A SYNERGISTIC FORMULATION FOR MANAGEMENT OF RESPIRATORY PATHOGENS INCLUDING CORONAVIRUSES

Abstract

A synergistic non-toxic formulation for management of respiratory pathogens including coronaviruses and the process of preparing the synergistic formulation. The effective, non-toxic composition comprises of biological peptides including calcium chelating peptide and zinc chelating peptides, protease inhibitor such as 9-Arginine-peptide and inhibitor of viral replication enzyme such as vitamin B12 to reduce the presence of respiratory viruses including coronaviruses. The formulation is a cocktail of ingredients having a synergistic effect which is capable of being administered through various means including by way of inhalation, as nasal spray or an oral pump or via nebulizer to inhibit the entry and replication of respiratory pathogens. The combination of the formulation is such that it can be customized for a number of viral infections. The formulation is economical, self-administrable and effective even in low doses.

Claims

1. A synergistic formulation for preventive management of respiratory pathogens including coronaviruses said formulation comprising at least one ion chelator at least one protease inhibitor and at least one inhibitor of viral replication enzyme in efficacious and non-toxic concentrations capable of being delivered orally, nasally and inhaled into lungs.

2. The synergistic formulation as claimed in claim 1, wherein, the said ion chelator(s) is selected from calcium chelating peptides and zinc chelating peptides.

3. The synergistic formulation as claimed in claim 2 wherein the calcium chelating peptides are selected from Phenylalanine-Aspartic acid-Histidine-Isoleucine-Valine-Tyrosine (FDHIVY).

4. The synergistic formulation as claimed in claim 3 wherein said calcium chelating peptides are at a concentration ranging from 0.1 g/mL to 2.0 g/mL.

5. The synergistic formulation as claimed in claim 2 wherein said zinc chelating peptides are selected from Asn-Cys-Ser, Ser-Met dipeptide and a Leu-Ala-Asn tripeptide at a concentration ranging from 0.9 g/mL to 1.1 g/mL.

6. The synergistic formulation as claimed in claim 1 wherein said protease inhibitor is preferably 9-L-Arginine-peptide at a concentration ranging from 5 uM to 400 uM.

7. The synergistic formulation as claimed in claim 1 wherein said inhibitor of viral replication enzyme is vitamin.

8. The synergistic formulation as claimed in claim 1 wherein said vitamin is selected from vitamin B.

9. The synergistic formulation as claimed in claim 1 wherein said vitamin B is preferably B.sub.12

10. The synergistic formulation as claimed in claim 1 wherein said vitamin B.sub.12 is at a concentration in the range between 0.5 mg/mL to 5.0 mg/mL

11. The synergistic formulation as claimed in claim 1 wherein said vitamin B.sub.12 is preferably vitamin B.sub.12 at a concentration in the range between 100 mg/mL to 1000 mg/mL.

12. The synergistic formulation as claimed in claim 1 wherein said formulation comprises a preservative at a concentration 0.01% w/v.

13. Process of preparing a synergistic formulation as claimed in claim 1 for preventive management of respiratory pathogens including coronaviruses said process comprising: synthesizing said peptides and purifying to >98% purity, storing said peptides in dry state, weighing said stored peptides in quantities corresponding to 100× to 1000× of the final concentration, combining said weighed peptides with vitamin B.sub.12 in one tube and storing them as a dry formulation, dissolving said dry formulation in phosphate-buffered saline at the time of use, for delivery of said synergistic formulation, orally, nasally and inhaled into lungs.

14. Synergistic formulation for preventive management of respiratory pathogens including coronaviruses said formulation comprising TABLE-US-00003 Ingredient Concentration Calcium chelating peptide 0.1 g/ml to 2.0 g/ml Zinc chelating peptide 0.1 g/ml to 2.0 gm/ml 9-L-Arginine-peptide 5 uM to 400 uM Vitamin B.sub.12 0.5 mg/ml to 5 mg/ml Alpha-A1-AT-RCL 5 uM to 200 uM benzalkonium chloride 0.01% w/v

15. The synergistic formulation as claimed in claim 1 wherein said formulation is capable of being delivered as a nasal spray, nebulizer or via personal oral pump.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0042] The foregoing and other features of embodiments will become more apparent from the following detailed description of embodiments, when read in conjunction with the accompanying drawings.

[0043] FIG. 1(a): Determination of IC-50 value of Positive Control (Remdesivir)

[0044] FIG. 1(b): Determination of IC-50 value of the formulation of the present invention referred to as EvoBO2020

[0045] FIG. 2: Depicts the anti-viral effect of formulation of the present invention referred to as EvoBO2020 in the plaque assay

[0046] FIG. 3: Depicts the plaque assay data for the positive control, Remdesivir.

[0047] FIG. 4: Depicts the plaque assay data for the formulation of the present invention referred to as EvoBO2020.

[0048] FIG. 5: Depicts the bar graph of absorbance 540 nm in the MTT Quantitation assay for lung cells exposed to EvoBO2020 formulation

DETAILED DESCRIPTION OF THE OF THE PREFERRED EMBODIMENT

[0049] In order to more clearly and concisely describe and point out the subject matter of the claimed invention, the following definitions are provided for specific terms, which are used in the following written description.

[0050] The term “Protease” refers to an enzyme that catalyzes proteolysis, the breakdown of proteins into smaller polypeptides or single amino acids. The term “Viral Replication” refers to a process of formation of biological viruses during the infection in the target host cell. The term “Furin” refers to an enzyme responsible for the proteolytic cleavage of viral envelope polyprotein precursor prior to viral infections into human airway cells. Furin is expressed in cells that line the human airways and in lung tissues, both extracellularly and in intracellular compartments.

[0051] The term “Neuropilin-1” refers to are transmembrane glycoproteins that serve as cell surface receptors for various ligands involved in angiogenesis. The term “ion chelator” refers to small molecules that bind very tightly to metal ions. The term “Polymerase” refers to an enzyme that synthesizes long chains of polymers or nucleic acids.

[0052] Generally, viruses are not capable of self-replication and require a living host cell for replication and multiplication. The viruses exploit host cell mechanisms to survive. Proteases plays an important role in the binding of virus to human cells, leading to endocytic uptake of the viruses and replication of viruses. The viral RNA is translated into a polypeptide sequence, which is assembled in a long chain that includes several individual proteins such as reverse transcriptase, protease, integrase etc. These enzymes must be cleaved from the longer polypeptide chain before they become functional. Viral protease cleaves the long chain into its individual enzyme components that facilitate the production of new viruses. Hence, viral proteases play an important role in replication of viral genome and multiplication of virus.

[0053] Furin is a human protease involved in proteolytic cleavage of viral envelope polyprotein and associated with the development of infection. The use of Furin protease inhibitor 9-L-Arginine-peptide inhibits the ability of the protease to cleave the viral S-glycoprotein and entry of the virus into lungs thus interfering with continued infection. The inhibition of Furin also interferes with the processing of viral proteins and packaging into new virus particles within infected cells.

[0054] The entry of SARS-CoV-2 in cells depend on binding of the spike (S) coat protein to angiotensin-converting enzyme2 (ACE2), which is present on the plasma membrane of airway epithelial cells, goblet secretory cells, and type II pneumocytes, and on S protein priming by the serine protease transmembrane serine protease 2 (TMPRSS2). In the coronavirus S protein priming, the transmembrane serine protease 2 (TMPRSS2) is regarded as one of the main priming proteases, which is an important step for binding of the S protein to the angiotensin-converting enzyme 2 (ACE2) receptor before cell entry. Therefore, targeting the host extracellular proteases, such as TMPRSS2, and other proteases has several advantages over targeting viral proteins. Serine residues have been found to be important catalytic residues present in all extracellular transmembrane anchored proteases expressed in lung cells.

[0055] The protease inhibitors of the present formulation are effective in inhibiting the binding of respiratory viruses to the airway epithelial tissues in the respiratory pathway which, in case of an infection process, is facilitated through the action of proteases. The specific peptides of the formulation also prevent the endocytosis of viruses especially SARS-CoV-2 which is enabled by host protein mediated cleavage of viral proteins.

[0056] The formulation of the present invention provides for a poly-lysine peptide of 7-10 residue length to temporarily inactivate extracellular proteases by binding to their catalytic sites. The poly-lysine peptide is preferably of molecular size of 0.1-1 nm. A possible longer or shorter version of this peptide may also be included in the formulation,

[0057] It is significant to note that other extracellular proteases expressed in lung cells include a class of proteins called Meprin proteases. The inclusion of zinc chelating peptides like: Asn-Cys-Ser tripeptide, Ser-Met dipeptide, and Leu-Ala-Asn tripeptide temporarily inhibits the activities of the Meprin class proteases, for as long as the formulation is present on the extracellular surface of lungs.

[0058] Neuropilin-1 (NRP-1) is a cell-surface receptor that plays an essential role in angiogenesis, regulation of vascular permeability, and the development of the nervous system. VEGF-A165 and other ligands of NRP-1 possess a C-terminal CendR sequence that interacts with the b1 domain of NRP-1 and causes cellular internalization and vascular leakage. CendR peptides bind to neuropilin-1 (NRP-1) on the target cells.

[0059] Neuropilin-1 is a co-receptor for SARS-CoV-2-highly expressed in lungs. Neuropilin binds to R-XX-R peptide motifs at the C-terminal end using its VEGF binding domain. R-X-XR- is produced by Furin cleavage of SARS-CoV-2 S-glycoprotein cleavage and resultant S-peptide. This CendR (C end Rule -R-X-X-r/K at the C-terminus) pathway stimulated transcytosis of extracellular large molecules independent of known endocytic paths (clathrin/wortmannin and other pinocytic inhibitors). The intracellular signaling/binding of other proteins to NRP-1 are prevented by the components of the present invention. Also, prevention of formation of the R-XX-R peptide fragment from the S-glycoprotein of SARS-CoV-2 by the action of proteases is achieved by the protease inhibitors used in the formulation of the present invention. The inactivation of such extracellular proteases is relieved only when the peptide and protease complex is degraded by natural cellular processes.

[0060] Compounds that inhibit viral replication target the enzymes used in viral replication. Virus replicates inside the host cell by manipulating the host enzymes to make multiple copies of itself. The use of viral replication inhibitor plays an important role in the efficacy of the formulation. The composition of the present invention also comprises of inhibitors of viral replication which play an important role in the efficacy of the formulation. The replication inhibitors of the present formulation, Vitamin B.sub.12 can stop the steps in the infection process of respiratory pathogens by preventing the multiplication of viruses facilitated by the activity of the enzyme RNA-dependent RNA polymerase, a key enzyme in viral replication. The inhibition of RNA-dependent RNA polymerase reduces both the production of new viruses as well as lateral spread of the SARS-CoV-2 virus from cell to cell.

[0061] The formulation of the present invention is a synergistic formulation for management of respiratory pathogens including coronaviruses. The formulation of the present invention comprises of a mixture of specific combination of biological, non-toxic peptide molecules, designed to be administered in multiple ways including by way of inhalation through nasal spray or oral pump or via nebulizer, for the prevention of respiratory infections.

[0062] In one embodiment, this synergistic formulation comprises of ion chelator selected from zinc chelating peptide and calcium chelating peptides. These ion chelators inhibit the activities of proteases for as long as the formulation is present on the extracellular surface of lungs. This inhibition is likely to prevent the priming of pathogenic proteins by the proteases.

[0063] Calcium, a secondary metabolite plays an important role in signaling of calcium channels or calcium pumps. The use of ion chelator i.e. calcium chelating peptide selected from FDHIVY and other similar calcium-chelating peptides in the formulation helps in chelating calcium, thereby inhibiting activity of human inflammatory protein called C Reactive Protein (CRP) and SARS-CoV-2 protein E, which is a calcium channel protein required to maintain infectivity of SARS-CoV-2 virus.

[0064] Neuropilin also has 2 calcium binding motifs at positions 195, 209 250. Cytoplasmic tails of neuropilin-sequence S-E-A-COOH binds to NIP which in turn mediates Clathrin mediated endocytosis. Calcium bound to these residues are further chelated by the addition of calcium chelating peptides-FDHIVY and other similar calcium-chelating peptides. Hence, the addition of a peptide with the sequence -Serine-Glutamic Acid-Alanine- (-S-E-A-) is able to stop the endocytosis of the SARS-CoV-2 virus that is enabled by the Furin-dependent cleavage of the S-glycoprotein.

[0065] The synergistic formulation of the present invention has been tested for antiviral activity on SARS-CoV-2 and for its non-toxicity on lung cells. Positive Control used in the experiments is Remdesivir. The formulation of the present invention is referred to as Test Material also referred to as EvoBO2020.

[0066] FIG. 1(a) depicts the results of IC-50 plaque assay against SARS-CoV-2, with the positive control Remdesivir, which is a known antiviral drug. The IC-50 value of Remdesivir was calculated by considering the top and baseline value 100 and 0 respectively. As is depicted in the figure, the percentage cytotoxicity of Remdesivir increased with the increase in its concentration. The IC-50 reported for Remdesivir was 2.14 μM, which is acceptable based on internal and literature data.

[0067] FIG. 1(b) depicts the results of IC-50 plaque assay against SARS-CoV-2, with the formulation of the present invention also referred to as EvoBO2020 in test reports. The IC-50 of the test material was calculated by considering the top and baseline value 100 and 0 respectively. The absolute IC-50 was calculated to be 0.02 dilutions against SARS-CoV-2.

[0068] FIG. 2 depicts the anti viral effect of the formulation of the present invention also referred to as EvoBO2020 in test reports. The formulation of the present invention was tested for its efficacy in reducing the SARS-CoV-2 infectivity. 1:200, 1:40, 1:20 and 1:10 dilutions of the test formulation were tested against the virus.

[0069] The green bars in FIG. 2 depict log viral particles and the blue bars depict the percentage reduction of viral particles by different dilutions of the test material. Viral RNA was extracted by MagMAX™ Viral/Pathogen Extraction Kit Fosun COVID-19 RT-PCR Detection Kit.

[0070] As can be inferred from the FIG. 2, with increase in concentration of the formulation of the present invention, the viral load decreases. From the FIG. 2, it is clear that the 1:10 dilution of the test material drastically reduces the viral load up to 90%.

[0071] The same result can be explained with reference to FIGS. 3 and 4. The table in FIG. 3 depicts the plaque assay data for the positive control, Remdesivir. The highest concentration of Remdesivir tested (25 μM) caused reduction in viral load up to 86.84%. This is in contrast to the results of the plaque assay conducted with EvoBO2020, as disclosed in FIG. 4, wherein, the highest concentration of EvoBO2020 tested (1:10 dilution) caused a drastic reduction in viral load up to 94.74%. Thus, it can be inferred from the viral plaque assay that the synergistic formulation of the present invention showed 15%, 61%, 84% and 90% viral reduction at 1:200, 1:40, 1:20 and 1:10 dilutions respectively. The viral particle number reduced from 10.sup.6.0 to 10.sup.5.0 at 1:10 dilution. [0072] The regression equation for viral particles Vs Ct value of the N-gene specific to SARS-CoV2 virus (y=−4.9474x+39.723, R.sup.2=0.9964) [0073] (X=Number of viral particles, y=Ct value) [0074] Number of viral particles are calculated using X=(39.723−Ct.sub.N−gene.sub.@different conc.)/4.9474. [0075] Percentage Viral reduction=N.sub.c−N.sub.t×100/N.sub.c [0076] where, N.sub.c denotes number of viral particles without drug (control) and N.sub.t denotes number of viral particles with drug (test)

[0077] Therefore, it can be inferred that the synergistic formulation of the present invention reduces viral load of SARS-CoV-2 significantly.

[0078] The formulation of the present invention comprises of a combination of ion chelator, protease inhibitor and inhibitor of viral replication enzyme. The formulation comprises a non-toxic synergistic cocktail of ion chelator, protease inhibitor and inhibitor of viral replication enzyme at a specific concentration for management of infections caused by the respiratory viruses.

[0079] Table 1 depicts the concentration of the biological, non-toxic peptide molecules included in the formulation of the present invention. The synergistic formulation of the present invention is prepared in various combinations of the constituents including protease inhibitor, 9 Arg, ion chelator, FDHIVY, Poly-Lys (L8), Anti-HAT, and vitamin B.sub.12. Each combination may include some of the above mentioned constituents depending on the use of the formulation. These concentrations of the components of the synergistic formulations of the present invention have been tested to be effective in 200 μL of the formulation.

TABLE-US-00001 TABLE 1 Effective concentrations of components of the formulation of the present invention Actual concentrations that were effective in 200 uL 1:30 dilution 1:20 dilution 9 Arg 1.3 mM 2 mM A1-AT-RCL 11.9 μM 17.85 μM FDHIVY 0.16 μg/ml 0.25 μg/ml Poly-Lys(L8) 1.3 mM 2 mM Anti-HAT 13.33 μM 20 μM

[0080] Generally, benzalkonium chloride is used as a preservative. However, the formulation is not restricted to the use of benzalkonium chloride and any similar preservative can be used in the formulation. The formulation of the present invention is a combination therapy in the form of a solution capable of being administered either by way of inhalation through nasal spray or oral pump or via nebulizer.

[0081] The formulation administered orally or as nasal drops or nebulizers reduce the binding of the respiratory virus to the airway epithelial tissues. Such a formulation is non-toxic when used on cultured lung cells, is unlikely to cross the lung membrane and is biodegradable.

[0082] FIG. 5 depicts the results of MTT Assay-Quantitation-Experiment on 1-A549 lung cells when exposed to the formulation of the present invention, EvoBO2020. The cultured lung cells were exposed to PBS, DOLO, and different concentrations namely 100×, 50×, 25× and 10×, of the formulation of the present invention. Lung cell viability of was checked by addition MTT to all the test wells and reading absorbance at 540 nm. FIG. 5 denotes the absorbance of cells at 540 nm. The Absorbance values have been given in Table 1.

TABLE-US-00002 TABLE 2 Absorbance at 540 nm after addition of MTT Sample Absorbance at 540 nm Control 1 0.26 Control 2 0.268 Control 3 0.294 PBS 1 0.189 PBS 2 0.164 PBS 3 0.229 DOLO 1 0.218 DOLO 2 0.237 DOLO 3 0.239 100× 1 0.202 100× 2 0.27 100× 3 0.255  50× 1 0.174  50× 2 0.22  50× 3 0.223  25× 1 0.231  25× 2 0.188  25× 2 0.198  10× 1 0.193  10× 2 0.176  10× 3 0.208

[0083] The absorbance at 540 nm is linearly related to the cell activity. The average absorbances at 540 nm for control wells and subsequently treated wells with the test formulation are close. This indicates that the lung cells remain viable even after addition of 50-75 times the active concentration of the test formulation for extended periods (10-12 hours). Therefore, it is concluded that the test formulation of the present invention is non-toxic to the lung cells.

[0084] From the lung cell viability experiment it can be concluded that the formulation of the present invention can be delivered up to 50-75 times the active concentration without inducing toxicity.

[0085] The formulation of the present invention is safe and effective against activation of coronaviruses as well as other viruses due to combination of known biochemicals, which are integral part of the biochemical processes and hence does not induce any toxic effects. As seen in FIG. 5, the formulation has been shown to reduce infection with virus in in-vitro testing while at the same time being non-toxic to lung cells.

[0086] The ingredients of the formulation of the present invention are specifically selected to inhibit the activity of respiratory viruses especially coronavirus which, in turn, reduces the infection events. The Biomolecules used in the formulation have been synthesized and prepared to 98% purity.

[0087] The formulation of the present invention is prepared as solution, which can be administered in multiple ways including inhalation as nasal drops or through an oral pump or through nebulizer. This makes the formulation economically significant since it provides for effective and easy administration of the required dose directly into the site of action without requiring high dose of the drugs and is safe due to lower concentration of the drug. The administration of the formulation through inhalation is associated with effective and ease of administration or self-administration without the requirement of any specific skill or training for the medical care or health care staff.

[0088] This is a unique formulation that has been designed against SARS-CoV-2 but can be further developed to reduce respiratory infections significantly. This can be a significant addition to the personal protection strategy of people exposed to respiratory infections.