furin level to prevent high inhibition of ACE2.

[0012] The present disclosure provides an aerosol to inhibit the cleaving of the COVID-19 vims at the furin stage. In one embodiment, the aerosol of the present application may include HTRE, BMC concentrate and water. Such combination has been found to provide inhibition at both the furin and ACE2 level, but preferably inhibits the attachment of the vims at the furin level. HTRE is a known resin in the cosmetic field. As understood by one of skill in the art, HTRE includes helianthus annuus seed oil and Canarium luzonicum gum nonvolatiles. Further, Protium heptaphyllum resin may also be used as it has similar effects of Canarium luzonicum gum nonvolatiles in the same concentration. The helianthus annuus seed oil is included as the Solubility product quotient (QSP), and the Canarium luzonicum gum nonvolatiles may be included between about 25% and about 35% in the HTRE based on a total weight of the HTRE composition. The Canarium luzonicum gum nonvolatiles are extracted from a tree, Canarium luzonicum. These compounds have been found to have anti-nociceptive (anti-pain), anti-inflammatory and antioxidant properties. Therefore, the Canarium luzonicum gum nonvolatiles has been used in the cosmetic industry as an anti-inflammatory for the skin and scalp and to protect the hair from UV radiation.

[0013] The BMC concentrate of the aerosol includes various compounds and minerals. For example, the compounds and minerals include aqua, glycerin, propanediol, magnesium chloride, sodium magnesium silicate, salicylic acid, methyl sufonyl methane, gluconolactone, sodium benzoate, xanthan gum, montmorihonite extract, illite extract, kaolinite extract and citric acid. In the present application, aqua refers to mineral water that includes at least about 0.15% of a salt, where the salt may be sodium chloride, potassium chloride, magnesium chloride or calcium chloride.

[0014] In one example of the BMC concentrate, the compounds and minerals may be included in the following amounts presented in Table 1.

Table 1: BMC concentrate composition [0015] The BMC concentrate is described in FR 2,952,814, FR 2,976,503 and CA 3,002,120, which are herein incorporated by reference. The BMC concentrate is typically produced as a delaminated clay and when water is added to the delaminated clay, forms a lotion.

[0016] Because of the anti-inflammatory characteristics of HTRE, the inventors suspected that this material may be useful for inhibiting the attachment of the vims. As will be described further herein, experiments were conducted to confirm that HTRE could effectively inhibit the attachment of the COVID-19 vims at both furin and ACE2 level. However, it is suspected that COVID-19 is spread through the air and respiratory systems. Therefore, to effectively inhibit the spread of the vims, an aerosol composition is desired. Because HTRE is a resin, it is not suitable on its own as an effective treatment to stop the spread of the vims. Thus, the inventors surprisingly found that combining HTRE and BMC in an aerosol would be an effective and efficient composition to inhibit the spread of the vims.

[0017] In one embodiment, the aerosol of the present application includes HTRE and BMC concentrate. HTRE may be included in the aerosol in an amount between about 0.5% and about 5%. More particularly, it may be included in an amount between about 1% and 3%. In the aerosol, BMC concentrate may be included in an amount between about 95% and about 99.5%. More particularly, it may be included in an amount between about 96% and about 98%. The amount of HTRE and BMC concentrate is based on a total weight of the aerosol. For example, in one variation, the composition includes about 97% BMC concentrate and about 3% HTRE.

[0018] The aerosol is prepared by mixing BMC concentrate and HTRE in a mixing system.

Specifically, the BMC concentrate is added to a mixer system at room temperature under vacuum, and HTRE is added to the mixer system for a time period between about 5 minutes to about 10 minutes. The time period of mixing depends on the amount of aerosol to be produced. If a greater amount of aerosol is being produced, the time period of mixing will be longer, for example, the time period would be about 15 minutes. The temperature of the mixing is preferably performed at room temperature, but may be from about 15°C to about 30°C. The mixing system may be a high shear mixing system including a rotor or stator agitator.

[0019] The aerosol is prepared such that the amount of BMC concentrate in the aerosol is about 95% to about 99.5% based on a total weight of the aerosol, the amount of HTRE is about 0.5% to about 5% based on a total weight of the aerosol, and the water is the remaining amount.

[0020] The aerosol may be used to inhibit the infection of COVID-19 in a human body. To be most effective, the aerosol (e.g, the aforementioned composition of HTRE, BMC concentrate and water) may be used by a person prior to going out in public, to being in a large crowd or any situation in which the exposure risk could be high. Prior to encountering such a situation, a person may apply one dose of the aerosol in each nostril. Each dose of the aerosol is effective for a time period. For instance, in one example, the dose may be effective for up to four hours and should be reapplied as needed throughout the day. Further, such a dose of the aerosol should not be applied more than four times a day. Further as to this example, each application of the aerosol applies about 0.25 mL to each nostril. [0021] The inhibition of furin and ACE2 has been evaluated with BMC and HTRE. The inventors surprisingly found that using both in combination provided more efficient delivery of the composition to the patient and improved inhibition of the COVID-19 virus. The present invention will be described by the following non-limiting examples.

[0022] Hereinafter, examples will be described to provide additional detail as to certain aspects of the present disclosure. However, the present disclosure may be embodied in many different forms and should not be construed as being limited to the examples set forth herein. Examples of the present disclosure are provided to more completely explain the present disclosure to a person with ordinary skill in the art.

Experimental Examples relating to BMC concentrate

Example 1 : Evaluation of ACE2 inhibition using BMC concentrate

[0023] The modulation of the activity of ACE2 was evaluated in an acellular in vitro model using an analysis kit known as an “ACE2 Inhibitor screening assay kit.” This assay kit is designed to measure ACE2 exopeptidase activity for screening and profiling applications.

[0024] A solution using BMC concentrate 33% in water was tested in triplicate at 3 different concentrations, 2%, 1% and 0.3%. This corresponded to having a BMC concentrated of 0.66%; 0.33% and 0.1%, respectively. To prepare the evaluation, a buffered solution of ACE2 reacted with ACE2 Fluorogenic substrate to form a fluorogenic compound. Fluorescence intensities were collected using an excitation filter passing the wavelengths of 544nm, using an Exc544 filter, and an emission filter passing the wavelengths from 580 to 600 nm, using an EM590-10 filter. A reference inhibitor, DX600, was brought into contact with ACE2 and the enzyme substrate was then incorporated.

[0025] The Examples tested are presented in Table 2 below. A blank sample, reference product (T+) and control (T-) were also tested.

Table 2. Examples evaluated for ACE2 activity

[0026] After the enzyme substrate was incorporated, the microplate was incubated at room temperature for 60 minutes. At the end of the incubation period, the activity of ACE2 with and without a sample or reference product was evaluated by measuring the fluorescence intensities, expressed in relative fluorescence unit (RFU). For each concentration tested, the modulation of ACE2 activity by the test product was calculated according to Formula E If the result was negative, the percentage is expressed as inhibition of the enzyme and if positive, expressed as activity of the enzyme. The results are presented in Table 3. Formula 1:

Percentage modulation of ACE2 activity

( Optical density of Reference — Optical density of ACE 2) x 100 Optical density of ACE2

Table 3. Average Results of ACE2 activity

[0027] From these results, the activity of ACE2 was inhibited by 71.02% in the presence of the reference inhibitor, Control +. When BMC is included in Examples 1-3, ACE2 activity is activated. This test clearly shows that the BMC concentrate has no ability to inhibit the ACE2.

Example 2: Evaluation of Furin Activity using BMC concentrate

[0028] The inhibition effect of BMC on furin activity was also evaluated. Decanoyl-Arg-Val-Lys-

Arg-CMK at lOOnM was used as a reference inhibitor of furin activity. Three different examples were prepared using different BMC concentrates as can be seen in Table 4 below. To prepare the Examples, a solution made of 33% BMC Concentrate was tested at 3 different concentrates, 2%; 0.5% and 0.1%. Examples 4-6 were prepared by solubilized directly in the assay buffer and then diluted in order to achieve the desired concentration.

Table 4. [0029] Furin was pre-incubated for 10 minutes at room temperature in the absence of BMC concentrate (control), in the presence of the reference product and Examples 4-6. After pre-incubation, furin substrate was added to the control, reference product and Examples 4-6 and each sample was incubated again at room temperature protected from light for 5 minutes. Each sample was tested three times.

[0030] The cleavage of furin fluorescent substrate was followed after adding the substrate and was evaluated by reading the fluorescence at 485nm/535nm. The results of the inhibition of furin activity are represented in Table 5 below.

Table 5. Results of Furin Activity

[0031] From this test, it was confirmed that BMC concentrate was a furin inhibitor. For example,

Example 4 having a BMC Concentrate of 0.66% was able to inhibit 56.1% of the furin activity. Because BMC Concentrate was not able to inhibit ACE2 activity, and was able to inhibit furin activity, it was confirmed that BMC Concentrate is a specific furin inhibitor, and not a global enzyme convertase inhibitor. Therefore, by inhibiting at the furin level only, the side effects may be lowered. This is because BMC appears to have little activity relative to other biological activity in a patient. Thus, the use of BMC Concentrate is better to avoid any side effects. As discussed above, ACE2 allows for COVID-19 to enter a host cell after the spike protein of COVID-19 is activated within the host cell. Furin may activate the spike protein of COVID-19. Therefore, there is a need to inhibit at the furin level, which indirectly also inhibits ACE2, locking the infective pathway of COVID-19.

Comparative Example 1 : Evaluation of Furin Activity using a Second BMC concentrate

[0032] The inhibition effect of a second BMC concentrate on furin activity was also evaluated using a SensoLyte RhllO Furin Assay Kit. The SensoLyte RhllO Furin Assay Kit screens enzyme inhibitors or continuous assay of furin activity using a fluorogenic substrate. Upon cleavage by furin, the fluorogenic substrate generates the RhllO (rhodamine 110) fluorphore with a fluorescence that is detected at excitation/emission=490nm/520 nm. The detection limit may be as low as 0.02 ng/mL. A buffered solution of Furin reacts with the RhllO furin substrate to form a fluorogenic compound. The sample or reference inhibitor is brought into contact with furin solution and the enzyme substrate is also incorporated to evaluate the activity of furin. Three different examples were prepared using different BMC concentrates as can be seen in Table 6 below. To prepare the Examples, a solution made of 100% BMC Concentrate was tested at 3 different concentrates, 7.5%; 5% and 1%. Comparative Examples 1-3 were prepared by solubilized directly in the assay buffer to achieve the desired concentration.

Table 6.

[0033] A solution of furin was added to the Rhl 10 furin substrate. The microplate is slightly agitated for 30 seconds and is then incubated at room temperature for 60 minutes. Each sample was tested three times. [0034] The cleavage of furin fluorescent substrate was followed after adding the substrate and was evaluated by reading the fluorescence at 490nm/520nm. The modulation of furin activity is calculated by Formula 2. The results of the furin inhibition are represented in Table 7 below.

Percentage modulation of Furin activity

( Optical density of Reference — Optical density of Furin ) x 100

Optical density of Furin

Table 7.

Experimental Examples Relating to HTRE

Example 3: Evaluation of ACE2 activity using HTRE [0035] The modulation of the activity of the ACE2 was evaluated in an acellular in vitro model using an analysis kit known as “ACE2 Inhibitor screening assay kit.” This assay kit is designed to measure ACE2 exopeptidase activity for screening and profiling applications.

[0036] HTRE was tested in triplicate at 3 different concentrations, 1%, 0.3% and 0.1%. To prepare the evaluation, a buffered solution of ACE2 reacted with ACE2 Fluorogenic substrate to form a fluorogenic compound. Fluoroscence intensities were collected using an excitation filter passing the wavelengths of 544nm, using an Exc544 filter, and an emission filter passing the wavelengths from 580 to 600 nm, using an EM590- 10 filter. A reference inhibitor, DX600, was brought into contact with ACE2 and the enzyme substrate is then incorporated.

[0037] The Examples tested are presented in Table 8 below.

Table 8. Examples evaluated for ACE2 activity

[0038] After the enzyme substrate was incorporated, the microplate was incubated at room temperature for 60 minutes. At the end of the incubation period, the activity of ACE2 with and without a sample or reference product was evaluated by measuring the fluorescence intensities, expressed in relative fluorescence unit (RFU). For each concentration tested, the modulation of ACE2 activity by the test product was calculated according to the following formula. If the result was negative, the percentage is expressed as inhibition of the enzyme. The results are presented in Table 9.

Percentage modulation of ACE2 activity

( Optical density of Reference — Optical density of ACE 2)

= - x 100

Optical density of ACE2

Table 9. Average Results of ACE2 activity

[0039] From these results, the activity of ACE2 was inhibited by 71.02% in the presence of the reference inhibitor, Control +. Further, the activity of ACE2 was inhibited by 43.25% in Example 7. When HTRE is included in Examples 8 and 9, ACE2 activity is activated. Therefore, a relatively higher concentration of HTRE is needed to inhibit ACE2.

Example 4: Evaluation of Furin Activity using HTRE

[0040] The inhibition effect of HTRE on furin activity was also evaluated. Decanoyl-Arg-Val-Lys-

Arg-CMK at lOOnM was used as a reference inhibitor of furin activity. Three different examples were prepared using different concentrations of HTRE as can be seen in Table 10 below. Examples 10-12 were prepared by solubilized directly in the assay buffer and then diluted in order to achieve the desired concentration.

Table 10.

[0041] Furin was pre-incubated for 10 minutes at room temperature in the absence (control), in the presence of the reference product and Examples 10-12. After pre-incubation, furin substrate was added to the control, reference product and Examples 10-12 and each sample was incubated again at room temperature protected from light for 5 minutes. Each sample was tested three times.

[0042] The cleavage of furin fluorescent substrate was followed after adding the substrate and was evaluated by reading the fluorescence at 485nm/535nm. The results of the furin activity are illustrated in Figure 1 and are represented in Table 11 below. As can be observed by these results, HTRE is capable of inhibiting furin activity, particularly as comparatively higher amounts as in Comparative Examples 4 and 5.

Table 11. Results of Furin Activity

Example 5: Evaluation of anti-infectious activity of HTRE against the SARS-CoV-2-like virus [0043] An assay was run to evaluate the effect HTRE has on the SARS-CoV-2-like virus. To run the assay, a screening assay kit was used as follows: Spike (SARS-CoV-2) Luc-reporter, Pseudotyped

Lentivirus (Ref.79942) infection of ACE2-HEK recombinant cells(Ref.79951) from BPS bioscience. [0044] The assay used two main components. The first component included recombinant clonal stable HEK293 cells constitutively express full length human ACE2 5 (SARS-CoV-2-like virus), Genbank#NM_021804.3), with surface expression of ACE2 confirmed by flow cytometry. The second component included a SARS-CoV-2 Spike Pseudotyped Lentivirus producedwith SARS-CoV-2 Spike (Genbank Accession#QHD43416.1) as the envelope glycoproteins instead ofthe commonly used VSV- G. These pseudovirions also contain the firefly luciferase gene driven by a CMVpromoter, therefore, the spike-mediated cell entry can be conveniently measured via luciferase reporter activity. The SARS-CoV- 2 Spike pseudotyped lentivirus can be used for screening applications in a Biosafety Level 2 facility. [0045] The key materials of the assay are presented below:

[0046] To perform the assay, ACE2-HEK cells were thawed in Thaw medium 1 , amplified in Growth medium IN, then harvested and plated in white, clear flat bottom 96-well culture plates at 10.000 cells/well in 50pl of Thaw medium 1. The cells were incubated overnight at 37°C. After one day, visual control of cell layer homogeneity and integrity was validated using inverted microscope, and the following test ingredients were prepared.

[0047] An extract of HTRE was tested at three different concentrations, 0.5%, 0.25% and 0.1%. To prepare the samples tested, DMSO was added with the different concentrations of HTRE and formed two different phases, termed “up” and “down.” Each phase was separately tested, where Examples 13-15 represent the HTRE “down” phases, while Examples 16 and 17 represented the HTRE “up” phase. The HTRE “up” phase forms an upper clear phase upon reconstitution, and contains a lesser amount of HTRE concentration. This is caused because of the affinity of the DMSO and the HTRE resin when combined. The affinity of DMSO and HTRE also forms a HTRE “down” phase, which has a greater concentration of the HTRE. [0048] The extracts were diluted at an intermediary llx concentration in Thaw medium 1, for subsequent transfer of 5 mΐ in the assay plate. After a 30 minute incubation period at 37°C, 5 mΐ of undiluted Pseudotyped lentivirus (Bald or SI -Spike) were added to the corresponding wells. Supplementary negative controls were also prepared including 5 mΐ of Thaw medium 1 containing either 5.5% of DMSO or PBS. A positive control was prepared including ACE2 blocking mAh at a final concentration of 0.5 mM in the assay wells.

[0049] Examples 13-17 were then spiked with the SARS-CoV-2-like virus to analyze the signal induced by the blocking antibody.

Table 12.

[0050] The results of the assay run is presented in Table 13 below. As can be seen in the Table, the

HTRE “down” phase had a drop of over 96% in RLU signal induced by the blocking antibody; the RLU signal was correlated to the presence of the SARS-CoV-2-like virus into host cells. The HTRE “down” phase showed a strong anti-infective effect with a maximum score of 98% inhibition of viral infection at a concentration of 0.25%. In contrast, the HTRE “up” phase had inferior blocking ability, which also confirmed that there was a lower concentration of HTRE in the “up” phase as compared to the “down” phase.

Table 13 Experimental Example relating to both BMC and HTRE

Example 6: Evaluation of Furin Inhibition using BMC concentrate and HTRE

[0051] The inhibition effect of BMC concentrate and HTRE was evaluated. A solution was prepared starting with a composition including BMC concentrate at 97% based on a total weight of the composition, and the amount of HTRE at 3% based on the total weight of the composition. Three different concentrations of the solution were then prepared for evaluation by mixing the composition with water such that the concentrations were 7.5%, 5% and 1% based on the total weight of the composition in the solution, corresponding to Examples 18-20 as shown in Table 14.

Table 14.

[0052] These compositions were then tested to examine furin inhibition properties. For this example, furin was pre-incubated for 10 minutes at room temperature in the absence of BMC concentrate (control), in the presence of the reference product and Examples 18-20. After pre-incubation, furin substrate was added to the control, reference product and Examples 18-20 and each sample was incubated again at room temperature protected from light for 5 minutes. Each sample was tested three times.

[0053] The cleavage of furin fluorescent substrate was followed after adding the substrate and was evaluated by reading the fluorescence at 485 nm/535nm. The results of the furin activity are represented in Table 15 below.

Table 15. Results of Furin Activity

[0054] It was found that including both BMC concentrate and HTRE inhibited furin activity more effectively than when using either BMC concentrate or HTRE individually. These experiments demonstrated a synergistic effect of a composition including both BMC and HTRE together in inhibiting furin. Therefore, though BMC concentrate is able to inhibit furin on its own, when mixed with HTRE, the furin activity was greater than 90% as can be seen in Examples 18 and 19.

[0055] These results were also promising because, as discussed above, HTRE can cause a number of side effect to a person using the composition if HTRE is included at comparatively high amounts since it has more global activity, as opposed to BMC with activity focused on furin. Thus, this composition, which only requires HTRE in a small amount, is beneficial for the additional reason that HTRE provides benefits to the user while its presence in such a small amount limits any side effects it may cause.