The present invention relates to virus deactivating compositions having a high viscosity, and methods and uses therefor. In particular, the compositions of the invention comprise dry powder hydroxypropyl methylcellulose (HPMC) particles and a chemical agent selected from dry powder signalling agents wherein the composition has a mean viscosity of greater than 24000 mPa.S.

It is known from USP 8,202,550 that HPMC powders, and the gel that forms when they are administered to the nasal tract, represent an effective means of intranasally administering therapeutic agents, and in particular, herbal, or homeopathic agents. This prior art invention teaches that a therapeutic agent included in a composition as described therein does produce a therapeutic effect. The gel formed when the HPMC is administered to the nasal tract slowly releases moisture and any therapeutic agent which is co-administered. This results in a lasting therapeutic effect, with beneficial effects of the therapeutic agent reported for up to 24 hours following administration. However, there is no disclosure of an hydroxypropyl-methylcellulose containing powder composition having a mean viscosity of from 26000 mPa.s +/- 2000 mPa.S to 40000 mPa.s. +/-5000mPa.S.

A study in Russia by Erofeeva et al (https://medi.ru/info/7023/) points to HPMC being useful in preventing influenza in children. However, there was no indication of the mean viscosity of the dry powder HPMC containing compositions used in the study.

An article by Popov T.A. et al on HPMC powder for the prevention and management of nasal symptoms (Popov T.A. et al Expert Review of Respiratory Medicine, 2017 Vol. 1 1. No. 1 1. 885-892 (https://doi.org/10.1080/17476348.2017.1375408) reported that HPMC provided a natural barrier to pollen allergens and noxious agents. The article itself and a number of studies cited therein involved various plant pollens and house dust mite allergens but did not refer to the mean viscosity of the powdered formulations employed and there is no mention of the compositions defined therein as being able to deactivate viruses in accordance with the definition of virus deactivation provided herein.

HPMC powder per se and HPMC powder containing peppermint and wild garlic has been shown to decrease viral titre of H5N1 in SPEV cell culture in an in vitro study when compared to controls (Lvov DK and Deryabin PG Virucidal Activity of Nasaleze (Nasaval) and Nasaleze Travel (Nasaleze Plus) in Cell Cultures Infected with Pathogenic Avian Flu virus (H5N1 ), 2010, European Journal for Nutraceutical Research 1 -8, www.phytomedcentral.org). There is no reference to mean viscosity and what effect it may have on the efficacy of the HPMC powder as a blocking agent to viral uptake.

Powdered compositions of HPMC and powdered signalling agents having a defined mean particle size as described in international PCT application No. PCT/GB2021/000013 to Nasaleze Patents Limited are considered to be more efficient at controlling or containing inter alia viral infection (size range of virus particles from 0.005pm to 0.3pm) than compositions of the prior art. However, there is no indication that the compositions described therein can deactivate viruses according to the definition of virus deactivation provided herein. Furthermore, there is no indication of the mean viscosity of total compositions described in international PCT application No. PCT/GB2021/000013.

Earlier prior art, EP 3154515B1 teaches the provision of HPMC powder having a viscosity of between 10-20 Pa.S in a 2% aqueous solution at 20 °C. There is no mention of the mean viscosity of whole or complete compositions therein.

The current pandemic of covid-19 disease due to the causative agent SARS-CoV-2 has resulted in tens of millions of people being vaccinated worldwide in an effort to save lives in many countries. While vaccination is the best current means available to save lives in countries having access thereto is ongoing, countries having limited access to vaccines and containing large populations of the total human population worldwide, numbering in the billions, remain at risk of contracting the disease and of possibly dying from it. The vaccination programs take time, considerable effort, and cost to deploy to very large numbers of people. As a result, many tens of millions of people continue to miss out on vaccinations as demand rapidly exhausts current supply.

In order to combat the spread of the disease many alleged safeguards have been put in place, such as compulsory face mask wearing in crowded places, limiting the numbers of people legally permitted to attend events put on for public consumption, and introducing so-called ‘social distancing’ rules at sporting events held at cricket, rugby, and football match stadia and the like, and at entertainment industry venues such as for pop concerts, ballet, opera, night clubs, and hostelry venues such as public houses, inns, restaurants, hotels and the like. Furthermore, ‘lockdown’ rules have been imposed on large sections of the population from time to time in attempts to limit the spread of the virus. All such safeguards have met with varying levels of success, including failure as is evident from the numbers of people who have allegedly contracted the virus which includes those who have allegedly caught the virus from both the unvaccinated and the vaccinated population pools.

The wearing of face masks comes with inherent problems that have become apparent over time. One issue with the wearing of face masks is that the continual supply and demand for them has meant that the numbers of single use face masks has burgeoned into the billions and this is reflected in a concomitant and growing litter problem. The litter issue brings with it a building social and economic problem relating to the cost of supplying face masks in such high numbers and a growing environmental issue as face masks are discarded in landfill sites and often, in the street.

The wearing of face masks is also linked to skin irritations and rashes caused inter alia by bacteria in some people and in others mask material gives rise to allergies resulting in unsightly rashes on the face. In certain activities involving physical effort such as in exercising and in occupations requiring heavy manual labour, the wearing of face masks is also impractical.

There exists a long felt need for a better or at least a complementary means to prevent virus uptake and virus spread that does not come with many of the drawbacks associated with safeguards that have been, and continue to be put in place, and in particular in the long term use of face masks by a public that appears reluctant to give them up. It has now surprisingly been found that viral infection of cells and virus transmission between cells can be substantially blocked by dry homogenised powder compositions of the invention containing hydroxypropyl-methyl cellulose taking up moisture from the nasal linings and so forming a gel. Compositions of the invention possess a mean viscosity of from about 24000 mPa.S to 40000 mPa.S. Such a finding is unexpected because hitherto it was thought that such high viscosity compositions would give rise to a gel that would form gel layers that would be unable to expand sufficiently with the uptake of moisture to be of any effective physiological use, not least because such a gel would not be expected to spread effectively enough over the nasal mucosa cell lining so as to form a disruptive layer to virus movement over a substantial portion thereof. The present inventors have found through in vitro and in vivo studies described herein that compositions possessing such high viscosities are not only useful for preventing virus infection but also prevent virus transmission between infected cells and to a very high degree of efficacy.

Furthermore, as shown in the examples section herein, the employment of respiratory virus deactivating compositions of the invention is surprisingly efficient at blocking infection and virus transmission between cells looking at different strains of coronavirus. This is unexpected because new strains of coronavirus display an improved ability to spread from person to person and hence through populations. Hitherto it was unknown if a physical barrier in accordance with that of the present invention could be useful in preventing such a spread.

It is an object of the invention to provide a composition that is able to substantially prevent infection of cells in the nasal passages, particularly infection of cells of the nasal mucosa by aerial born respiratory viruses, such as coronaviruses, influenza viruses and the like.

It is a further object of the invention to provide a composition that is also able to prevent respiratory viruses from being released from already infected cells in the nasal passageways, such as from cells of the nasal mucosa.

The advantages alluded to above and other advantages will become apparent from the following description.

According to the present invention, there is provided a respiratory virus deactivating composition in the form of a dry homogenised powder comprising: i) dry powder hydroxypropyl methylcellulose (HPMC) particles; and ii) at least one chemical agent selected from dry powder signalling agents, wherein the said dry homogenised powder has a mean viscosity within the range 26000 mPa.s +/- 2000 mPa.S to 40000 mPa.s. +/-5000mPa.S at 20°C in a 3.6% aqueous solution.

Respiratory virus deactivating compositions according to the invention are useful against respiratory virus species selected from influenza virus, respiratory syncytial virus, parainfluenza viruses, metapneumovirus, rhinovirus, adenoviruses, bocaviruses and particularly against coronaviruses as demonstrated herein. In a preferment respiratory virus deactivating compositions according to the invention are active against coronavirus species. Coronaviruses that compositions of the present invention may be used prophylactically against include SARS-CoV, MERS-CoV, SARS-COV-2, HCov-NL63, HCov-OC43, CoV-HKLH , HCov-229E; and mutant strains thereof. In a further preferment, virus deactivating compositions of the invention have virus deactivating activity against the SARS-COV-2 virus and mutants thereof. Influenza species that virus deactivating compositions of the present invention can be used prophylactically against include influenza viruses: A, such as A(H1 N1 ) and A(H3N2) and the like, B, and C; and mutant strains thereof.

For the purposes of the present invention ‘a virus deactivating composition’ is defined as one that i) substantially or completely blocks infection of cells of the nasal mucosa by respiratory viruses, for example SARS-CoV2 and mutant strains thereof; and ii) substantially or completely blocks the release of respiratory viruses from nasal mucosal cells previously infected with viruses, such as SARS-CoV-2. Nasal mucosal cells are placed in contact with powder compositions of the invention that then form an expansive gel on contact with moisture from such cells. The gel is physically overlaid on the nasal mucosal cell lining and thereby substantially covers it. It is to be understood that a virus deactivating composition of the invention does not act so as to kill or render a respiratory virus physiologically dysfunctional. Respiratory viruses are physically blocked or prevented from infecting mucosal cells with which the composition comes into contact and substantially slows down respiratory virus release from respiratory virus-infected mucosal cells.

The ‘dry homogenised powder’ is typically made up of HPMC particles and at least one signalling agent. The HPMC particles can be of any size provided always that when admixed with other components, such as at least one signalling agent, the resultant mixture possesses a mean viscosity within the range 26000 mPa.s +/- 2000 mPa.S to 40000 mPa.s. +/-5000mPa.S at 20°C in a 3.6% aqueous solution. The viscosities of compositions of the invention are measured using standard procedures as taught in the European Pharmacopeia Chapter 2.2.10 on Rheology Analysis and may be performed on a TA Discovery Hybrid Rheometer 1 (TA DHR1 ) from TA Instruments Inc., Wilmington, USA), or comparable Rheometer, and the mean viscosity of several samples calculated therefrom. A suitable mean particle size for HPMC particles of use in the invention has been found to be from 60um to 140um, although it is to be understood that the mean particle size of HPMC particles may also lie outside of this range, for example, between about 50um and about 150um as long as the virus deactivating composition of the invention has a mean viscosity as defined herein.

In a preferment, the mean viscosity of the dry homogenised powder of the invention lies within the range 29000 +/- 5000 mPa.S to 38000 +/-.5000 mPa.S. In a further preferment the mean viscosity is 32900 mPa.S +/- 5500 mPa.S

The compositions of the present invention are designed for application to the nasal mucosa through insufflation via the nose.

Compositions of the invention must be able to form gels on contact with moisture. The compositions of the invention should not contain additives that may or could substantially interfere with their ability to form gels on contact with moisture, such as additives that can significantly lower the pH of the nasal mucosa. On contact with the nasal mucosa, the dry powder particles of the invention absorb moisture and thereby form a gel matrix on the surface thereof. The function of the gel is considered to be at least twofold: firstly, it acts as a physical barrier to the uptake of viruses through the nasal mucosa and secondly it physically blocks transfection between cells. During the hydration of dry powdered compositions of the invention a gel matrix is formed through contact with moisture in which larger particles and smaller particles of the gel matrix combine to form a molecular net or molecular matrix wherein the smaller particles occupy spaces or gaps between larger particles and so contribute to gel formation, helping the larger particles to subsume together more easily. Viral particles become trapped in the gel and are substantially unable to infect cells of the nasal mucosa.

Compositions of the invention include dry powder signalling agents. A signalling agent for the purposes of the present invention is one which once inhaled imparts a substantial olfactory and/or a buccal cavity sensation to the user. Such a sensation indicates to the user that the composition of the invention has been applied successfully and/or time to apply more composition. The olfactory sensation, i.e. detection of a smell or scent, provides the user with the knowledge that the composition has reached the nasal mucosa. A buccal cavity sensation may indicate that it is time to apply more of the composition to the nasal mucosa because the taste sensation within the buccal cavity may indicate that the cilia of the nasal mucosa have removed at least a portion of the applied composition from the nasal passages and into the throat and/or buccal cavity, from where it is then swallowed.

Suitable signalling agents of use in compositions of the invention are ones that do not affect, or do not substantially affect, the ability of the homogenised powder compositions of the invention to form expansive gels on contact with moisture from the nasal mucosa. Typically, signalling agents are selected from menthol, strawberry, mint, spearmint, peppermint, eucalyptus, lavender, citrus, (such as lemon, lime, cumquat, clementine, and tangerine), garlic (such as European wild garlic, cultivated garlic, and allicin, a stabilised garlic fraction), and any combination of two or more thereof.

The signalling agent may make up from 1.5% to <7% of the total weight of the composition, depending on design. Examples of compositions of the invention possessing an appropriate mean viscosity include those compositions having a mean viscosity of (i) 33328 +/- 300 mPa.S. with signalling agent present as a combination of 2% peppermint powder and 3% garlic powder making up 5% of the total weight of the composition; (ii) 29414 +/- 70. mPa.S with signalling agent present as a combination of 2% peppermint powder and 3% allicin making up 5% of the total weight of the composition (iii) 37453 +/-1 100 mPa.S and the signalling agent is peppermint powder making up 1.5% of the total weight of the composition. In the three examples listed, HPMC makes up the rest of the proportion of the virus deactivating composition. A further example of a composition of the invention is iv) having a mean viscosity of 36224 +/-1 100 mPa.S and the signalling agent is lemon powder making up 5% of the total weight of the composition.

Other components which may be present in compositions of the invention may further comprise kali bichromicum (potassium bichromate); a thickening agent such as a gum or starch; a disintegrant, such as sodium starch glycolate or cross-linked povidone; a release agent such as magnesium stearate; an emulsifying agent; a surfactant; pharmaceutically acceptable excipients; anti-caking agents; granulating agents; preservatives; such colorants as may be desired or any combination thereof.

In preferred embodiments of the present invention, the powder compositions do not include components which are often used in intranasal compositions (dry powders or solutions) which can cause irritation or affect ciliary movement, for example, solvents, such as propylene glycol, absorption enhancers, such as cyclodextrins or glycosides, muco-adhesives such as chitosan, or preservatives such as citric acid, citrates and benzalkonium chloride. The use of such additives can be undesirable, as they may cause discomfort and interfere with the normal functioning of the gel and/or nasal mucosa, which may in turn, adversely affect breathing.

In a further embodiment of the invention there is thus provided a respiratory virus deactivating composition as herein defined consisting of: i) hydroxypropyl methylcellulose particles; and ii) at least one chemical agent selected from dry powder signalling agents, wherein the said dry homogenised powder has a mean viscosity within the range 26000 mPa.s +/- 2000 mPa.S to 40000 mPa.s. +/-5000mPa.S at 20 °C in a 3.6% aqueous solution.

In a preferment, respiratory virus deactivating compositions according to the invention may be employed against virus species selected from influenza virus, respiratory syncytial virus, parainfluenza viruses, metapneumovirus, rhinovirus, coronaviruses, adenoviruses, and bocaviruses. In a preferment respiratory virus deactivating compositions according to the invention are active against coronavirus species and influenza species. Coronaviruses that compositions of the present invention may be used prophylactically against include SARS-CoV, MERS-CoV, SARS-COV-2, HCov- NL63, HCov-OC43, CoV-HKlH , HCov-229E; and mutant strains thereof. In a further preferment, virus deactivating compositions of the invention have virus deactivating activity against the SARS-COV-2 virus and mutants thereof. Influenza species that virus deactivating compositions of the present invention can be used prophylactically against include influenza viruses: A, such as A(H1 N1 ) and A(H3N2) and the like, B, and C; and mutant strains thereof.

In a still further preferment there is provided a respiratory virus deactivating composition as herein defined for use as a nasally administered prophylactic composition. Again, it is emphasised that respiratory virus deactivating compositions of the invention act solely as a physical barrier to respiratory virus movement as defined herein. Thus, compositions of this preferment of the invention are for use in preventing respiratory viral infection caused by respiratory virus species selected from influenza virus, respiratory syncytial virus, parainfluenza viruses, metapneumovirus, rhinovirus, coronaviruses, adenoviruses, and bocaviruses. In this preferment, respiratory virus deactivating compositions according to the invention are active against coronavirus species and influenza species. Coronaviruses that compositions of the present invention may be used prophylactically against include SARS-CoV, MERS-CoV, SARS-COV-2, HCov-NL63, HCov-OC43, CoV-HKU1 , HCov-229E; and mutant strains thereof. Preferably, respiratory virus deactivating compositions of this preferment of the invention have virus deactivating activity against the SARS-COV-2 virus and mutants thereof, and influenza species such as influenza A, for example, A(H1 N1 ) and A(H3N2) and the like, influenza B, influenza C and mutant strains thereof.

Thus, there is provided a composition in the form of a dry homogenised powder for use in deactivating respiratory viruses as defined herein, wherein the respiratory viruses are selected from the group coronaviruses, influenza viruses, respiratory syncytial viruses, parainfluenza viruses, metapneumoviruses, rhinoviruses, adenoviruses, and bocaviruses. Preferably, the respiratory viruses are selected from influenza viruses, rhinoviruses, and coronaviruses. More preferably, the respiratory virus is a rhinovirus, or an influenza virus or a coronavirus. Thus, in a further preferment there is provided a respiratory virus deactivating composition of the invention for use in preventing SARS-COV-2 viral infection.

In a further embodiment, there is provided a respiratory virus deactivating composition of the invention, wherein the said composition is for use in prophylaxis of covid-19 disease.

In yet another embodiment of the invention there is provided a method of making a dry powder respiratory virus deactivating composition as defined herein for use in limiting the spread of respiratory virus related disease, such as covid-19 disease, comprising:

1 ) adding signalling agent powder to hydroxypropyl methylcellulose powder; and

2) diffusively blending the two ingredients of 1 ) in a blending machine, wherein the resultant dry homogenised powder has a mean viscosity within the range 26000 mPa.s +/- 2000 mPa.S to 40000 mPa.s. +/-5000mPa.S at 20 °C in a 3.6% aqueous solution.

As a further embodiment of the invention there is provided use of a respiratory virus deactivating composition as defined herein, in the prophylactic treatment of an individual against contracting SARS-COV-2 disease.

As a further embodiment of the invention there is provided a method of treating a patient prophylactically against contracting a respiratory virus disease, such as coronavirus disease comprising: administering intranasally to a patient a virus deactivating composition of the invention in homogenised dry powder form comprising: i) micronized hydroxypropyl methylcellulose particles; and ii) at least one chemical agent selected from signalling agents, wherein the homogenised dry powder has a mean viscosity within the range 26000 mPa.s +/- 2000 mPa.S to 40000 mPa.s. +/-5000mPa.S at 20 °C in a 3.6% aqueous solution.

The composition of the invention may be administered as a single puff or squirt from a squeezy bottle as defined and described in either of EP1368090B1 or EP3183022B1.

In a preferment of this embodiment of the invention the composition comprises at least 93% hydroxypropyl-methylcellulose particles and at least 2% signalling agent by total weight of the composition.

The dosage of the composition used in this embodiment of the invention is between 1 mg to 10mg per nostril of a patient or wherein the dosage is between 2.5mg to 7.5mg, between 3mg to 7mg, between 4mg to 6mg, or 5mg per nostril of a patient.

The mean viscosity of the virus deactivating composition in this embodiment of the invention can be in the range 29000 +/- 5000 mPa.S to 38000 +/-.5000 mPa.S; is 32900 mPa.S +/- 5500 mPa.S; is 29414 +/- 7O.mPa.S; is 37224 +/- 300 mPa.S; or is 33328 +/-300 mPa.

The signalling agent in this embodiment of the invention may be selected from menthol, strawberry, mint, spearmint, peppermint, eucalyptus, lavender, citrus, (such as lemon, lime, cumquat, clementine, and tangerine), garlic (such as European wild garlic, cultivated garlic, and allicin, a stabilised garlic fraction), and any combination of two or more thereof.

In preferments of this embodiment of the invention, the signalling agent may be present in an amount of up to 7% of the total weight of the composition; 1.5% of signalling agent, such as peppermint, of the total weight of the composition; 5% of signalling agent, such as lemon, of the total weight of the composition ; 7% of signalling agent of the total weight of the composition consisting of two signalling agents, such as 2% peppermint and 5% garlic; or 5% of signalling agent of the total weight of the composition consisting of two signalling agents, such as 2% peppermint powder and 3% allicin powder.

Compositions of the invention can be applied with devices which are suitable for dispensing the virus deactivating compositions of the present invention as disclosed in, for example, EP1368090B1 and EP3183022B1 , the teaching of which is incorporated herein in its entirety. The bottles disclosed therein use a very simple mechanism for restricting the amount of powder which is dispensed thus ensuring that blockage of the nasal tract is unlikely to occur.

According to a further embodiment of the present invention there is provided a kit of parts for a respiratory virus deactivating composition comprising i) a first receptacle containing dry homogenised hydroxypropyl methylcellulose powder; ii) a second receptacle containing powder signalling agent. The kit of parts may also comprise a third receptacle for admixing the powders of i) and ii). Preferably, one or both of the receptacles of i) and ii) comprise deformable and resilient surfaces that are depressible with the finger and thumb so as to permit the administration of powder to the nasal mucosa. Preferably still, the third receptacle also comprises deformable and resilient sides that are depressible with the finger and thumb so as to permit the administration of powder to the nasal mucosa.

The powders may be first admixed and then applied to the nasal mucosa or each powder may be administered sequentially. In either method of administration, the receptacle of choice for administering each powder whether admixed or independently administered can be a squeezy bottle as defined and described in either of EP1368090B1 and EP3183022B1 . The signalling agent and powdered HPMC are admixed together so as to make up a virus deactivating composition as defined herein that includes from 1 .5% to <7% of signalling agent, preferably from 2.0% to 5% of the total weight of the composition. Such kits are also aimed at being used prophylactically to protect against viral attack from respiratory viruses as defined herein, such as influenza A viruses, for example, H1 N1 , H5N1 , and H3N2; and coronaviruses, such as MERS-CoV, SARS-CoV, HCoV-229E, HCov-NL63, HCoV-OC43, CoV-HKU1 , and SARS-COV-2; and mutants thereof. As a further embodiment of the invention there is provided a composition as herein defined, wherein the said composition is for use as a nasally administered medicament.

As a yet further embodiment of the invention there is provided a composition as herein defined, wherein the said composition is for use in preventing the onset of a viral respiratory disease such as covid-19 disease or influenza.

As a still further embodiment of the invention there is provided a composition as herein defined for use in prophylaxis of a viral respiratory disease as defined herein such as covid-19 disease or influenza.

Naturally, the skilled artisan will appreciate that all compositional embodiments of the invention detailed herein are for delivery to the nasal mucosa via insufflation through the nose.

There now follow Figures and examples of the invention. It is to be understood that the teaching of the Figures and examples is not to be construed as limiting the invention in any way.

FIGURES

Figure 1

Hydroxypropyl methylcellulose inhibits SARS-CoV-2 release in a dose-dependent manner. Vero A/T cells in 24-well plates were infected with SARS-CoV-2 (England-2) at an MOI of 0.01 (upper panels) or 1 (lower panels) prior to addition of product A, B, or C, at 1 mg, 3 mg, or 6.4 mg per well. Supernatant samples were taken at 24, 48 and 72 h post-infection and released virus quantified by plaque assay. Virus titres (pfu/mL) are shown for each product at each time point. Data points below the assay detection limit of 2x10 ¹ pfu/mL are shown with open circles. Error bars represent the standard deviation of two experiments each carried out in triplicate.

Figure 2

Hydroxypropyl methylcellulose inhibits SARS-CoV-2 infection. Vero A/T cells in 24- well plates were pre-treated with product A, B, or C, at 6.4 mg per well, followed by infection with (A) England-2, (B) Alpha, or (C) Beta strain of SARS-CoV-2 at MOI 1 . Cell monolayer survival was determined at 24, 48 and 72 h post-infection and expressed as a percentage of uninfected control cells. Error bars represent the standard deviation of two experiments, each with 5 replicates.

EXAMPLES SECTION

1. Introduction

SARS-CoV-2 is a large, enveloped virus belonging to the coronavirus family and is the causative agent of the COVID-19 pandemic that has, to date, infected over 194 million people and caused more than 4 million deaths worldwide (https://covid19.who.int/). The transmission of respiratory pathogens, including SARS-CoV-2, from person to person occurs during activities such as sneezing, coughing, and talking, via the generation of aerosols that contain droplets of varying sizes [1 -4], Transmission can occur in one of two ways: (1 ) virus-loaded droplets exhaled from an infected person coming into contact with the eyes, nose, or mouth of an uninfected person, or (2) droplets that have settled on surfaces and are subsequently transferred to an uninfected person’s hands, who then touches their eyes, nose, or mouth. Current evidence suggests that airborne and droplet particle transmission via direct contact with the eyes, nose, or mouth is a likely form of transmission of SARS-CoV-2 [5-7], This has formed the scientific basis for many of the non-pharmaceutical interventions in the fight against COVID-19 such as mask wearing and maintaining social distancing of at least 2 metres. Over-the-counter nasal sprays offer an additional measure for both the prevention of infection, and the spread thereafter, of respiratory pathogens. While a number of products incorporate small drug molecules or reactive species, for example reactive oxygen and nitric oxide[8], that actively target the virus, many rely on the creation of a physical barrier capable of blocking virus uptake. These passive barriers are generated via semisynthetic or natural gelling agents such as hydroxypropyl methylcellulose (HPMC) [9], or carrageenan [10]. Carrageenan has been well studied in vitro, with formulations demonstrating an ability to block SARS- CoV2 [1 1 -14] and Influenza [14, 15] infection. Furthermore, clinical studies have demonstrated the potential to reduce disease burden, revealing the potential of nasal sprays in real-world scenarios [16]. HPMC has been less well studied, but has recently shown efficacy against coronavirus 229E [17], and the ability to block allergens such as pollen [18].

To improve the evidence base for HPMC based products, we assessed three HPMC- based nasal sprays from Nasaleze International Limited for their ability to block both infection with SARS-CoV2, and the release of virus from cells previously infected with SARS-CoV-2. We show that virus infection and release from cells is inhibited in a dose-dependent manner, with the optimum dose of 6.4 mg/3.5cm ² inhibiting virus release over a 72-hour period. This efficacy is purely physical, and thus is independent of virus variant.

2. Materials and Methods

2.1 Cells and virus

VeroE6 cells expressing ACE2 and TMPRSS2 (Vero A/T) were maintained at 37°C, 5% CO2 in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% heat inactivated FBS. All SARS-CoV-2 work was carried out in an approved Category 3 facility. SARS-CoV-2 stocks were grown in standard VeroE6 cells in the presence of 2% FBS. All assays were conducted using DMEM supplemented with 2% heat- inactivated FBS and 50 pg/mL Gentamycin (2%-DMEM).

For plaque assays, Vero A/T cells were seeded at 1 x10 ⁵ /well in 12-well plates 18 hours prior to use. Samples were 10-fold serially diluted in 2%-DMEM to give a dilution series from 10’ ¹ to 10’ ⁶ and cells infected for 1 h at 37°C, 5% CO2. Virus inoculum was removed and cells overlayed with a 1 :1 mix of 2.4% Avicel® and 2X MEM (20% 10X MEM; 2% L-Glutamine; 4% FBS; 5.4% sodium bicarbonate (7.5% soln.)). Cells were incubated for 72 h at 37 °C, 5% CO2 at which point overlay was removed, cells were washed once with PBS and fixed with 1 mL/well methanol for at least 5 minutes. Following removal of methanol, cells were stained with 0.1 % (w/v) crystal violet, and plaques counted.

Virus: This study utilised the England-2 strain of SARS-CoV-2 along with the Alpha (B1 .1 ,7/Kent) and Beta (B.1 ,351/South Africa) variants, all originally obtained from Public Health England. 2.2 Products

Test products were provided blind as Product A, B, and C from Nasaleze International Limited. Upon completion of the study product details were provided to aid data analysis. Product A: Nasaleze Cold/Travel Original (93% HPMC, 2% peppermint powder and 5% European wild garlic powder). Product B: Nasaleze Cold/Travel Allicin (95% HPMC, 2% peppermint powder and 3% allicin powder). Product C: Nasaleze Allergy (98.5% HPMC and 1 .5% peppermint powder).

2.3 Inhibition of Virus Release

Vero A/T cells were seeded at 5x10 ⁴ /well in 24-well plates 18 hours prior to use. Cells were infected with SARS-CoV-2 (England-2) at an MOI of 1 or 0.01 and incubated for 30 min on a platform rocker at 37°C, 5% CO2. Virus inoculum was removed, and cells washed twice with PBS and 150 pL/well of fresh 2%-DMEM added. To each well either 1 mg, 3 mg, or 6.4 mg of product was evenly distributed across the cell monolayer and incubated at room temperature for 15 min for gel-matrix formation to occur. Triplicate replicates were carried for each test conditions, and duplicate experiments were conducted. A no-product/plus virus control well was also established on each plate. Once gel-matrix had formed, 1 .5 mL of 2%-DMEM was added to each well and cells were incubated at 37°C, 5% CO2 for the duration of the experiment. At 24, 48, and 72 h post-infection 100 pL of supernatant was removed from each well and replaced with 100 pL of fresh 2%-DMEM. Samples were stored at -80°C until virus quantification by plaque assay as above.

2.4 Inhibition of Virus Infection

Vero A/T cells were seeded at 5x10 ⁴ /well in 24-well plates 18 hours prior to use. Three types of assay plates were established: (1 ) Test plate of product plus virus, (2) Novirus control plate with product only, and (3) No-product control plate with virus only. For test and no-virus control plates, growth media was removed from cells, replaced with 150 pL of fresh 2%-DMEM and 6.4 mg of product was evenly distributed within each well. Five replicate wells were established for each product tested. Plates were incubated at room temperature for 15 min for gel-matrix formation. Once the gel-matrix had formed, to each well was added 1.5 mL of 2%-DMEM containing SARS-CoV-2 (England-2, Alpha or Beta variant) at MOI 1. For the no-product control plate 1.5 mL of 2%-DMEM containing virus as above was added to each of 5 replicate wells per virus strain. Additionally, 5 wells were established containing 1 .5 mL of media only, to act as an uninfected control. Cells were incubated at 37°C, 5% CO2, for the duration of the experiment.

At 24, 48, and 72 h plates were assayed for cell monolayer survival as a measure of the level of virus infection. Supernatant was removed from all wells, 1 mL/well of methanol added and cells incubated at room temperature for 1 h to fix. Methanol was removed and 0.5 mL/well of 0.1 % crystal violet stain added for 20 min. Crystal violet was removed, and cells washed repeatedly with water until all product was removed from the cell monolayer. Crystal violet was added for a second incubation of 5 min to ensure all cells were evenly stained following removal of product. Crystal violet was again removed, cells washed once more with water and plates left to dry.

Monolayer survival was determined by the level of crystal violet staining observed. To each stained well 200 pL of 10% acetic acid was added and incubated for 15 min on a platform rocker to re-solubilise crystal violet. Samples were diluted 1 :20 in water and the OD 595 determined. Monolayer staining was expressed as a percentage of uninfected controls.

2.5 Suspension Test

6.4 mg of product was resuspended in 100 pl of DMEM to generate the gel-matrix. To this was added 100 pl of virus inoculum containing approximately 2x10 ⁶ pfu of the England-2 strain of SARS-CoV-2. The gel/virus mix was incubated for 1 h at room temperature before the addition of 1 mL of DMEM to diffuse the gel matrix. Residual virus was quantified by plaque assay as above.

3. RESULTS

3.1 Hydroxypropyl methylcellulose inhibits release of SARS-CoV-2 in a dosedependent manner

To determine if HPMC could inhibit the release of virus from cells previously infected with SARS-CoV-2 we blind-tested three different powder nasal spray formulations from Nasaleze International Limited, referred throughout as products A, B, and C (see Materials and Methods for details). VeroE6 cells expressing ACE2 and TMPRSS 2 were first infected with the England-2 strain of SARS-CoV-2 at a multiplicity of infection (MOI) of 0.01 or 1 , in 24-well plates. Following infection cells were coated with each of the three test products to form a gel matrix that covered the cell monolayer (see Materials and Methods). Products were added at either 1 mg, 3 mg, or the manufacturers recommended dose of 6.4 mg per well, where one well represents a surface area of 3.5 cm ² . The gel matrices were overlaid with 1 .5 mL of media/well and incubated at 37°C for 72 h. At 24-, 48-, and 72-hours post-infection (hpi) 100 pL of supernatant was removed from each well and the amount of virus released was quantified by plaque assay (Figure 1 ).

We observed a clear dose response in the level of virus release. In the absence of product, virus concentrations reached 10 ⁶ -10 ⁷ pfu/mL by 24 hpi, and were similar regardless of the MOI used in infection, highlighting the rapid growth of SARS-CoV-2 in cell culture. In the presence of 1 mg of product, and following infection at MOI 1 , virus concentrations reached an average of 10 ⁴ -10 ⁵ plaque-forming units (pfu)/mL by 72 hpi, an approximate 2-logw reduction in titre. In comparison, in the presence of 6.4 mg of product virus levels were generally below the detection limit of the assay (2x10 ¹ pfu/mL), which corresponded to a >5-log (or >99.999%) reduction in virus release when compared to the no-product control (Figure 1 ). No difference in efficacy was observed between the three products at the highest concentration tested demonstrating that 6.4 mg of a powder nasal spray was sufficient to cover a surface area of 3.5 cm ² in a manner that effectively inhibited SARS-CoV-2 release from infected cells.

3.2 Hydroxypropyl methyl cellulose block infection with SARS-CoV-2

Having demonstrated that all three HPMC-containing products could effectively inhibit the release of virus from previously infected cells with a dose of 6.4 mg/3.5cm ² , we next investigated whether the nasal sprays could also inhibit de novo infection with SARS-CoV-2. To assess the inhibition of infection, formation of the product gel-matrix was first required to cover the cell monolayer. Due to the nature of the gel-matrix, its subsequent removal from cell monolayers is difficult without the removal process itself resulting in damage to the cell monolayer. As any damage to the monolayer would negatively bias the measurement of virus infection, we established a quantifiable assay that allowed cells to be fixed prior to determining the level of virus infection. SARS-CoV-2 infection of Vero A/T cells leads to destruction of the cell monolayer and thus could be used as a proxy measure for the level of infection by normalising monolayer survival - measured by residual crystal violet staining - of infected cells to uninfected cells. Additional controls were set up to measure monolayer survival in the absence of product, with or without SARS- CoV-2 infection (see Materials and Methods). Having established from the previous experiments that 6 mg was the most effective dose in a 24-well format we proceeded with testing at this concentration, limiting infections to MOI 1. In addition, to determine whether virus strain had any impact on product effectiveness we conducted infections with both the Alpha and Beta strains of SARS-CoV-2, as well as England-2, as tested above.

In the no-product, virus-infected controls cells, monolayer staining was reduced to <30% for all strains by 72 hpi (Figure 2; and S1 [available online at www.mdpi.com/xxx/s1 , Figure S1 : Plate images of inhibition of infection]). In comparison, we observed no reduction in cell monolayer staining if virus infections were carried out on cells pre-coated with any one of the three nasal spray products (Figure 2). The ability to block infection of the cell monolayer was also independent of the virus strain used for infection, with similar results obtained for all three SARS-CoV- 2 strains tested.

3.3 Hydroxypropyl methylcellulose is not virucidal

As all three Nasaleze products had demonstrated an ability to inhibit both virus release from infected cells and de novo infection, we next sought to determine whether any of the products had direct virucidal activity against SARS-CoV-2 that could be contributing to the inhibition observed. In a modified suspension assay 6.4 mg of each product was first resuspended in 100 pl of media to mimic the gel-matrix created during the cell-based assays. A virus inoculum containing approximately 2x10 ⁶ pfu of SARS- CoV-2 (strain England-2) was added to the gel in a 1 :1 (v/v) ratio and incubated at room temperature for 1 h, after which virus was recovered for quantification. No reduction in virus titre was observed for products B and C when compared to a media only control. A small reduction in virus titre of 2-logw was observed for product A. This could suggest weak virucidal activity, however this product formed a noticeably denser gel-matrix plug in this test, thus a more likely explanation is simply that some virus remained trapped, leading to reduced recovery for this product. These results show that, under the conditions tested, HPMC-based products are not virucidal, and overall are highly suggestive that the mechanism of action is via the formation of a passivebarrier that physically blocks interaction of the virus at the cell surface.

4. Discussion

One of the primary drivers in the spread of respiratory pathogens is airborne transmission via aerosols generated from infected individuals. The importance of preventative measures to block such transmission routes has been highlighted during the ongoing COVID-19 pandemic. While vaccination programs against SARS-CoV-2 are proving highly successful at preventing severe disease they do not completely stop infection and transmission, and worldwide vaccine uptake is highly variable due to a number of socio-political and economic factors. For this reason, additional preventative measures are likely to be required for some time. One such option is the use of nasal sprays that prevent uptake and release of virus. By functioning as a physical barrier, these products function irrespective of mutations that alter viral transmissibility, and do not need to be reformulated for different virus variants or strains; as a result, they may also form part of infection control measures in future viral pandemics, prior to the generation of specific vaccines.

In this study we blind-tested three over-the-counter nasal sprays, based on a powder form of HPMC, for their ability to block infection and spread of SARS-CoV-2 in an in vitro cell culture system. We observed a dose-dependent response in the ability of all products tested to inhibit virus release from cells previously infected with SARS-CoV- 2 (Figure 1 ). As these products function via the formation of a passive, physical barrier (Figure 3) this was not unexpected as the greater the barrier the less virus would be able to penetrate into the surrounding media. There were some indications that product A was able to inhibit virus release to a greater degree than products B and C. For example, following infection at MO1 1 , and in the presence of 3 mg of product, <10 ² pfu/mL of virus was released by 72 hpi from product A compared to approximately 10 ⁵ pfu/mL from product C. This did not correlate with higher HPMC concentrations - 93% and 98.5% for A and C respectively - suggesting that additional components in the nasal-spray may be contributing to antiviral activity, potentially by altering the physical nature of the gel matrix. We subsequently demonstrated that, at the highest dose tested, all products were similarly able to block de novo infection of cells with SARS- CoV-2 (Figures 2 and S1 ).

This study demonstrates high efficacy in an in vitro cell culture system, however there are additional challenges in the use of nasal sprays in vivo. The nasal cavity is composed of a dual chamber measuring 5 cm high and 10 cm long, with a resulting total surface area of around 150 cm ² for both cavities [19]. When used as instructed Nasaleze products deliver a dose of 6.4 mg to each nostril. Although viruses can potentially infect, and be excreted from, both the mouth and the nose, as well as in droplets originating in the lungs, not all sources of virus excretion would be inhibited by a nasal spray powder.

The nose remains the primary route by which air is inhaled, making the nasal cavity the predominant environment in which pathogens and other foreign particles are first targeted for removal, and a likely site for initial infection [19], Limiting pathogen uptake via the nasal cavity therefore represents a valid target for the inhibition of viruses such as SARS-CoV-2 [1 1 ], In support of this, clinical studies have demonstrated that carrageenan-based nasal barrier products reduce the duration of symptoms from coronavirus, influenza, and rhinovirus [16]. Our data suggests that HPMC-based nasal sprays may similarly play a role in limiting the spread and infection of SARS-CoV-2 via the nasal route. It also supports the development of clinical studies to evaluate the real-world efficacy of HMPC based products.

Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1 , Figure S1 : Plate images of inhibition of infection.

References

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Experiment 3 - Regular Use of Nasally Applied Powder Hydroxypropylmethyl cellulose (pHPMC) During the Pollen Season Protects Against SARS-COV-2 Infection

Background: Puffing powder hydroxypropyl methylcellulose (pHPMC) into the nasal cavity of allergy sufferers creates a gel barrier which has been proven an effective means to control the symptoms of allergic rhinitis. This mechanical protective layer would also stay in the way of other noxious agents like microbes and fine particulate pollutants. The aim of our study was to find out whether regular use of pHPMC in patients with seasonal allergic rhinitis (SAR) may also offer protection against SARS- CoV-2 infection.

Methods: This was a real life retrospective study during the pollen season in Bulgaria (between March and July 2021 ). Seventy nine subjects, 43 men, aged 35 years /median/, range 17-55 years, who had been prescribed a sample of Product C (mean viscosity within the range 26000 mPa.s +/- 2000 mPa.S to 40000 mPa.s. +/- 5000mPa.S at 20°C in a 3.6% aqueous solution) for seasonal allergic rhinitis were identified in the log book of the Medical Centre Excelsior in Sofia. They were compared with 79 gender-matched patients (43 men, aged 39, range 16-55 years, who have been consulted for different other allergic conditions not requiring nasal treatment. All subjects were contacted after the end of the grass pollen season and asked if they had been identified with SARS-CoV-2 infection.

Results: A third wave of COVID-19 affected Bulgarians between March and June 2021 . Out of 79 subjects using pHPMC during that period, there were two cases of the infection, while among the patients not using pHPMC the number of cases was 6 (P=0.032. Chi-square analysis).

Conclusions: The pHPMC used as a mechanical barrier against pollen in subjects with SAR also offers them protection against SARS-COV-2 infection.