FIELD OF THE INVENTION

The present invention relates to the field of pharmaceuticals and provide products, compounds and compositions useful for the treatment of viral infections. Particularly, the present invention relates to compounds and compositions which are capable of treating infections of epithelial tissues including, without limitation, respiratory tract, gastrointestinal tract, oropharyngeal tract, skin, eye, genitourinary tract; and dengue.

BACKGROUND OF THE INVENTION

Respiratory tract infections are considered to be one of the most prevalent cause of disease, worldwide (Connors et al., 2016). Globally, acute lower respiratory tract infections are an important cause of morbidity and mortality in children below 5 years of age. Scientific studies have identified respiratory syncytial virus (RSV), as the most common viral cause of death due to such infection; other prominent viruses being human metapneumovirus, parainfluenza viruses, influenza viruses A and B, adenoviruses and of recent origin coronavirus. Several such infections are reported every year leading to more than half a million deaths in children below the age of five, largely in low and middle income households.

More recently, SARS-CoV-2 (COVID-19) has caused immense suffering in the world. One of the symptoms of COVID-19 infection is found to be lung related issues. Another of the symptoms of COVID 19 infection is found to be stomach related issues. Other than COVID 19, there are other enveloped viruses which causes similar problems and which disrupt organ physiology.

Viruses, as discussed above, are known to transmit through respiratory aerosol droplets, by ingesting food contaminated with viruses, or by touching with infected hands and even through inanimate surfaces.

Although human bodies can resist such viruses by producing Antimicrobial Peptides (AMPs) which is known to be human body’s own defense mechanism, a few interventions can boost the body’s ability to enhance production and secretion of such AMPs. There is thus a need for compounds and compositions that are mild on the respiratory tract, gastrointestinal tract, skin and other organs but are sufficiently effective in killing or inactivating virus to prevent, treat or mitigate infection. There is also a need for boosting the synthesis, secretion and activity of Antimicrobial Peptides (AMPs). The present invention provides such compounds or compositions and offers solution to the existing problem.

SUMMARY OF THE INVENTION

The present invention relates to a pharmaceutical composition comprising (i) a peroxisome proliferator-activated receptor (PPAR) activating fatty acid and/or a cathelicidin (LL37) compound, its precursors or analogue thereof; (ii) a vitamin B3 compound, its precursor or analogue thereof; along with pharmaceutically acceptable additives and carriers. The composition of the invention is useful in the treatment of a viral infection. Preferably, the composition is useful for treating viral infection caused by a coronavirus or an influenza virus or dengue.

An aspect of the invention relates to a pharmaceutical composition comprising (i) a peroxisome proliferator-activated receptor (PPAR) activating fatty acid and/or a cathelicidin (LL37) compound, its precursors or analogue thereof; (ii) a vitamin B3 compound, its precursor or analogue thereof; along with pharmaceutically acceptable additives and carriers for use in the treatment of a viral infection.

An aspect of the invention also relates to a peroxisome proliferator-activated receptor (PPAR) activating fatty acid for use in the treatment of a viral infection.

A preferred aspect of the invention relates to a peroxisome proliferator-activated receptor (PPAR) activating fatty acid for use in the treatment of an infection of the respiratory tract, gastrointestinal tract, oropharyngeal tract, skin, eye, or genitourinary tract.

Another aspect of the present invention relates to a pharmaceutical composition comprising (i) a peroxisome proliferator-activated receptor (PPAR) activating fatty acid and (ii) a vitamin B3 compound, its precursor or analogue thereof. Another aspect of the present invention relates to a pharmaceutical composition comprising (i) a peroxisome proliferator- activated receptor (PPAR) activating fatty acid and (ii) a vitamin B3 compound or its precursors or analogue thereof for use in the treatment of a viral infection.

A preferred aspect of the present invention relates to a pharmaceutical composition comprising

(i) a peroxisome proliferator-activated receptor (PPAR) activating fatty acid and (ii) a vitamin B3 compound or its precursors or analogue thereof for use in the treatment of an infection of the respiratory tract, gastrointestinal tract, oropharyngeal tract, skin, eye, or genitourinary tract.

Another aspect of the present invention relates to a composition comprising

(i) 0.01 to 5.0 wt% a peroxisome proliferator-activated receptor (PPAR) activating fatty acid;

(ii) 0 to 5.0 wt% vitamin B3 compound, its precursor or analogue thereof,

(iii) 0.001 to 5 wt% a preservative; and

(iv) 70 to 99.9 wt% water.

Yet another aspect of the present invention relates to a composition for inhalation for inactivating enveloped virus comprising

(i) 0.01 to 5.0 wt% a PPAR activating fatty acid;

(ii) 0 to 5.0 wt% a vitamin B3 compound or its precursors or analogue thereof;

(iii) 70 to 99 wt% water;

(iv) 1 to 20 wt% humectant; and

(v) less than 1.5 wt% surfactant chosen from non-ionic or amphoteric surfactant or mixtures thereof.

Yet another aspect of the present invention relates to a nasal drops composition comprising

(i) ) 0.01 to 5.0 wt% a peroxisome proliferator-activated receptor (PPAR) activating fatty acid;

(ii) 0 to 5.0 wt% vitamin B3 compound or analogue thereof,

(iii) 0.001 to 5 wt% a preservative; and

(iv) 70 to 99.9 wt% water.

A further aspect of the present invention relates to a nasal spray device comprising a spray pump capable of spraying a composition from a container containing a nasal drops composition of the invention. An aspect of the present invention relates to a pharmaceutical composition comprising (i) a peroxisome proliferator-activated receptor (PPAR) activating fatty acid and (ii) a vitamin B3 compound, its precursor or analogue thereof for use in treatment of dengue virus.

Yet another aspect of the present invention relates to a pharmaceutical composition comprising (i) LL37 compound or its precursors or analogue thereof; and (ii) vitamin B3 compound or its precursors or analogue thereof for use in the treatment of a viral infection.

Yet another aspect of the present invention relates to a pharmaceutical composition comprising (i) LL37 compound or its precursors or analogue thereof ; and (ii) vitamin B3 compound or its precursors or analogue thereof and for use in the treatment of an infection of the respiratory tract, gastrointestinal tract, oropharyngeal tract, skin, eye, or genitourinary tract.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1: Antiviral activity of 12-hydroxystearic acid (12-HSA) against SARS-CoV-2. (A) Human epidermal keratinocytes (HaCaT cell line) were treated with increasing concentrations of 12-hydroxystearic acid (12-HSA) for 72 hours. The secretome of these treated cells was then incubated with SARS-CoV-2 and the neutralization of the virus was determined by assessing its ability to infect Caco-2 cells by quantifying the expression of a viral gene (E-gene) by qPCR; (B) Effect of 12-HSA (20 pM) on LL37 gene expression in epithelial cell lines HaCaT (human keratinocytes), Calu3 (human lung cells), and Caco2 (human colon cells).

Figure 2: Effect of 12-HAS, vitamin B3 and their combination on epithelial cells preinfected with SARS-CoV-2. (A) Effect of the treatment of Calu 3 cells with 12-hydroxystearic acid (10 pM), Niacinamide (16.4 mM) and their combination on SARS-CoV-2 neutralization, determined by plaque formation assay. (B) Human primary epidermal keratinocytes were treated with 12-hydroxystearic acid (20 pM), Niacinamide (16.4 mM) and their combination for 72 hours. The secretome of the treated epidermal keratinocytes was then incubated with SARS-CoV-2 and the neutralization of the virus was determined by plaque formation assay.

Figure 3: Antiviral activity of LL37 against SARS-CoV-2. (A) Effect of increasing LL37 concentrations on SARS-CoV-2’s ability to infect Caco-2 cells measured by viral gene expression; (B) Effect of LL37 (5 pM) on the ability of SARS-CoV-2 variant’s [B.1.1.7 (alpha), B.1.617.1 (kappa) and B.1.617.2 (delta)] ability to infect Caco-2 cells measured by viral gene expression.

Figure 4: Niacinamide enhances the antiviral activity of LL37. (A) Effect of pre-treatment of SARS-CoV-2 with LL-37 (with or without niacinamide) on the ability of the virus to infect Caco2 cells as measured by detection of the viral E gene by qPCR; (B) Effect of pre-treatment of SARS-CoV-2 variants [B.1.1.7 (alpha), B.1.617.1 (kappa) and B.1.617.2 (delta)] with LL- 37 (with or without niacinamide) on the ability of the virus to infect VeroE6 cells measured by TCID50 assay

Figure 5: Antiviral activity of LL37 and Niacinamide (Vit.B3) against SARS-CoV-2 as measured by plaque forming units assay. Effect of pre-treatment of SARS-CoV-2 with LL- 37 (with or without niacinamide) on the ability of the virus to infect VeroE6 cells as measured by plaque forming units

Figure 6: Membrane disruption assay upon exposure to LL37 and niacinamide. Synthetic vesicles with increasing negative charge (vesicle 1 is low and vesicle 3 is highest negative charge). Vesicle 3 closely mimics the features of the coronavirus family envelope. Vesicles were incubated with either LL37 or LL37 + niacinamide and membrane disruption was measured as a time-dependent percentage of fluorescence recovery (higher percentage of fluorescence recovery = higher membrane disruption).

Figure 7: Effect of 12HSA, niacinamide and their combination on DENV2 infection in A549 host cells.

Figure 8: Cell viability assay with 12HSA, Niacinamide and combination thereof.

DETAILED DESCRIPTION OF THE INVENTION

These and other aspects, features and advantages will become apparent to those of ordinary skill in the art from a reading of the following detailed description and the appended claims. For the avoidance of doubt, any feature of one aspect of the present invention may be utilized in any other aspect of the invention. The word “comprising” is intended to mean “including” but not necessarily “consisting of’ or “composed of.” In other words, the listed steps or options need not be exhaustive. It is noted that the examples given in the description below are intended to clarify the invention and are not intended to limit the invention to those examples per se. Similarly, all percentages are weight/weight percentages unless otherwise indicated. Except in the operating and comparative examples, or where otherwise explicitly indicated, all numbers in this description and claims indicating amounts of material or conditions of reaction, physical properties of materials and/or use are to be understood as modified by the word “about”. Numerical ranges expressed in the format "from x to y" are understood to include x and y. When for a specific feature multiple preferred ranges are described in the format "from x to y", it is understood that all ranges combining the different endpoints are also contemplated. The term “a”, “an” is intended to include plurality of the material and intended to include “one or more”.

The present invention relates to compounds and compositions, useful for treatment of viral infections and dengue.

Accordingly, the present invention provides a pharmaceutical composition comprising (i) a peroxisome proliferator-activated receptor (PPAR) activating fatty acid and/or a cathelicidin (LL37) compound, its precursors or analogue thereof; (ii) a vitamin B3 compound, its precursor or analogue thereof; along with pharmaceutically acceptable additives and carriers.

In an embodiment, the vitamin B3 compound, its precursor or analogue thereof is selected from a group comprising tryptophan, niacin, nicotinic acid, isonicotinamide, picolinamide, niacinamide (nicotinamide), derivatives of nicotinamimide.

In another embodiment, the PPAR activating fatty acid contains a hydroxyl and/or methyl side chain. The hydroxyl containing fatty acids are having 6 to 30 carbon atoms, preferably 8 to 30 carbon atoms, more preferably 14 to 20 carbon atoms.

In an embodiment, the PPAR activating fatty acid is selected from cis-parinaric acid, cis-9- trans-11 conjugated linoleic acid, columbinic acid, docosahexaenoic acid, eicosapentaenoic acid, hexadecatrienoic acid, linolenelaidic acid (isomer of linolenic acid), petroselinic acid, pinolenic acid, punicic acid, ricinoleic acid, ricinolaidic acid (isomer of ricinoleic acid), stearidonic acid, trans- 10-cis- 12 conjugated linoleic acid, 7-trans octadecanoic acid, vaccenic acid, octadecene dioic acid, hydroxystearic acid. In yet another embodiment, the PPAR activating fatty acid is selected from 2 -hydroxymyristic acid, 3-hydroxymyristic acid, 2-hydroxypalmitic acid, 3-hydroxypalmitic acid, 10- hydroxystearic acid (10-HSA), 12-hydroxystearic acid (12-HSA), 17-hydroxy stearic acid, trihydoxystearic acid or trihydroxy stearin or compounds that yield one or more molecules of hydroxystearic acid or hydroxy stearate on their breakdown like mono, di or tri ester of glycerol with hydroxy stearic acid.

In a preferred embodiment, the PPAR activating fatty acid is 12-hydroxystearic acid (12-HSA).

In a preferred embodiment, the PPAR activating fatty acid is a hydrolysable PPAR precursors.

In an embodiment, the pharmaceutically acceptable additives of the composition are preservatives, humectants, surfactants, water.

In an embodiment, he the pharmaceutical composition of tinvention comprises any pharmaceutically acceptable excipient that is required to prepare the desired formulation and dosage form.

In another embodiment, the composition comprises 0 to 5.0 wt% a vitamin B3 compound or its precursors or analogue thereof.

In yet another embodiment, the composition comprises 0.01 to 5.0 wt% a PPAR activating fatty acid.

In yet another preferred embodiment, the composition comprises 0.0001 to 2 wt% LL37.

In an embodiment, the invention comprises

(i) 0.01 to 5.0 wt% a peroxisome proliferator-activated receptor (PPAR) activating fatty acid;

(ii) 0.0001 to 2 wt% LL37, its precursors or analogue thereof;

(iii) 0.01 to 5.0 wt% vitamin B3 compound, its precursor or analogue thereof; and

(iv) pharmaceutically acceptable additives and carriers. In a preferred embodiment, the pharmaceutical composition comprises:

(i) 0.01 to 5.0 wt% a peroxisome proliferator- activated receptor (PPAR) activating fatty acid;

(ii) 0.01 to 5.0 wt% vitamin B3 compound, its precursor or analogue thereof,

(iii) 0.001 to 5 wt% a preservative; and

(iv) 70 to 99.9 wt% water.

In yet another preferred embodiment, the pharmaceutical composition comprises:

(i) 0.01 to 5.0 wt% a PPAR activating fatty acid;

(ii) 0 to 5.0 wt% a vitamin B3 compound or its precursors or analogue thereof;

(iii) 70 to 99 wt% water;

(iv) 1 to 20 wt% humectant; and

(v) less than 1.5 wt% surfactant chosen from non-ionic or amphoteric surfactant or mixtures thereof.

The present invention also relates to a pharmaceutical composition comprising (i) a PPAR activating fatty acid or a cathelicidin (LL37) compound, its precursors or analogue thereof; and (ii) a vitamin B3 compound, its precursor or analogue thereof; along with pharmaceutically acceptable additives and carriers for use in the treatment of a viral infection.

In an embodiment, the present invention also provides a pharmaceutical composition comprising (i) a peroxisome proliferator-activated receptor (PPAR) activating fatty acid and/or a cathelicidin (LL37) compound, its precursors or analogue thereof; (ii) a vitamin B3 compound, its precursor or analogue thereof; along with pharmaceutically acceptable additives and carriers for use in the treatment of a viral infection.

The composition of the invention can be formulated in any form depending upon the requirement. In an embodiment, the composition is in the form selected from a group comprising tablets, capsules, oral preparations, powders, granules, pills, injectable or infusible liquid solutions, spray, drops, suspensions, emulsions, suppositories, ointments, creams, lotions, gels, pastes and transdermal delivery devices. In another embodiment, the invention provides a PPAR activating fatty acid for use in the treatment of a viral infection.

The present invention further relates to a vitamin B3 compound, its precursors or analogue thereof for use in the treatment of a viral infection.

In yet another embodiment, the invention provides a cathelicidin (LL37) compound, its precursors or analogue thereof for use in the treatment of a viral infection.

LL37 is a cationic, amphiphilic, antimicrobial peptide, composed of 37 amino acids, with the sequence described by Tossi and collaborators (Zelezetsky et al., 2006). It is also known as cathelicidin peptide derived from human, and it is known to be an antimicrobial peptide. LL37 peptide can be produced by a method for synthesizing peptide that is well known in the pertinent art, and the production method is not particularly limited. As for the method of synthesis, it is preferably carried out according to a method for chemical synthesis of a peptide which is commonly employed in the pertinent art. More preferably, synthesis is carried out by a solution phase peptide synthesis, a solid-phase peptide synthesis, a fragment condensation method, or F-moc or T-BOC chemical method. Most preferably, synthesis is carried out by a solution phase peptide synthesis (Merrifield, 1964), but it is not limited thereto.

The inventors have found that naturally occurring LL37 has the potential to neutralise the SARS-CoV-2 viral infection by targeting its outer lipid coating and that vitamin B3 further enhances this antiviral activity of the peptide. Therefore, either exogenous administration of the AMP with niacinamide or other strategies to boost the endogenous production of the peptide (e.g. a PPAR activating fatty acid) in combination with niacinamide is a potent method to not only block viral transmission, but also an effective therapy to limit viral load and disease severity of a patient post infection.

Thus, in an embodiment, the present invention provides a pharmaceutical product or composition for boosting the synthesis, secretion and activity of Antimicrobial Peptides (AMPs), in particular LL37, in the epithelial cells.

In another embodiment, the present invention provides a pharmaceutical product or composition for inactivation or killing of enveloped viruses that infect epithelial cells. In yet another embodiment, the present invention provides a pharmaceutical product or composition for boosting Antimicrobial Peptides (AMPs) in the epithelial cells thereby inactivating or killing enveloped viruses.

In yet another embodiment, the present invention provides a pharmaceutical product or composition for use in the treatment of infections of epithelial tissues including, without limitation, respiratory tract, gastrointestinal tract, oropharyngeal tract, skin, eye, and genitourinary tract.

In an embodiment, the invention provides a method of treatment of viral infections.

In a preferred embodiment, the invention provides a method of treatment of viral infection caused by coronavirus or influenza virus or dengue virus.

In yet another preferred embodiment, the invention provides method of treatment of viral infections, preferably an infection of the respiratory tract, gastrointestinal tract, oropharyngeal tract, skin, eye, or genitourinary tract. The viral infection is preferably a respiratory tract infection. The viral infection is preferably caused by a coronavirus or influenza virus. The viral infection is preferably caused by SARS-CoV-2 or HINT.

In an embodiment, the method of treatment comprises administering to a subject in need thereof a composition comprising a PPAR activating fatty acid, a vitamin B3 compound or its precursors or analogue thereof along with pharmaceutically acceptable additives and carrier.

In another embodiment, the method of treatment comprises administering to a subject in need thereof a composition comprising a LL37 compound, its precursors or analogue thereof; a vitamin B3 compound or its precursors or analogue thereof; along with pharmaceutically acceptable additives and carrier.

In another embodiment, the method of treatment comprises administering to a subject in need thereof a composition comprising a a PPAR activating fatty acid and LL37 compound, its precursors or analogue thereof; a vitamin B3 compound or its precursors or analogue thereof; along with pharmaceutically acceptable additives and carrier. The dosage of the treatment regime is dependent upon various factors such as the severity of the infection, age and wight of the subject etc.

In an embodiment, the invention provides use of the composition comprising a PPAR activating fatty acid and/or a LL37 compound, its precursors or analogue thereof; a vitamin B3 compound or its precursors or analogue thereof; along with pharmaceutically acceptable additives and carrier for treating viral infections.

In yet another embodiment, the invention provides use of a PPAR activating fatty acid for treating viral infections.

In yet another embodiment, the invention provides use of LL37 compound, its precursors or analogue thereof for treating viral infections.

In an embodiment, the compound and compositions of the invention are specifically capable of treating viral infection caused by a coronavirus or an influenza virus or dengue virus.

In another embodiment, the compound and compositions of the invention are also capable of treating infection caused by a dengue virus.

In an embodiment, the viral infections treatable by the compound or composition of the present invention is preferably an infection of the respiratory tract, gastrointestinal tract, oropharyngeal tract, skin, eye, or genitourinary tract. The viral infection is preferably a respiratory tract infection. The viral infection is preferably caused by a coronavirus or influenza virus. The viral infection is preferably caused by SARS-CoV-2 or HINT.

In another embodiment, the compound and compositions of the present invention is used to inactivate or kill virus especially enveloped virus in the respiratory tract of a human body or animal body.

The present invention encompasses in its scope the compound and compositions for preventing reinfection. The compound and composition of the present invention are capable of preventing reinfection through use of one or more of the actives claimed in the present invention. By this is meant that such actives will prevent respiratory infections or infections caused by other viruses such as dengue virus in the future for several hours after such treatment.

Viral infections as per the present invention includes infections caused by a coronavirus or an influenza virus. Preferred coronavirus which may be treated as per the present invention is SARS-CoV-2. Preferred influenza virus which may be treated as per the present invention is H1N1. Other enveloped virus which may be treated as per the present invention include RSV (respiratory syncytial virus) and HSV (herpes simplex virus).

The inventors have found that a treatment based on combination of PPAR activated fatty acids and vitamin B3 compounds, precursors or analogues thereof can inhibit the widespread tropical viral infection - Dengue.

Thus, in a specific embodiment, the invention encompasses in its scope a composition comprising (i) PPAR activated fatty acids; (ii) vitamin B3 compounds, precursors or analogues thereof; (iii) pharmaceutically acceptable additives and carriers, wherein said composition is useful for treating Dengue viral infection.

In a specific embodiment, the invention relates to a composition comprising (i) 12HSA; and (ii) Niacinamide. Said composition is useful for treating Dengue viral infection.

In another embodiment, the invention provides a combination of PPAR activated fatty acids such as 12HSA and vitamin B3 compound such as Nicinamide which induces a class of antiviral peptides (A VPs) that can act on variety of enveloped viruses.

In a preferred aspect, combination of PPAR activated fatty acids such as 12HSA and vitamin B3 compound such as Nicinamide inhibits DENV2 strain infection.

In an embodiment, the present invention provides a pharmaceutical product which is able to boost AMP generation and secretion from epithelial cells and thereby protecting against viral infection. This effect is synergistically enhanced in presence of a combination of a vitamin B3 compound or its precursors or analogues thereof and a PPAR activating fatty acid. Furthermore, the antimicrobial effect of LL37 is also synergistically enhanced by the combination with vitamin B3 compound or its precursors or analogues thereof. In an embodiment, the invention provides a method of boosting AMP production.

In yet another embodiment, the invention provides a method of boosting AMP production using a composition comprising a PPAR activated fatty acid, vitamin B3 compounds or analogues or precursors thereof along with pharmaceutically acceptable additives and carrier.

In yet another embodiment, the invention provides a method of boosting AMP production in a subject in need thereof, said method comprising administering to the subject a composition comprising a PPAR activated fatty acid, vitamin B3 compounds or analogues or precursors thereof along with pharmaceutically acceptable additives and carrier.

One of the aspect of the invention is a PPAR activated fatty acid for use in treatment of viral diseases. Peroxisome proliferator-activated receptors (abbreviated herein to PPAR) are transcription factors that control lipid metabolism. There are three isotypes - PPARa, PPARp/6 and PPARy all of which have been localised in the skin and in the epithelial cells of the lungs (Belvisi and Mitchell, 2009). A range of specific fatty acids activates these factors resulting in anti-inflammatory action to reduce irritation responses and pro-differentiation/antiproliferation responses. PPAR activating fatty acid as per this invention includes fatty acids which have a PPAR activating action and also includes their corresponding mono, di and triglyceride forms.

It is particularly desirable to select PPAR activating fatty acids containing a hydroxyl and/or methyl side chain. Many such acids contain 14 to 30 carbons.

Hydroxy fatty acids are derivatives of fatty acids containing a hydroxyl group at one or more positions. Preferably, the fatty acids are saturated or unsaturated and branched or unbranched. The hydroxy fatty acids used in the present invention having 6 to 30 carbon atoms, preferably 8 to 30 carbon atoms, more preferably 14 to 20 carbon atoms. Hydroxy fatty acids as per this invention includes esters or precursors thereof. Esters may be of the C1-C6 alkyl esters. Precursors may be trihydroxy stearin or compounds that yield one or more molecules of hydroxystearic acid or hydroxy stearate on their breakdown like mono, di or tri ester of glycerol with hydroxy stearic acid.

Preferably, the hydroxy fatty acid is selected from 2-hydroxymyristic acid, 3-hydroxymyristic acid, 2-hydroxypalmitic acid, 3-hydroxypalmitic acid, 10- hydroxy stearic acid, 12- hydroxystearic acid, 17-hydroxystearic acid, trihy doxy stearic acid (e.g. 9,10,13- trihydroxy stearic acid) or trihydroxy stearin or compounds that yield one or more molecules of hydroxystearic acid or hydroxy stearate on their breakdown like mono, di or tri ester of glycerol with hydroxy stearic acid.

Preferably the hydroxy fatty acid is hydroxy stearic acid. The most preferred hydroxystearic acids are 10-hydroxystearic acid (10-HSA) and 12-hydroxystearic acid (12-HSA).

Examples of PPAR activating fatty acids with demonstrated PPAR activating activity are, but not limited to, cis-parinaric acid, cis-9-trans-l l conjugated linoleic acid, columbinic acid, docosahexaenoic acid, eicosapentaenoic acid, hexadecatrienoic acid, linolenelaidic acid (isomer of linolenic acid), petroselinic acid, pinolenic acid, punicic acid, ricinoleic acid, ricinolaidic acid (isomer of ricinoleic acid), stearidonic acid, trans- 10-cis- 12 conjugated linoleic acid, 7-trans octadecanoic acid, vaccenic acid, octadecene dioic acid and hydroxystearic acid.

A PPAR activating fatty acid as per this invention also includes hydrolysable PPAR precursors. Potential source of hydroly sable PPAR precursors include, but not limited to, triglycerides such as coriander seed oil for petroselinic acid, impatiens balsimina seed oil, Parinarium laurinarium kernel fat or Sabastiana brasilinensis seed oil for cis-parinaric acid, dehydrated castor seed oil for conjugated linoleic acids, and aquilegia vulgaris oil for columbinic acid. If a single hydrolysable precursor of a PPAR activating fatty acid is employed, it specifically excludes borage oil, castor oil and sunflower seed oil. Desirably, the PPAR activating fatty acid contains 16 or 18 carbon atoms.

An especially preferred PPAR activating fatty acid is hydroxystearic acids or esters thereof. Most preferred PPAR activating fatty acid is hydroxy stearic acid.

It is preferred that the hydroxy stearic acid is 10-hydroxystearic acid, 12-hydroxystearic acid or trihydoxystearic acid (e.g. 9,10,13-trihydroxystearic acid) or trihydroxy stearin or compounds that yield one or more molecules of hydroxy stearic acid or hydroxy stearate on their breakdown like mono, di or tri ester of glycerol with hydroxystearic acid. Of these, 10-hydroxystearic acid, 12-hydroxystearic acid and 9,10,13-trihydroxystearic acid are more preferred, 12- hydroxystearic acid (12-HSA) being most preferred. 12-HSA has the structure as given below:

It is preferred that the PPAR-activating fatty acid for use in any aspect of the present invention is also a Caspase 8 regulator.

A preferred aspect of the present invention relates to a pharmaceutical composition comprising (i) a peroxisome proliferator-activated receptor (PPAR) activating fatty acid and (ii) a vitamin B3 compound, its precursor or analogue thereof.

A preferred aspect of the present invention relates to a pharmaceutical composition comprising (i) a PPAR activating fatty acid and (ii) a vitamin B3 compound, its precursor or analogue thereof for use in the treatment of a viral infection.

A preferred aspect of the present invention relates to a pharmaceutical composition comprising (i) a PPAR activating fatty acid and (ii) a vitamin B3 compound, its precursor or analogue thereof for use in the treatment of a viral infection caused by coronavirus or influenza virus or dengue virus.

A preferred aspect of the present invention relates to a pharmaceutical composition comprising (i) a PPAR activating fatty acid and (ii) a vitamin B3 compound, its precursor or analogue thereof for use in the treatment of a viral infection caused by SARS-CoV-2 or H1N1 influenza virus or DENV2 dengue virus.

The vitamin B3 compound, its precursor or analogue thereof is selected from a group comprising tryptophan, niacin, nicotinic acid, isonicotinamide, picolinamide, and niacinamide (which is also known as nicotinamide) or combinations thereof. In one embodiment, vitamin B3 is preferably niacinamide. Niacinamide also known as pyridine-3-carboxamide is the active, water soluble form of vitamin B3. Analogues of vitamin B3, as per this invention, also includes derivatives like cyclo alkyl nicotinamide with the cyclo alkyl group having 3 to 6 carbon atoms.

Vitamin B3 is essential to the coenzymes NADH and NADPH and therefore for over 200 enzymatic reactions in the body including ATP formation. Without wishing to be bound by theory, the inventors believe that the actives claimed in the present invention viz. Vitamin B3, its precursors and analogues thereof and the PPAR activating fatty acid e.g. 12-HSA activates host cells to help drive the defense against viruses like SARS-Cov2. The inventors believe that this mode of action is different from direct antivirals like low boiling alcohols, bleaches and cationic surfactants like quaternary ammonium compounds which act directly on the virus particle to inactivate them thereby delivering the anti-viral benefit.

Yet another aspect of the present invention relates to a PPAR activating fatty acid for use in the treatment of a viral infection caused by coronavirus or influenza virus or dengue virus.

Yet another aspect of the present invention relates to a PPAR activating fatty acid for use in the treatment of a viral infection caused by SARS-CoV-2 or H1N1 influenza virus or DENV2 dengue virus.

Yet another aspect of the present invention relates to a pharmaceutical composition comprising (i) a PPAR activating fatty acid and (ii) a pharmaceutically acceptable carrier.

Yet another aspect of the present invention relates to a pharmaceutical composition comprising (i) a PPAR activating fatty acid and (ii) a pharmaceutically acceptable carrier for use in the treatment of a viral infection.

Yet another aspect of the present invention relates to a pharmaceutical composition comprising (i) a PPAR activating fatty acid and (ii) a pharmaceutically acceptable carrier and additives.

Yet another aspect of the present invention relates to a composition comprising (i) a PPAR activating fatty acid and (ii) a pharmaceutically acceptable carrier and additives, for use in the treatment of an infection of the respiratory tract, gastrointestinal tract, oropharyngeal tract, skin, eye, or genitourinary tract.

Yet another aspect of the present invention relates to a pharmaceutical composition comprising (i) a PPAR activating fatty acid, (ii) a vitamin B3 compound, its precursors or analogue thereof and (iii) a pharmaceutically acceptable carrier. Yet another aspect of the present invention relates to a pharmaceutical composition comprising (i) a PPAR activating fatty acid, (ii) a vitamin B3 compound, its precursors or analogue thereof and (iii) a pharmaceutically acceptable carrier for use in the treatment of viral infections.

Yet another aspect of the present invention relates to a pharmaceutical composition comprising (i) a PPAR activating fatty acid, (ii) a vitamin B3 compound, its precursors or analogue thereof and (iii) a pharmaceutically acceptable carrier, for use in the treatment of viral infections.

Preferably, the pharmaceutically acceptable carrier in above composition comprises solvents. Examples of solvents in the composition include, but not limiting to, water, ethyl alcohol, isopropanol, acetone, ethylene glycol mono ethyl ether, diethylene glycol mono butyl ether, diethylene glycol mono ethyl ether and mixtures thereof. The composition may comprise polyhydric alcohols selected from, but not limiting to, glycerine, 1,3 -butylene glycol, propylene glycol, 1,3 -propanediol, pentylene glycol, hexylene glycol, and sorbitol.

Preferably, the pharmaceutically acceptable carrier in the above composition comprises powders. Examples of powders that may be used in the composition include chalk, talc, fullers earth, kaolin, starch, gums, colloidal silica sodium poly aery late, tetra alkyl and/or trialkyl aryl ammonium smectites, chemically modified magnesium aluminium silicate, organically modified montmorillonite clay, hydrated aluminium silicate, fumed silica, carboxyvinyl polymer, sodium carboxymethyl cellulose, ethylene glycol monostearate and mixtures thereof.

By way of non-limiting example, said pharmaceutically acceptable carriers may comprise binders, diluents, lubricants, glidants, disintegrants, solubilizing (wetting) agents, stabilizers, colorants, anti-caking agents, emulsifiers, thickeners and gelling agents, coating agents, humectants, sequestrants, and sweeteners.

The pharmaceutical compositions can be chosen on the basis of the treatment requirements. Such compositions are prepared by blending and are suitably adapted to oral or parenteral administration, and as such can be in the form of, but not limiting to, tablets, capsules, oral preparations, powders, granules, pills, injectable or infusible liquid solutions, suspensions, emulsions, suppositories, ointments, creams, lotions, gels, pastes and transdermal delivery devices. Tablets and capsules for oral administration are normally presented in unit dose form and contain conventional excipients such as, but not limiting to, binders, fillers (including cellulose, mannitol, lactose), diluents, tableting agents, lubricants (including magnesium stearate), detergents, disintegrants (e.g. polyvinylpyrrolidone and starch derivatives such as sodium glycolate starch), coloring agents, flavoring agents, and wetting agents (for example sodium lauryl sulfate).

The oral solid compositions can be prepared by conventional methods of blending, filling or tableting. The blending operation can be repeated to distribute the active principle throughout compositions containing large quantities of fillers. Such operations are conventional.

Oral liquid preparations can be in the form of, for example, but not limiting to, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or can be presented as a dry product for reconstitution with water or with a suitable vehicle before use. Such liquid preparations can contain conventional additives such as, but not limiting to, suspending agents, for example sorbitol, syrup, methyl cellulose, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, aluminium stearate gel, or hydrogenated edible fats; emulsifying agents, such as lecithin, sorbitan monooleate, or acacia; non-aqueous vehicles (which can include edible oils), such as almond oil, fractionated coconut oil, oily esters such as esters of glycerine, propylene glycol, or ethyl alcohol; preservatives, such as methyl or propyl p-hydroxybenzoate or sorbic acid, and if desired, conventional flavoring or coloring agents. Oral formulations also include conventional slow-release formulations such as enterically coated tablets or granules.

Pharmaceutical preparation for administration by inhalation can be delivered from an insufflator or a nebulizer pressurized pack.

For parenteral administration, fluid unit dosages can be prepared, containing the compound and a sterile vehicle. The compound can be either suspended or dissolved, depending on the vehicle and concentration required to be administered. The parenteral solutions are normally prepared by dissolving the compound in a vehicle, sterilising by filtration, filling suitable vials and sealing. Advantageously, adjuvants such as local anaesthetics, preservatives and buffering agents can also be dissolved in the vehicle. To increase the stability, the composition can be frozen after having filled the vials and removed the water under vacuum. Parenteral suspensions are prepared in substantially the same manner, except that the compound can be suspended in the vehicle instead of being dissolved, and sterilized by exposure to ethylene oxide before suspending in the sterile vehicle. Advantageously, a surfactant or wetting agent can be included in the composition to facilitate uniform distribution of the compound of the invention.

For buccal or sublingual administration, the compositions may be tablets, lozenges, pastilles, or gel.

The compounds can be pharmaceutically formulated as suppositories or retention enemas, e.g. containing conventional suppositories bases such as cocoa butter, polyethylene glycol, or other glycerides, for a rectal administration.

Another means of administering the compounds of the invention regards topical treatment. Topical formulations can contain for example, but not limiting to, ointments, creams, lotions, gels, solutions, pastes and/or can contain liposomes, micelles and/or microspheres. Examples of ointments include oleaginous ointments such as vegetable oils, animal fats, semisolid hydrocarbons, emulsifiable ointments such as hydroxy stearin sulfate, anhydrous lanolin, hydrophilic petrolatum, cetyl alcohol, glycerol monostearate, stearic acid, water soluble ointments containing polyethylene glycols of various molecular weights. Creams, as known to formulation experts, are viscous liquids or semisolid emulsions, and contain an oil phase, an emulsifier and an aqueous phase. The oil phase generally contains petrolatum and an alcohol such as cetyl or stearic alcohol. Formulations suitable for topical administration to the eye also include eye drops, wherein the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent for the active ingredient.

A further method of administering the compounds of the invention regards transdermal delivery. Typical transdermal formulations comprise conventional aqueous and non-aqueous vectors, such as creams, oils, lotions or pastes or can be in the form of membranes or medicated patches.

A reference for the formulations is the book by Remington (“Remington: The Science and Practice of Pharmacy”, Lippincott Williams & Wilkins, 2000). The compounds of the invention may be presented in a liposome or other micro particulate or other nanoparticle designed to target the compound. Acceptable liposomes can be neutral, negatively, or positively charged, the charge being a function of the charge of the liposome components and pH of the liposome solution. Liposomes can be normally prepared using a mixture of phospholipids and cholesterol. Suitable phospholipids include, but not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid, phosphotidylglycerol, phosphatidylinositol. Polyethylene glycol can be added to improve the blood circulation time of liposomes. Acceptable nanoparticles include albumin nanoparticles and gold nanoparticles.

According to a preferred aspect, the present invention relates to an inhalation composition for inactivating enveloped virus comprising

(i) 0 to 5.0 wt% a vitamin B3 compound or its precursors or analogue thereof;

(ii) 0.01 to 5.0 wt% a PPAR activating fatty acid;

(iii) 70 to 99 wt% water;

(iv) 1 to 20 wt% humectant; and

(v) less than 1.5 wt% surfactant chosen from non-ionic or amphoteric surfactant or mixtures thereof.

According to a preferred aspect, the present invention provides a nasal drop composition comprising

(i) 0.01 to 5.0 wt% vitamin B3 compound, its precursor or analogue thereof,

(ii) 0.01 to 5.0 wt% a peroxisome proliferator-activated receptor (PPAR) activating fatty acid;

(iii) 0.001 to 5 wt% a preservative; and

(iv) 70 to 99.9 wt% water.

Nasal drop compositions of the present invention are generally applied on to the nasal cavity using a nasal spray device. Thus, the present invention also provides for a nasal spray device comprising a spray pump capable of spraying the nasal drops composition of the invention from a container containing a nasal drops composition. The preservative is preferably benzalkonium chloride (BKC) or benzathonium chloride (BZC), preferably BKC. The nasal drops composition of the invention may additionally comprise 0.01 to 10% surfactant which is preferably a non-ionic surfactant. Non-ionic surfactants as detailed hereinabove may be used. Yet another aspect of the present invention relates to a pharmaceutical composition comprising (i) a LL37 compound, its precursors or analogue thereof and (ii) a vitamin B3 compound, its precursors or analogue thereof.

Yet another aspect of the present invention relates to a pharmaceutical composition comprising (i) a LL37 compound, its precursors or analogue thereof and (ii) a vitamin B3 compound, its precursors or analogue thereof for use in the treatment of viral infections.

Yet another aspect of the present invention relates to a pharmaceutical composition comprising (i) a LL37 compound, its precursors or analogue thereof and (ii) a vitamin B3 compound, its precursors or analogue thereof for use in the treatment of an infection of the respiratory tract, gastrointestinal tract, oropharyngeal tract, skin, eye, or genitourinary tract.

Yet another aspect of the present invention relates to a pharmaceutical composition comprising (i) a LL37 compound, its precursors or analogue thereof, (ii) a vitamin B3 compound, its precursors or analogue thereof and (iii) a pharmaceutically acceptable carrier.

Yet another aspect of the present invention relates to a pharmaceutical composition comprising (i) a LL37 compound, its precursors or analogue thereof, (ii) a vitamin B3 compound, its precursors or analogue thereof and (iii) a pharmaceutically acceptable carrier and additives, for use in the treatment of viral infections.

Yet another aspect of the present invention relates to a pharmaceutical composition comprising (i) a LL37 compound, its precursors or analogue thereof, (ii) a vitamin B3 compound, its precursors or analogue thereof and (iii) a pharmaceutically acceptable carrier and additives, for use in the treatment of an infection of the respiratory tract, gastrointestinal tract, oropharyngeal tract, skin, eye, or genitourinary tract.

The present invention will now be exemplified by way of the following non-limiting examples.

EXAMPLES

Example 1: Effect of 12-hydroxystearic acid (12-HSA) on virus kill on skin keratinocyte cell line HaCaT Skin keratinocyte cell line HaCaT were grown in 24 well plate at 100% confluency (approx. 2xl0 ⁵ cells) and then differentiated with 2.8 mM Ca ²⁺ . These monolayers of cells were treated with 10 pM, 20 pM and 40 pM of 12-hydroxystearic acid (12-HSA) and treatment was done for 72 hours, following standard protocols. Post this treatment the supernatant that contains the secreted anti-microbial peptides (AMPs) was collected and was incubated with SARS- CoV-2 virus for 4 hours. After the 4 hours of incubation, the virus kill was enumerated by using quantitative PCR based methods. The data on the viral gene expression as a percentage of control is shown in Figure 1 and in Table 1 below:

Table 1. Effect of the secretome of skin keratinocyte cell line HaCaT cell lines differentiated and treated with increasing 12-hydroxystearic acid (12-HSA) concentrations on SARS-CoV-2 neutralization

Sample

Control .

The data in the Figure 1 and Table 1 above indicates that the secretome generated in the presence of 12HSA is capable of killing/inactivating the virus.

Example 2: Effect of 12-hydroxystearic acid (12-HSA) and Niacinamide (Vit. B3) and their combination on lung epithelial cells (Calu-3 cells):

Treatments of compounds and plaque formation assay.

0.1 MOI SARS-CoV-2 was allowed to adsorb on Calu-3 cells, plated in a 48 well plate with about 120,000 cells/well, for one hour. After washing, fresh media was added along with niacinamide (16.4 mM), 12-hydroxystearic acid (10 pM), their combination and their respective vehicle controls. After 48 hours, the conditioned media (containing the virus particles) were collected and plaque forming unit (pfu/ml) was determined for each treatment by performing plaque formation assay on Vero-E6 cells (48 well plate).

The data is summarized in Figure 2 A and Table 2 below:

Table 2. Effect of 12-hydroxystearic acid (10 pM), Niacinamide (16.4 mM) and their combination on SARS-CoV-2 neutralization

The data in Figure 2A and Table 2 indicates that 12-HSA and Niacinamide are both capable of inhibiting the SARS-CoV-2 as evident from the reduced plaque forming units in the assay used above. The inhibition is significantly improved with a combination of 12-HSA and Niacinamide.

Example 3: Effect of human keratinocytes supernatant on SARS-CoV2 infectivity:

Human primary skin keratinocyte were grown in 24 well plate at 100% confluency (approx. 2xl0 ⁵ cells) and then differentiated with 1.5 mM Ca ²⁺ . Differentiated primary human keratinocytes were incubated in serum free Epilife media with Niacinamide (Vit. B3), 12- hydroxystearic acid (12-HSA) and their combination at the concentrations mentioned in Table

3 for 72 hours and conditioned media was harvested. The SARS-Cov-2 virus particles were incubated for about 4 hours with the above-mentioned conditioned media. The pfu/ml of these treated virus particles was determined using VeroE6 reporter cells through Plaque or TCID50. The % infection with respect to the control is shown in Figure 2B and Table 3 below: Table 3. Effect of 12-hydroxystearic acid (20 pM), Niacinamide (16.4 mM) and their combination on SARS-CoV-2 neutralization The data in Figure 2B and Table 3 indicates that effect similar to that in Figure 2 A and Table 2 could be obtained with primary human keratinocytes also.

Example 4: Effect of 12-hydroxystearic acid (12-HSA) on the LL37 gene expression of epithelial cell lines HaCaT, Calu3, Caco2

Epithelial cell lines originating from skin (HaCaT), lung (Calu3), and colon (CaCo2) were utilized to see the effect of 12-HSA treatment on their LL37 gene expression levels. The cells were grown in 24 well plate at 100% confluency (approx. 2xl0 ⁵ cells) and then taken forward for the treatments.

The cells were treated with 20 pM 12-HSA for 48 hours. The cells were harvested and RNA was utilized to quantify the LL37 gene expression through qPCR method. The data on the gene expression as a fold change to respective control is shown in Figure IB and Table 4:

Table 4. Effect of 12-HSA (20 pM) on LL37 gene expression in epithelial cell lines HaCaT, Calu3, and Caco2

The data in Figure IB and Table 4 indicates that 12HSA treatment induces LL37 gene expression in epithelial cell lines HaCaT, Calu3, and Caco2.

Example 5: Effect of LL37 on the infectivity of SARS-CoV-2

To ascertain whether LL37 has any effect on SARS-CoV-2, the inventors incubated the virus particles (0.01-0.5 MOI) with 0 to 15 pM of LL37 for 1 hour at 37° C and then assessed virus capability to infect CaCo2 cells by incubating cells together with virus for 1 hour. The media was replaced with fresh media and cells were kept for another 24 hours incubation period. The RNA was isolated, and the viral infectivity was measured by performing qPCR to detect the relative viral gene expression in CaCo2 cells. The inventors observed a dose-dependent decrease in viral gene expression (Fig. 3A). In a similar experiment, treatment of multiple SARS-CoV-2 strains (alpha, kappa, delta with 5 pM LL37 likewise showed a decrease in the viral gene expression in CaCo2 cells (Fig. 3B).

Example 6: Effect of LL37 and its combination with Niacinamide (Vit. B3) on the infectivity of SARS-CoV-2

The SARS-CoV-2 particles were incubated at 37° C for 1 hour with indicated concentrations and combinations of LL37 and niacinamide (Fig. 4A).The virus particles (0.01-0.5 MOI) were then allowed to get adsorbed on Caco2 cells for 1 hour. The media was replaced with fresh media and cells were kept for another 24 hours incubation period. The RNA was isolated, and the viral infectivity was measured by performing qPCR to detect the relative viral gene expression in CaCo2 cells (Fig. 4A).

In a similar experiment, the SARS-CoV-2 particles were incubated at 37° C for 1 hour with indicated concentrations and combinations of LL37 and niacinamide. The virus particles were then allowed to get adsorbed on VeroE6 reporter cell line for 1 hour to determine pfu/ml as per the standard plaque forming assay protocol. The effect of such combinations on the viral pfu/ml is shown in Figure 5 and Table 5:

Table 5. Effect of LL37, Niacinamide and their combination on SARS-CoV-2 neutralization

Example 7: Effect of LL37 and its combination with Niacinamide on the infectivity of multiple variants of SARS-CoV-2

To ascertain the effect of LL37 and its combination with niacinamide (B3) the inventors incubated alpha, kappa, and delta strains of SARS-CoV-2 with 5 pM LL37, or 16.4mM B3, or 5 pM LL37 + 16.4mM B3. The virus particles were incubated with this compounds at 37° C for 1 hour and then added on VeroE6 reporter cell line to check for virus-induced cytopathy. Viral neutralisation with LL37 and LL37+B3 was enumerated using TCID50 method. The results are described in Figure 4. The inventors observed that LL37 supplemented with niacinamide exhibited an enhanced potency against multiple variants of SARS-CoV-2 (Fig. 4B).

Example 8: Effect of LL37 and its combination with Niacinamide on the corona virus mimicking membrane

The virus like particles that mimic surface membrane of the corona virus family were generated using various combinations of synthetic lipids l,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), l,2-dioleoyl-sn-glycero-3- phospho-L-serine (DOPS), and fluorescently labelled FRET pair lipids. The resulting negative charge on the vesicles are as given here: Vesicle 1 = 0.3 mV, Vesicle 2 = -18.9 mV, Vesicle 3 = -36.4mV. The membrane disruption of these vesicle by LL37 would result in fluorescence recovery. As shown in Figure 6, incubation of these vesicles with LL37 show time dependent membrane disruption, where higher negative charge leads to more disruptions. Also, addition of niacinamide with LL37 leads to higher membrane disruption for Vesicle 3, which has highest negative membrane charge. This suggests that addition of niacinamide to LL37 can enhance its virucidal effect, particularly for the virus carrying negative membrane charge.

Example 9: Effect of 12HSA, niacinamide and their combination on DENV2 strain of dengue virus

• 24 well plate of A549 cells were prepared with 10 ⁵ cells per well and kept at 37° C, 5% CO ₂ for 24 hr.

• 1ml culture media having following chemicals were prepared:

1. Control (no treatment), 2. Niacinamide (2mg/ml), 3. 12HSA (20mM), 4. 12HSA (20mM) + Niacinamide (2mg/ml) [Note: The concentrations were decided based on the toxicity assay and were found to be safe for in-vitro assay.]

• In each tube, known amount of DENV2 virus was added [Note: The added amount of DENV2 particles was decided so that it gives the Ct value of ~20 in the control RT- qPCR reaction.]

• The media with chemicals and DENV2 mixture was added on the A549 cells and incubated for 3 hr.

• After 3 hr adsorption, media were removed from all the wells and fresh media with same chemicals, except DENV2, were added in the respective wells. • After 48 hr, RNA was isolated from A549 cells and DENV2 quantity was measured through RT-qPCR.

The results of the experiment are provided in Figure 7.

Exmaple 10: Studying cell viability with 12HSA, Niacinamide and combination thereof

The calu3 cells were plated at 20000 cells/well in a 96 well plate. After 48 hr, the cells were treated with varying concentrations of niacinamide, 12HSA, and their combination. After 48 hr, WST-1 based cell viability assay was performed as per the standard protocol and manufacturer’s instruction.

The cell viability assay predicted 50% cell death with 5.6 mg/ml of niacinamide. The utilized 0-80 pm range of 12HSA did not show any significant toxicity. Similarly 0.4 mg/ml of niacinamide in presence or absence of 12HSA found to be safe for in-vitro cell viability. The results of the experiment are provided in Figure 8.

The results achieved from the examples clearly indicate a synergistic effect of PPAR activated fatty acid and vitamin B3 compounds. The results also clearly indicate the synergistic effect of LL37 and vitamin B3 compounds. Thus the compositions of the invention are synergistic.

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