DESC:Field of the invention
The present invention provides a composition comprising peptide(s) with protease activity. The present invention further provides use of the said composition for pharmaceutical purpose. The said composition can also be used for cosmetic purpose. Preferably, the composition of the current invention provides treatment and prevention of a coronavirus infection in a subject. In particular, the invention provides treatment or prevention of a coronavirus infection in a mammal using porcine trypsin.

Background of the invention
Three highly pathogenic human coronaviruses (CoVs) have been identified so far, including Middle East respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV), and a 2019 novel coronavirus (2019-nCoV), as previously termed by the World Health Organization (WHO). Among them, SARS-CoV was first reported in Guangdong, China in 2002. SARS-CoV caused human-to-human transmission and resulted in the 2003 outbreak with about 10% case fatality rate (CFR), while MERS-CoV was reported in Saudi Arabia in June 2012. Even though with its limited human-to human transmission, MERS-CoV showed a CFR of about 34.4%. The 2019-nCoV was first reported in Wuhan, China in December 2019 from patients with pneumonia, and it has exceeded both SARS-CoV and MERS-CoV in its rate of transmission among humans. 2019-nCoV was renamed SARS-CoV-2 by Coronaviridae Study Group (CSG) of the International Committee on Taxonomy of Viruses (ICTV), while it was renamed HCoV-19, as a common virus name, by a group of virologists in China. The disease and the virus causing it were named Coronavirus Disease 2019 (COVID-19) and the virus responsible for COVID-19 or the COVID-19 virus, respectively, by WHO. The outbreak of a novel coronavirus (2019-nCoV) represents a pandemic threat that has been declared a public health emergency of international concern (PHEIC). There is an urgent need for the immediate development of vaccines, antiviral drugs and other therapeutics for prevention and treatment of COVID-19. More than 100 pre-clinical or clinical trials are going on involving various therapeutics, which include novel therapies, vaccines as well as repurposing of already approved drugs, but with different indications such as anti-malarial, anti-viral, anti-parasitic drugs, cytokine or complement targeting antibodies, etc. Huge progresses were made in last one year for bringing effective vaccines against SARS-CoV-2. As per WHO draft landscape of vaccines, currently 172 vaccine candidates are in pre-clinical development, 63 are in clinical development. Three candidates including two mRNA based candidate from Pfizer and Moderna and Chimpanzee adenovirus vectored based candidate from AstraZeneca got emergency use authorization globally. In India, Covaxin and COVISHILED vaccines are approved under emergency use authorization. Though vaccination remains one of the cornerstone of immediate approaches to prevent the spread of this dreaded disease, still, it is difficult to vaccinate such a huge population of India as well as globally in a near future. Thus, there is a need of a product which is ready-to-use and easy to administer for large population and can still effectively prevent viral infection. Current invention provides a composition comprising peptide(s) with protease activity which is ready-to-use and can be administered without need of medical practitioner or hospitalization. The composition prepared according to the current invention prevents viral infection by reducing viral load including viral load of SARS-CoV-2. There is an enzyme based product available in the market with brand name ‘COLDZYME’ for the treatment of common cold. The said product contains cod trypsin. Current invention provides a composition of porcine trypsin for the treatment or prevention of virus infection by reducing viral load, preferably 99% of viral load including SARS-CoV-2 in a subject.

Summary of the invention
The present invention provides a composition comprising peptide(s) with protease activity. The said composition is used for pharmaceutical purpose. It can also be used for cosmetic purpose. The peptide(s) with protease activity according to the current invention is a trypsin or chymotrypsin. Preferably, the composition of the current invention is used for the treatment and prevention of a coronavirus infection in a subject. In particular, the invention provides treatment or prevention of a coronavirus infection in a mammal using a trypsin, preferably porcine trypsin.

Definitions
The term “peptide(s) with protease activity” as used herein refers to any peptide (both naturally occurring and non-naturally occurring) which is capable of catalysing proteolysis in vivo, in the mammalian (e.g. human) body. Thus, any type of protease may be utilised in the invention, including but not limited to serine proteases (such as trypsins/chymotrypsins), threonine proteases, cysteine proteases, aspartate proteases, glutamic acid proteases and metalloproteases.
The term “coronavirus infection” as used herein refers to an infection caused by or otherwise associated with growth of coronavirus in a subject, in the family Coronaviridae (subfamily Coronavirinae).
The term “treatment” as used herein refers to the alleviation, partially or fully, of the symptoms of the virus infection, preferably here coronavirus infection (e.g. the sore throat, blocked and/or runny nose, cough and/or elevated temperature associated with a common cold).
The term “prevention” as used herein refers to the reduction in risk of coronavirus infection in patients. Such prevention may not be absolute, i.e. it may not prevent all such patients developing a coronavirus infection, or may only partially prevent an infection. The terms “prevention” and “prophylaxis” may be used interchangeably.
The terms “peptide”, “polypeptide”, “protein” and “amino acid sequence” as used herein generally refer to a polymer of amino acid residues and are not limited to a minimum length of the product. Thus, peptides, oligopeptides, dimers, multimers, and the like, are included within the definition. Both full-length proteins and fragments thereof are encompassed by the definition.
The term “fragment” or “variant” as used herein refers to a functional part of a full-length peptide, polypeptide or protein, whose sequence is not identical to the respective full-length peptide, polypeptide or protein but retains the same function as the full-length peptide, polypeptide or protein. The said functional fragment or the functional variant may have more, less, or the same number of residues than the corresponding native molecule and / or may contain one or more amino acid or nucleotide substitutions.
The term “pharmaceutical formulation” refers to preparations, which are in such form as to permit the biological activity of the active ingredient(s) to be unequivocally effective. The term “pharmaceutical formulation”, “pharmaceutical composition” and “composition” can be used here interchangeably.
The term “excipient” refers to an agent that may be added to a formulation to stabilize the active drug substance in the formulated form to adjust and maintain osmolality and pH of the pharmaceutical preparations. Examples of commonly used excipients include, but are not limited to, polyvalent alcohol(s) / polyol(s), salt(s), buffer(s) or its salt(s), alkali metal ion(s), alcohol(s), flavouring agent(s), sugar(s), amino acid(s), acid(s), base(s), surfactant(s) and polymer(s). “Pharmaceutically acceptable” excipients are those which can reasonably be administered to a subject mammal to provide an effective dose of the active ingredient employed.
The term “effective amount” as used herein refers to the amount of ingredient of the composition, preferably the amount of an active ingredient (porcine trypsin as used herein) that provides a therapeutic effect of prevention or treatment for a given condition and administration regimen.
The terms “patient” and “subject” are used interchangeably and are used in their conventional sense to refer to a living organism suffering from or prone to a condition that can be prevented or treated by administration of a composition of the present invention, and includes both humans and non-human animals. Examples of subjects include, but are not limited to, humans, chimpanzees and other apes and monkey species; farm animals such as cattle, sheeps, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs; birds, including domestic, wild and game birds such as chickens, turkeys and other gallinaceous birds, ducks, geese, and the like. The term does not denote a particular age. Thus, adult, juvenile and new born individuals are of interest.
Abbreviations used in the current invention:
% : percentage
°C : degree Celsius
µL : micro liter
2019- nCoV : novel coronavirus
BSA : Bovine serum albumin
g / L : gram / Liter
HCl : Hydrochloric acid
Hrs : Hours
L : Liter
MEM : Minimal Essential Eagles Medium
mins : Minutes
mL : mili liter
NaOH : Sodium hydroxide
q.s. : Quantity sufficient
SARS : Severe acute respiratory syndrome
TCID : Tissue culture infective dose

Embodiments of the invention
In one embodiment, the current invention provides a composition comprising peptide(s) with protease activity. In a preferred embodiment, the peptide(s) with protease activity according to the current invention is trypsin or chymotrypsin. In a more preferred embodiment, the trypsin according to the current invention is porcine trypsin.
In a second embodiment, the current invention provides a composition comprising a peptide(s) with protease activity for cosmetic purpose or for pharmaceutical purpose.
In a third embodiment, the current invention provides a composition comprising a peptide(s) with protease activity and excipients which are acceptable for pharmaceutical and for cosmetic purpose.
In yet another embodiment, the current invention provides a composition comprising a peptide(s) with protease activity which can be used for the treatment or prevention of viral infection. In a preferred embodiment, the current invention provides a composition comprising a peptide(s) with protease activity for the treatment or prevention of coronavirus infection. In a more preferred embodiment, the current invention provides a composition comprising peptide(s) with protease for the treatment or prevention of SARS-CoV-2 infection.
In a fourth embodiment is provided a composition of a peptide(s) with protease activity which helps reduce viral load of certain viruses including corona virus and/or SARS-CoV-2. In one of the preferred embodiments, the current invention provides a composition comprising peptide(s) with protease activity which reduces viral load, including that of corona virus and/or SARS-CoV-2 by up to 99 %.
In a fifth embodiment, the current invention provides a composition comprising a peptide(s) with protease activity and excipient(s). In one of the embodiments, the current invention provides a composition comprising peptide(s) with protease activity and polyvalent alcohol (polyol) as an excipient. In a preferred embodiment, the current invention provides a composition comprising peptide(s) with protease activity and glycerol.
In one of the embodiments, the current invention provides a composition comprising peptide(s) with protease activity and a suitable solvent, optionally with other suitable excipient(s). In a preferred embodiment, the current invention provides a composition comprising peptide(s) with protease activity and alcohol as the solvent, preferably but are not limited to ethanol, optionally with other suitable excipient(s).
In one of the embodiments, the current invention provides a composition comprising peptide(s) with protease activity and a salt, preferably an inorganic salt, optionally with other suitable excipient(s). In a preferred embodiment, the current invention provides a composition comprising peptide(s) with protease activity and calcium chloride (CaCl2), preferably calcium chloride dihydrate, optionally with other suitable excipient(s).
In one of the embodiments, the current invention provides a composition comprising peptide(s) with protease activity and trometamol, optionally with other suitable excipient(s).
In one of the embodiments, the current invention provides a composition comprising peptide(s) with protease activity and menthol, optionally with other suitable excipient(s).
In certain embodiment, the current invention provides a composition comprising porcine trypsin, glycerol, ethanol, calcium chloride, trometamol, menthol and / or benzyl alcohol optionally, with an acid or a base in sufficient quantity to maintain suitable pH of the composition. In one of the embodiments, the current invention provides a composition comprising porcine trypsin with a pH in the range of pH 6.0 to pH 9.5. In certain preferred embodiments, the current invention provides a composition comprising porcine trypsin with flavouring agent(s) in addition to other suitable excipient(s).
In further certain embodiment, the current invention provides a composition comprising 2.7 units / dose to 32.4 units / dose, preferably 16.2 units / dose, more preferably, 8.1 units / spray porcine trypsin , 1 % w/v to 70 % w/v glycerol, 0.1 % w/v to 9 % w/v ethanol, 0.1 % w/v to 1.7 % w/v calcium chloride, 0.01 % w/v to 0.20 % w/v trometamol, 0.001 % w/v to 0.03 % w/v menthol and / or 0.01 % w/v to 0.6 % w/v benzyl alcohol optionally with an acid or a base in a sufficient quantity to maintain suitable pH of the composition.
In certain embodiment, the current invention provides a composition comprising 8.1 unit of recombinant porcine trypsin, 63 % w/v of glycerol, 0.9 % w/v of ethanol, 0.1722 % w/v of calcium chloride (calcium chloride dihydrate), 0.02 % w/v of trometamol, 0.003 % w/v of menthol, purified water, optionally with an acid or a base in a quantity sufficient amount to maintain suitable pH of the composition. The suitable pH of the composition according to the current invention is preferably pH 6.0 to pH 9.5.
In certain embodiment, the current invention provides a composition comprising 8.1 units of recombinant porcine trypsin, 1 % w/v of glycerol, 0.9 % w/v of ethanol, 0.1722 % w/v of calcium chloride (calcium chloride dihydrate), 0.02 % w/v of trometamol, 0.003 % w/v of menthol, 0.066 % w/v benzyl alcohol, purified water, optionally with an acid or a base in a quantity sufficient amount to maintain suitable pH of the composition. The suitable pH of the composition according to the current invention is preferably pH 6.0 to pH 9.5.
In one of the embodiments, the current invention provides a composition comprising peptide(s) with protease activity may further comprise excipient(s) selected from buffer(s), emulsifier(s), adjuvant(s), stabiliser(s), wetting agent(s), filler(s), sugar(s), flavouring agent(s).
In another embodiment, the compositions comprising a peptide(s) with protease activity according to the invention may be administered via any suitable route but are not limited to oral, buccal, topical, ocular, sub-lingual, nasal, pulmonary and parenteral, preferably oral or nasal.

Detailed description of the invention
The present invention provides a composition comprising peptide(s) with protease activity for cosmetic or for pharmaceutical purpose. The composition of peptide(s) with protease activity according to the present invention is used in the prevention of or in the treatment of virus infection, particularly corona virus infection, more preferably SARS-CoV-2 infection in a subject. The peptide(s) with protease activity according to the current invention is trypsin or chymotrypsin, preferably trypsin. Trypsin according to the current invention is either a porcine trypsin or a bovine trypsin. Amino acid sequence of porcine trypsin according to the current invention corresponds to that reported in Uniport database accession no. P00761 (9-229 amino acid residues). Amino acid sequence of porcine trypsin is well known to the skilled person (for e.g. Hermodson et al., Determination of the amino acid sequence of porcine trypsin by sequenator analysis, Biochemistry 1973, 12, 17, 3146–3153). The trypsin according to the present invention can be in an isolated form or in a recombinant form. The functional variant of fragment of trypsin or chymotrypsin can also be used to prepare composition of the current invention. In a preferred embodiment, the trypsin, preferably porcine trypsin is present in the range of 2.7 units / dose to 32.4 units / dose. In one of the preferred embodiments, the trypsin, preferably porcine trypsin is present in composition as 16.2 units / dose. In one of the preferred embodiments, the trypsin preferably porcine trypsin is present in composition as 8.1 units / spray. In one of the more preferred embodiments, the composition of porcine trypsin is in spray form and contains 8.1 units per spray. Trypsin may inhibit ability of virus to infect cells and thus trypsin may deactivate the virus. The composition according to the current invention may include other effective amount of peptide(s) with protease activity. This is a predetermined quantity of peptide calculated to produce a desired effect in association with the required excipient(s) as described herein. The desired effect according to the current invention is the treatment or prevention of viral infection. Preferably, the desired effect according to the current invention is prevention of viral infection including corona virus infection by reducing viral load including load of SARS-CoV-2. Reduction of viral load by peptide(s) with protease activity can be checked by suitable in-vitro methods. The virucidal activity of the composition of the present invention is measured using cytopathic effect as illustrated herein below examples. Further, composition according to the current invention may include polyvalent alcohol such as polyethylene glycol (PEG), propylene glycol, glycerol and the like. Preferably, polyvalent alcohol according to the current invention is glycerol. Glycerol may act as osmotically active barrier for the virus. Glycerol is also one of the critical diluents for oral spray composition or hydrogel formation or nasal spray formulation. In one of the embodiments, polyvalent alcohol, preferably glycerol according to the present invention is present in the range of 1 % w/v to 70 % w/v. The said range includes all integer and non-integer values in between. For example, it includes 1 % w/v, 2 % w/v, 5 % w/v, 10 % w/v, 20 % w/v, 30 % w/v, 50 % w/v, 63 % w/v, 70 % w/v and other integer and non-integer values between the given ranges. In one of the more preferred embodiments, polyvalent alcohol, preferably, glycerol according to the present invention is present in amount of 63 % w/v or 1 % w/v. The composition according to the current invention may further include ethanol as co-solvent. In one of the embodiments, alcohol, preferably ethanol according to the present invention is present in the range of ¬¬0.1 % w/v to 9 % w/v. The said range includes all integer and non-integer values. For example, it includes 0.1 % w/v, 0.2 % w/v, 0.9 % w/v, 1 % w/v, 2 % w/v, 3 % w/v, 5 % w/v, 6 % w/v, 9 % w/v and other integer and non-integer value between the given ranges. In one of the more preferred embodiments, alcohol, preferably ethanol according to the present invention is present in amount of 0.9 % w/v. The composition of peptide(s) with protease activity may further comprise inorganic salt or its hydrate form, preferably calcium chloride (calcium chloride dihydrate). Calcium chloride (calcium chloride dihydrate) according to the current invention may act as a stabilizer. In one of the embodiments, salt, preferably calcium chloride (calcium chloride dihydrate) according to the present invention is present in the range of 0.1 % w/v to 1.7 % w/v. The said range includes all integer and non-integer value. For example, it includes 0.1 % w/v, 0.1722 % w/v, 0.2 % w/v, 0.5 % w/v, 0.7 % w/v, 0.9 % w/v, 1 % w/v, 1.5 % w/v, 1.7 % w/v and other integer and non-integer value between the given ranges. In one of the more preferred embodiments, inorganic salt, preferably, calcium chloride according to the present invention is present in an amount of 0.1722 % w/v. Acid or base can be added in quantity sufficient amount to maintain desired pH of the final composition. The pH of the composition according to the current invention is in the range of pH 6.0 to pH 9.5. Desired pH of the formulation can be obtained by adding suitable acid, preferably HCl or a suitable base, preferably NaOH, as per requirement. Furthermore, the composition of the present invention may include menthol, specifically for oral administration. Menthol according to the current invention is a cooling agent as well as flavouring agent. In one of the embodiments, menthol according to the present invention is present in the range of ¬¬0.001 % w/v to 0.03 % w/v. The said range includes all integer and non-integer values. For example, it includes 0.001 % w/v, 0.002 % w/v, 0.003 % w/v, 0.01 % w/v, 0.02 % w/v, 0.03 % w/v and other integer and non-integer value between the given ranges. In one of the more preferred embodiments, menthol according to the present invention is present in an amount of 0.003 % w/v. Alternatively, the composition of peptide(s) with protease activity can be prepared for nasal administration. Said alternate nasal spray formulation may further include one or more preservatives. In one of the embodiments, the preservative according to the current invention is selected from benzyl alcohol, benzalkonium chloride, benzethonium chloride, potassium sorbate, parabens, chorobutanol, phenyl ethyl alcohol and the like. In one of the preferred embodiments, the preservative according to the current invention is benzyl alcohol. In one of the embodiments, preservative according to the present invention is present in the range of 0.01 % w/v to 0.6 % w/v. The said range includes all integer and non-integer value. For example, it includes 0.01 % w/v, 0.02 % w/v, 0.06 % w/v, 0.066 % w/v, 0.1 % w/v, 0.2 % w/v, 0.4 % w/v, 0.5 % w/v, 0.6 % w/v and other integer and non-integer value between the given ranges. In one of the more preferred embodiments, preservative preferably benzyl alcohol according to the present invention is present in an amount of 0.066 % w/v. The composition according to the present invention may include buffer preferably tris buffer or trometamol salt of tris buffer. In one of the embodiments, trometamol according to the present invention is present in the range of 0.01 % w/v to 0.20 % w/v. The said range includes all integer and non-integer value. For example, it includes 0.01 % w/v, 0.02 % w/v, 0.05 % w/v, 0.1 % w/v, 0.15 % w/v, 0.20 % w/v and other integer and non-integer value between the given ranges. In one of the more preferred embodiments, trometamol according to the present invention is present in an amount of 0.02 % w/v. The composition according to the present invention may include flavouring agent(s). The preferred flavouring agent(s) of the current invention may include but are not limited to bubble gum flavour. In one of the more preferred embodiments, flavouring agent(s) of the current invention is present in an amount of 0.02 % w/v.
The composition of peptide(s) with protease activity can be administered via any suitable route but are not limited to oral, buccal, topical, ocular, sub-lingual, nasal, pulmonary and parenteral. Preferred route of administration of composition of the present invention is oral or nasal. In a more preferred embodiment, the composition prepared according to the current invention is administered in spray form either mouth spray form or nasal spray form. Alternatively, the composition according to the current invention can be prepared in liquid or lyophilised or hydrogel or lozenge or pastille or tablet or syrup or chewing gum form.
The preferred mouth spray composition of porcine trypsin prepared according to the current invention can be administered as 2 puffs at one time as a single dose. It can be administered every other hour up to a maximum of 6 times daily.
The trypsin composition prepared according to the current invention can be used preventively to reduce the risk of viral infection. The trypsin composition prepared according to the current invention can be used in the early stages of infection to shorten the duration of a viral infection. The trypsin composition prepared according to the current invention is suitable for adults and children over 4 years.
Following non-limiting examples illustrate the described compositions of the present invention and the means of carrying out the invention to obtain a stable dosage form of said composition of peptide(s) with protease activity. It will be appreciated that the examples are illustrative and such other suitable modifications/additions etc. as are well within the scope of the persons skilled in the art are meant to be encompassed within the scope of the present invention. The efficacy of the trypsin in the treatment of SARS-CoV-2 infection or related clinical manifestations may be evaluated in-vitro as described in following examples.

Examples
Example 1: porcine composition preparation at manufacturing scale
Recombinant porcine trypsin was used as peptide with protease activity to prepare oral spray formulation for the treatment of viral infection. It was present in powder form. Detail of composition of porcine trypsin is given in table 1. The composition as described in table 1 was prepared for mouth spray form.
Table 1: Composition of porcine trypsin
Sr. No. Name of Ingredient Amount (%w/v)
1 Recombinant porcine trypsin powder 8.1 units / spray
2 Ethanol 0.90
3 Glycerol 63.00
4 Calcium Chloride Dihydrate 0.1722
5 Trometamol 0.02
6 Menthol 0.003
8 Hydrochloric acid q.s. to pH
9 Purified Water q.s. to 180 mcl

Manufacturing process of making porcine trypsin composition is given below as flow chart.
Transfer purified water into manufacturing vessel.
Add dispensed quantity of Calcium chloride dihydrate into manufacturing vessel. Rinse the container of Calcium chloride dihydrate with purified water and add into manufacturing vessel. Stir for 10 minutes.
Add dispensed quantity of Tromethamine into manufacturing vessel. Rinse the container of Tromethamine with purified water and add into above solution of manufacturing tank. Stir for 10 minutes.
Add dispensed quantity of Glycerin in the above solution of manufacturing tank. Rinse the container of glycerin with of purified water and add into above solution of manufacturing tank. Stir for 30 minutes.

Add and dissolve Menthol in the container of Ethanol. Add this solution into above solution of manufacturing vessel. Rinse the containers of Menthol and Ethanol with purified water and add into above solution of manufacturing tank. Stir for 10 minutes.

Add and dissolve Trypsin into 1mM HCl. Add this solution into above solution of manufacturing vessel. Rinse the containers of Trypsin and HCl with purified water and add into above solution of manufacturing tank. Stir for 10 minutes.
Make up the final volume with purified water and stir for 20 minutes.

Example 2: Analysis of anti-viral effect of porcine trypsin composition against SARS-CoV-2 virus
In this experiment, virucidal activity of porcine trypsin composition was demonstrated against SARS-CoV-2 virus using cytopathic effect. Porcine trypsin composition was prepared as described in table 1 for this experiment.
A. Virucidal activity of the test products
20 µL of 0.3 g/L BSA was pipetted in a 2 mL centrifuge tube. 20 µL of SARS-CoV-2 virus (TCID50:106.19/mL) was added carefully avoiding the sides of the centrifuge tubes. 160 µL of the porcine trypsin composition was added to the centrifuge tube. The tube was incubated at 20°C-25°C for 10 minutes. At the end of incubation, contents were mixed and 50 µL of the contents was added to 450 µL of cold maintenance media (2 % MEM media). Serial dilutions from 101 to 108 was prepared within 30 minutes. Prepared dilutions were added over pre-seeded VERO cells and the plate was incubated for 72 hrs. At the end of incubation, the plate for cytopathic effect was observed. The plate was scored on the basis of observation. (Score 4 represents all the 4 fields observed in the well got infected, and score 0 represents all the 4 fields in the well remain uninfected). The titer was calculated using Reed and Meunch equation.
B. Product test cytotoxicity
20 µL of 0.3 g/L BSA was pipetted in a 2 mL centrifuge tube. 20 µL of milli-Q water was added to the tube. 160 µL of the test product was added to the centrifuge tube. Serial dilutions from 101 to 108 was prepared. The prepared dilutions was added over pre-seeded VERO cells and the plate was incubated for 72 hrs. At the end of incubation, the plate for cytopathic effect was observed. The plate was scored on the basis of observation. (Score 4 represents all the 4 fields observed in the well got infected and score 0 represents all the 4 fields in the well remain uninfected). The cytotoxicity was calculated.
C. Titration of virus control (with interfering substance)
20 µL of 0.3 g/L BSA was pipetted in a 2 mL centrifuge tube. 160 µL of milli-Q water was added to the tube. 20 µL of SARS-CoV-2 virus (TCID50:106.19/mL) was added carefully avoiding the sides of the centrifuge tubes. The tube was incubated at 20 °C-25 °C for 10 minutes. At the end of incubation, the contents were mixed and 50 µL of the contents was added to 450 µL of media (2 % MEM media). Serial dilutions was prepared from 101 to 108 within 30 minutes. The prepared dilutions were added over pre-seeded VERO cells and the plate was incubated for 72 hrs. At the end of incubation, the plate was observed for cytopathic effect. The plate was scored on the basis of observation. (Score 4 represents all the 4 fields observed in the well got infected and score 0 represents all the 4 fields in the well remain uninfected). The titer was calculated using Reed and Meunch equation.
D. Titration of virus control (without interfering substance)
50 µL of SARS-CoV-2 (TCID50:106.19/mL) virus was pipetted to 450 µL of media (2 % MEM media) carefully avoiding the sides of the centrifuge tube. Serial dilutions was prepared from 101 to 108. The prepared dilutions were added over pre-seeded VERO cells and incubate the plate for 72 hrs. At the end of incubation, the plate was observed for cytopathic effect. The plate was scored on the basis of observation. (Score 4 represents all the 4 fields observed in the well got infected and score 0 represents all the 4 fields in the well remain uninfected). The titer was calculated using Reed and Meunch equation.

Results and observations of analysis of example 2 are provided in table 2.
Table 2: Results and observation
Virus log reduction
Product Concentration Interfering substance Test temp. Level of cytotoxicity Log TCID50 after 10 mins Log reduction compared to virus control % Reduction
Porcine composition Neat 0.3 g/L
BSA 20 °C - 25°C 0 3.25 2.25 99.43
Virus control
Name Concentration Interfering substance Test temp. Level of cytotoxicity Log TCID50 after 10 mins Log reduction compared to virus control % Reduction
SARS-CoV-2 virus Undiluted virus, titer : 106.19 TCID50/mL 0.3 g/L BSA 20 °C - 25°C NA 105.50 NA NA
SARS-CoV-2 virus Undiluted virus, titer : 106 TCID50/mL NA 20°C - 25°C NA 105.61 NA NA
Porcine trypsin composition prepared according to example 1 showed log reduction of 2.25 (99.43 % deactivation of SARS-CoV-2) logs at undiluted (ready-to-use) concentration at 20°C to 25°C for 10 minutes contact time under clean conditions (0.3 g/L Bovine Serum Albumin) against SARS-CoV-2 virus.

List of reference referred in this application
Hermodson et al., Determination of the amino acid sequence of porcine trypsin by sequenator analysis, Biochemistry 1973, 12, 17, 3146–3153

Incorporation by reference
The entire disclosure of each of the patent documents and scientific articles referred to herein is incorporated by reference for all purposes.

Equivalents
The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting the invention described herein. Scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

,CLAIMS:We claim:

1. A composition comprising effective amount of porcine trypsin and suitable excipient(s) for treatment or prevention of coronavirus infection, preferably SARS-CoV-2 infection.

2. The composition as claimed in claim 1 wherein the suitable excipient(s) are selected from polyol, solvent, salt, buffer, preservative, flavouring agent, cooling agent, acid, base and combination thereof.

3. The composition as claimed in claim 1 comprises porcine trypsin, glycerol, ethanol, calcium chloride, trometamol, menthol optionally with an acid or a base in a sufficient quantity to maintain suitable pH of the composition.

4. The composition as claimed in claim 1 comprises 2.7 units / dose to 32.4 units / dose, preferably 16.2 units / dose, more preferably, 8.1 units / spray porcine trypsin, 1 % w/v to 70 % w/v glycerol, 0.1 % w/v to 9 % w/v ethanol, 0.1 % w/v to 1.7 % w/v calcium chloride, 0.01 % w/v to 0.20 % w/v trometamol, ¬¬0.001 % w/v to 0.03 % w/v menthol and / or 0.01 % w/v to 0.6 % w/v benzyl alcohol optionally with an acid or a base in a sufficient quantity to maintain suitable pH of the composition.

5. The composition as claimed in claim 1 comprises 8.1 units of recombinant porcine trypsin, 63 % w/v of glycerol, 0.9 % w/v of ethanol, 0.1722 % w/v of calcium chloride (calcium chloride dihydrate), 0.02 % w/v of trometamol, 0.003 % w/v of menthol, purified water, optionally with an acid or a base in a quantity sufficient amount to maintain suitable pH of the composition.

6. The composition as claimed in claim 1 comprises 8.1 units of recombinant porcine trypsin, 1 % w/v of glycerol, 0.9 % w/v of ethanol, 0.1722 % w/v of calcium chloride (calcium chloride dihydrate), 0.02 % w/v of trometamol, 0.003 % w/v of menthol, 0.066 % w/v benzyl alcohol, purified water, optionally with an acid or a base in a quantity sufficient amount to maintain suitable pH of the composition.

7. The composition as claimed in claim 1 to 6, wherein the suitable pH of the composition is preferably pH 6.0 to pH 9.5.

8. The composition as claimed in any preceding claims wherein the composition reduces 99 % of SARS-CoV-2.

9. The composition as claimed in claim 1 to 8 is administered in mouth spray form or nasal spray form.

Dated this 27th day of April 2022.

HARIHARAN SUBRAMANIAM
IN/PA-93
Of SUBRAMANIAM & ASSOCIATE
ATTORNEYS FOR THE APPLICANTS