A NASAL DROP COMPOSITION AND DEVICE THEREOF

Field of the invention

5 The present invention relates to use of actives and compositions and devices comprising them for treating a mucosal surface for inactivation or killing enveloped virus.

Background of the invention

Respiratory infections are considered to be one of the most prevalent cause of disease, worldwide. Globally, acute lower respiratory 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 A5 and B, adenoviruses and of recent origin coronavirus. Several tens of millions of 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.

Virus as discussed above, are known to transmit through respiratory aerosol drops or by touching with infected hands and even through inanimate surfaces. As a consequence, hand0 hygiene is a commonly recommended method of killing such germs and thereby reducing the risk of not only respiratory infection but also gastrointestinal diseases. Hand hygiene includes use of soap and running water to wash the hands which makes it not only free of dirt but also in washing away and killing germs including bacteria and virus. Alternately, alcohol-based hand sanitizers which contain very little water may be rubbed on the hands as another effective5 means of removing virus and bacteria from hands. While such methods are very good in instantaneously killing the germs they are sometimes considered harsh. There is thus a need for actives and compositions that are mild on mucosal surfaces but are sufficiently effective in killing or inactivating virus to prevent or mitigate infection.

The present inventors when looking for such actives hit upon specific type of fatty acid which0 are known to be PPAR (peroxisome proliferator-activated receptor) activating fatty acid which they have now found to kill or inactivate enveloped virus, which heretofore was not known before. Further, they found that it synergizes with a specific vitamin viz. Vitamin B3 or its precursors or analogue thereof to deliver enhanced antiviral activity. Further they also found that vitamin B3 or its precursors or analogues thereof in itself can also deliver such activity. PPAR fatty acid as per this invention includes fatty acids which have a PPAR action and also includes their corresponding mono, di and triglyceride forms.

It is thus an object of the present invention to provide for novel actives that kill or inactivate enveloped virus.

It is another object of the present invention to provide for such benefit from using commonly used actives that are mild on mucosal surfaces of the body.

Summary of the Invention

The first aspect of the present invention relates to use of a peroxisome proliferator-activated receptor (PPAR) activating fatty acid for inactivation or kill of enveloped virus on mucosal surface of a human or animal body.

A preferred aspect of the present invention relates to use of a composition comprising (i) a vitamin B3 compound or its precursors or analogue thereof and (ii) a peroxisome proliferator- activated receptor (PPAR) activating fatty acid for inactivation or kill of enveloped virus on mucosal surface of a human or animal body.

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

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

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

(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. .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 present invention may be used to inactivate or kill virus especially enveloped virus on mucosal surface of a human body or animal body. The surface is therefore an animate surface.

Animate surfaces include the external surfaces of living organisms like plants and animals including humans. Due to the rich availability of nutrients and water on most animate surfaces, germs reside and are capable of multiplying on such surfaces. The present invention is used against virus infection on mucosal surfaces. Mucosal surface as per the present invention includes surfaces in the nasal cavity, the throat, and the retronasal cavity.

The present invention may be used through a leave-on composition By a leave-on composition is meant a composition which is applied on the mucosal surface of the human body and allowed to remain thereon

The present invention relates to use of a PPAR activating fatty acid for inactivation or kill of enveloped virus on mucosal surface of a human or animal body. The use is preferably non- therapeutic. The enveloped virus is preferably SARS-Cov2. The present invention is also capable of preventing reinfection of the mucosal surface through use of one or more of the actives claimed in the present invention for inactivation or kill of enveloped virus on these surfaces. By this is meant that surfaces treated with such actives will prevent infection of the surface in the future for several hours after such treatment.

Enveloped virus as per the present invention includes a coronavirus or an influenza virus. Preferred coronavirus which may treated as per the present invention is SARS-Cov2. Preferred influenza virus which may be treated as per the present invention is H1 N1. Other enveloped virus which may be treated as per the present invention include RSV (respiratory syncytial virus) and HSV (herpes simplex virus).

Peroxisome proliferator-activated receptors (abbreviated herein to PPAR) are transcription factors that control lipid metabolism. There are three isotypes PPARa PPARp/8 and PPARYy all of which have been localised in the skin. A range of specific fatty acids activates these factors resulting in anti-inflammatory action to reduce cutaneous irritation responses and pro- differentiation/antiproliferation responses to normalise skin metabolism and provide additional skin-care benefits. It is particularly desirable to select PPAR fatty acids containing a hydroxyl and/or methyl side chain. Many such acids contain from 14 to 30 carbons. Examples of PPAR fatty acids with demonstrated PPAR activating activity are cis-parinaric acid, cis-9-trans-11 conjugated linoleic acid, columbinic acid, docosahexaenoic acid, eicosapentanoic 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 acids.

A PPAR fatty acid as per this invention also includes hydrolysable PPAR precursors. Potential source of hydrolysable PPAR precursors include 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 acid contains 16 or 18 carbon atoms.

An especially preferred PPAR acid is hydroxy stearic acids or esters thereof. Most preferred PPAR acid is hydroxystearic acids. It is preferred that the hydroxystearic acid is 10- hydroxy stearic acid, 12-hydroxystearic acid or trihydoxystearic acid (e.g. 9, 10,13-tri hydroxy stearic acid) or trihydroxy stearin or compounds that yield one or more molecules of hydroxy stearic acid or hydroxystearate 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.

As per a preferred aspect, a composition as per the invention may additionally comprise an antimicrobial peptide (AMP). A further preferred aspect refers to use of a composition as per the invention which additionally comprises an anti-microbial peptide. The preferred AMP is LL37.

A preferred aspect of the present invention relates to use of a composition comprising (i) a vitamin B3 compound, its precursor or analogue thereof and (ii) a peroxisome proliferator- activated receptor (PPAR) activating fatty acid for inactivation or kill of enveloped virus on mucosal surface of a human or animal body. The use is preferably non-therapeutic.

Yet another aspect of the present invention relates to use of a PPAR-activating fatty acid as an enveloped virus killing or inactivating agent in a composition also comprising a vitamin B3 compound, its precursor or analogue thereof.

Yet another aspect relates to use of a combination of a PPAR-activating fatty acid and a vitamin B3 compound, its precursor or analogue thereof in a mucosal composition as an enveloped-virus-killing or inactivating system.

The vitamin B3 compound, its precursor or analogue thereof is selected from one or more of tryptophan, niacin, nicotinic acid, isonicotinamide, picolinamide, and nicotinamide (which is also known as niacinamide). It 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.

When included in the composition of the present invention comprises an effective amount of niacinamide, typically is in a concentration of 0.05 to 3%, preferably in 0.1 to 2% weight of the composition. In such a composition hydroxystearic acid is preferably included in 0.05 to 3%, more preferably 0.1 to 2%, by weight of the composition.

Without wishing to be bound by theory the inventors believe that the actives claimed in the present invention viz. the PPAR fatty acid e.g. 12-HSA activates host cells to help drive the defense benefit against non-enveloped viruses like SARS-Cov2. They believe that this mode of action is quite 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.

It is further understood by way of experiments that actives like the PPAR fatty acids deposit in sufficient quantities on to the topical surface even from a wash off composition. Further the actives are seen to move into the skin to activate the AMPs from the cells. The generation of AMPs is a biological process and this takes a long time. It has been seen that maximum amount of AMPs are generated in the order of 48 to 72 hours after the deposition of the active.

According to yet another aspect of the present invention there is provided use of a composition for care or cleansing of mucosal surfaces comprising (i) PPAR activating fatty acid and (ii) a mucosally acceptable carrier selected from one or more of water, a humectant, and an emulsifier, for inactivation or kill of enveloped virus on mucosal surface of a human or animal body. The use is preferably non-therapeutic. Such a composition may additionally comprise a vitamin B3 compound, its precursor or an analogue thereof.

The present invention also relates to a method of inactivating or killing an enveloped virus on a mucosal surface of a human or animal body comprising the step of exposing the desired surface to a PPAR activating fatty acid; or compositions thereof. The method also includes an aspect wherein the composition additionally comprises a vitamin B3 compound, its precursor or analogue thereof. The method as per the present invention is for cosmetic use i.e. it is for non-therapeutic applications.

According to a preferred aspect of the present invention there is provided a nasal drops composition composition comprising

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

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

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

(iv) 70 to 99.9 wt% water.

Nasal drops composition 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 which may be used are polyethoxylated fatty acid sorbitan esters, ethoxylated fatty acids, esters of polyethylene glycol, ethoxylates of fatty acid monoglycerides and diglycerides, and ethylene oxide/propylene oxide block polymers

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

Examples

Example A, 1-3: Effect of 12-HSA on virus kill on skin keratinocyte cell line HaCaT

Skin keratinocyte cell line HaCaT were differentiated and then treated with 10 .M, 20 .M and 40 .M of 12HSA and treatment was done for 72 hrs, following standard protocols. Post this treatment the secretome that contains the secreted anti-microbial peptides (AMPs) was collected and was incubated with SARS-CoV2 virus for 4hrs. After the 4hrs 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 table -1 below: Table -1

The data in the table above indicates that the secretome generated in the presence of 12HSA is capable of ki lling/inactivati ng the virus.

Example B-D.4-6: Effect of 12-HSA and vitamin B3 and a combination on 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), 12HSA (10 .M), and their respective vehicle controls. After 48 hrs, 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 Table -2 below:

Table - 2 The data in the above table 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 vitamin B3.

Example E-G,7-9: Effect of human keratinocytes supernatant on SARS-CoV2 infectivity:

Differentiated primary human keratinocytes were incubated in serum free Epilife media with the mentioned concentrations of niacinamide and/or 12HSA for 72 hr and conditioned media was harvested. The SARS-Cov-2 virus particles were incubated for about 4 hrs 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 Table - 3 below:

Table - 3

The data in the above table indicates that effect similar to that in Table -2 could be obtained with primary human keratinocytes also.

Example H-N: Effect of some vitamin B3 compounds its precursor or analogue thereof on virus kill on skin keratinocyte cell line

Skin keratinocyte cell line CRL4048 were differentiated and then treated with various concentrations of vitamin B3 compounds as shown in Table 4 below and treatment was done for 72 hrs, following standard protocols. Post this treatment the secretome that contains the secreted anti-microbial peptides (AMPs) was collected and was incubated with SARS-CoV2 virus for 4hrs. After the 4hrs of incubation, the virus kill was enumerated by incubating the virus mixture on the VeroE6 reporter cell line, and using quantitative PCR based method for measuring the infection. The data on the viral gene expression as a percentage of control is shown in table -4 below:

Table -4

The data in the table -4 together with the data in table - 1 above indicates that 12HSA is vastly superior to vitamin B3 compounds in that niacinamide (example I and J) is not effective at similar (micromolar) concentration. When other vitamin B3 compounds are used (Examples K-N) one needs orders of magnitude higher (millimolar instead of micromolar concentrations) to get virus kill as compared to 12 HSA (Example 1-3 of Table -1).

Example P, 10-14: Effect of other PPAR fatty acids on virus kill on skin keratinocyte cell line

Other PPAR fatty acids were used to study the viral gene expression as shown in Table - 5 below. The procedure used to generate the data was similar to the one used for Table - 4. The data is summarized in the table -5 below:

Table -5

In the table above CLA stands for conjugated linoelic acid

The data in the table -5 above indicates that other PPAR fatty acids (Examples 10-14) are as effective as 12HSA (Examples 1-3 in Table -1) at similar (micromolar) concentrations in viral gene expression.