Technical Field

[001 ] The present technology relates to a system and method for delivering a therapeutic agent to the airway. More particularly, the technology relates to delivery of a therapeutic agent to the airway for treatment of a respiratory disease or infection using a closed circuit nebulizer system.

Cross reference to related application

[002] This application claims priority to Australian Provisional Application No. 2020903159 filed 3 September 2020, which is hereby incorporated by reference in its entirety.

Background

[003] Oral or intravenous treatment of respiratory tract infections, particularly viral and other microbial infections, is limited by the potential side effects of the therapeutic to other organs.

[004] On the other hand, delivering therapeutic agents by inhalation for treatment of respiratory infections has the capacity to deliver relatively large doses directly to the target in the lung with low levels of absorption into the bloodstream thereby limiting concerns of the therapeutic agent producing side effects to other organs.

[005] Additional advantages of inhalation of therapeutic agents include rapid delivery, high bioavailability and limited metabolism.

[006] Delivery of soluble therapeutic agents to the respiratory tract may be achieved by inhaling aerosol, a fine mist of tiny droplets, generated by a nebulizer containing a solution of the therapeutic agent.

[007] Typically a jet nebulizer uses a stream of compressed air to draw a solution of the therapeutic agent through a small orifice to generate a cloud of droplets of 1 -10pm in diameter. The newer vibrating mesh nebulizers generate aerosol with a rapidly vibrating mesh containing many minute holes which causes the solution to pass through creating tiny droplets of around 1-5pm in diameter, smaller than jet nebulizers. Ultrasonic nebulizers create aerosol using a piezo-electrode.

[008] The aerosol is then inhaled through the mouth and/or nose of a patient to reach the lung where the droplets settle on the internal lining of the airway. Smaller droplets penetrate deeper into the lungs. [009] However, a major problem is that during use, all standard nebulizers produce aerosol continuously yet aerosol is directed towards the lung only during inhalation. During exhalation, all aerosol produced simply escapes to the atmosphere and is wasted. This makes them extremely inefficient with only around 10% of the dose of therapeutic agent placed in the nebulizer reaching the lungs and at least 60% ending up in the room. For expensive therapeutic agents this is a costly waste, and in the case of toxic therapeutic agents, the medication lost to the atmosphere is potentially harmful to nearby personnel.

[010] Other factors, such as the individual anatomy of a patient's airway, the physical conditions that the drug will encounter in the airway (e.g., humidity), the efficiency of the clearance mechanisms of the lung (e.g., mucociliary clearance and alveolar macrophages) and the pathophysiological effects of acute and chronic diseases (e.g., inflammation level or pulmonary tissue scarring resulting from chronic obstructive pulmonary disease or viral infections, including coronavirus infections such as Severe Acute Respiratory Syndrome (SARS-CoV), Middle East Respiratory Syndrome (MERS-CoV), and COVID-19) all impact on the final dose and concentration of the administered medication reaching the target in the patients lungs.

[011] Newer nebulizers have the capacity to reduce the amount of aerosol lost during inhalation although these are expensive, costing over a thousand dollars.

[012] The above factors are particularly critical when the medication being administered relies on adequate and accurate concentration and dose delivery and in order to achieve a clinical effect in the lung or part of the lung.

[013] The present inventors have identified alcohol (ethanol) as a suitable candidate for treatment of respiratory infections, including viral respiratory infections. While no reports can be found using ethanol delivered to the lower airway to treat infection, ethanol has previously been delivered directly into the lungs by intra-tracheal injection in patients who have experienced trauma and displaying Acute Respiratory Distress Syndrome (Takahashi and Endo. Improvement of Acute Respiratory Distress Syndrome with Ethyl Alcohol Infusion into the Airway: A Case Report. J Pulm Respir Med 2018, 8:3). However, for ethanol to be used regularly for treatment of respiratory conditions, an improved method of reliably delivering the ethanol as a therapeutic agent to the respiratory tract without requiring a general anaesthetic and intubation, is required. Summary

[014] The present inventors have developed a closed-circuit nebulizer device which maximizes dose delivery efficiency and concentration, and prevents environmental escape of aerosol from the system and any exhaled infectious organisms from the device during use.

[015] In a first aspect there is provided a closed circuit nebulizer system for delivery of a therapeutic agent to the airway of a subject, the system comprising: a) a nebulizer comprising a container for holding a solution of a therapeutic agent wherein, in use, the nebulizer generates an aerosol of the solution; b) a spacer having a collapsible reservoir for containing the aerosol; the reservoir having an inlet in fluid communication with the nebulizer wherein the inlet is configured to receive the aerosol from the nebulizer; c) an outlet in fluid communication with the reservoir, wherein the outlet comprises a valve assembly comprising at least one selectively activatable occlusion member that prevents the entrainment of exhaled breath into the reservoir; and d) an exhaust passage in fluid communication with an exhaust valve, wherein the exhaust passage directs the subject's exhaled breath towards an exhaust filter to receive the exhaled breath via the exhaust passage and prevent environmental release of the therapeutic agent.

[016] In use, the closed circuit nebulizer system prevents escape of the aerosol into the atmosphere while the user is inhaling and exhaling.

[017] In some embodiments the outlet is in fluid communication with a face mask, nasal mask, or mouthpiece configured to receive the valve assembly.

[018] In some embodiments the outlet may be connected to the face mask, nasal mask, or mouthpiece via a suitable conduit such as a flexible tube.

[019] The reservoir is configured to retain a volume of aerosol produced by the nebulizer while the user exhales, such that, in use, the volume of aerosol is available to be inhaled by the user.

[020] In some embodiments the system further comprises a solution of a therapeutic agent.

[021 ] In a second aspect there is provided a method of delivering a therapeutic agent to an airway of a subject comprising; a) generating an aerosol and/or vapour of a solution of a therapeutic agent using the closed circuit nebulizer system of the first aspect; and b) having the subject inhale the aerosol and/or vapour from the closed circuit nebulizer system to deliver the therapeutic agent to the airway.

[022] In a third aspect there is provided a method of treating a respiratory disease in a subject comprising delivering a therapeutic agent to an airway of the subject by; a) generating an aerosol and/or vapour of a solution of a therapeutic agent using the closed circuit nebulizer system of the first aspect; and b) having the subject inhale the aerosol and/or vapour from the closed circuit nebulizer system to deliver the therapeutic agent to the airway.

[023] In one embodiment the airway is at least one of the nasal cavity, nasopharyngeal airway, sinuses, oral cavity, pharynx, larynx, trachea, bronchi, lungs, bronchiole, and alveoli. For example, the airway may be at least one of the bronchi, lungs, bronchioles, and alveoli. Preferably, the therapeutic agent is substantially evenly distributed throughout the airway.

[024] In one embodiment the amount of therapeutic agent delivered to the airway is at least twice the amount delivered by a standard nebulizer alone.

[025] In one embodiment the concentration of the therapeutic agent delivered to the airway is not diluted by additional air entrained through the nebulizer during inhalation.

[026] Substantially all of the therapeutic agent in the solution may be delivered to the airway.

[027] The therapeutic agent may be an alcohol, antibiotic, antiviral, antifungal, steroid, bronchodilator, mucolytic, or antineoplastic agent.

[028] In some embodiments the alcohol is ethanol, for example the solution may comprise about 30% (v/v), about 40% (v/v), about 50% (v/v), about 60% (v/v), about 70% (v/v), about 80% (v/v), about 90% (v/v) ethanol, or about 95% (v/v) ethanol.

[029] In one embodiment the solution is about 40% (v/v), about 60% (v/v), or about 80% (v/v) ethanol in water.

[030] In some embodiments, the therapeutic agent is present in a diluent. The diluent may be water, saline, or other suitable diluent.

[031 ] In some embodiments, the therapeutic agent is present in a solution that is buffered or in which the pH is titrated with an acid or alkali.

[032] In some embodiments the pH of the solution may be below or above 7, preferably the pH is in the range of 7-8. [033] The solution may further comprise at least one of a local anaesthetic, chelator, surfactant, osmotic agent, nitric oxide donor, or D-amino acid.

[034] In some embodiments the subject has a respiratory disease, for example ARDS, cystic fibrosis, a neoplastic disease, a bacterial, viral, fungal disease, an allergic or other immune disease, an auto-immune disease, inflammation associated with any of these respiratory diseases, allergic disease, immune disease, or auto-immune disease, or inflammation occurring independently of disease.

[035] The viral disease may be influenza, respiratory syncytial virus, SARS, MERS, or COVID-19.

[036] The bacterial disease may be due to mycobacterium tuberculosis or a non- tuberculous mycobacterium such as, but not exclusively, Mycobacterium avium complex, Mycobacterium kansasii, and Mycobacterium abscessus.

[037] The inflammation may be associated with Acute respiratory Distress Syndrome (ARDS)

[038] In some embodiments the level of ethanol in the subject's breath becomes undetectable by about 15 minutes, or by about 20 minutes after the ethanol is delivered to the airway.

[039] In one embodiment the subject's blood ethanol does not exceed 0.02 g/dL, preferably the subject's blood ethanol does not exceed 0.01 g/dL.

[040] The methods may further comprise having the subject inhale the ethanol aerosol and/or vapour from the closed circuit nebulizer system over a period of about 1 min to about 30 mins, or about 45 mins, or about 60 mins.

[041 ] The subject may inhale the ethanol aerosol and/or vapour from the closed circuit nebulizer system multiple times, for example at intervals from about 15 mins to about 2 hours, or from about 3 hours to about 12 hours. Preferably, the interval is about 2 hours, about 4 hours, about 6 hours, or about 8 hours.

[042] In a fourth aspect there is provided a kit for delivery of a therapeutic agent to the airway of a subject, the kit comprising the closed circuit nebulizer system of the first aspect and a solution comprising the therapeutic agent. The kit may further comprise a mouthpiece or mask adapted to connect to the outlet of the closed circuit nebulizer system, either directly or via a suitable tube or conduit. Definitions

[043] Throughout this specification, unless the context clearly requires otherwise, the word 'comprise', or variations such as 'comprises' or 'comprising', will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[044] Throughout this specification, the term 'consisting of' means consisting only of.

[045] Throughout this specification the term 'consisting essentially of' means that any elements listed after the term are mandatory, but other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements may be included.

[046] Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present technology. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present technology as it existed before the priority date of each claim of this specification.

[047] Unless the context requires otherwise or specifically stated to the contrary, integers, steps, or elements of the technology recited herein as singular integers, steps or elements clearly encompass both singular and plural forms of the recited integers, steps or elements.

[048] In the context of the present specification the terms 'a' and 'an' are used to refer to one or more than one (i.e., at least one) of the grammatical object of the article. By way of example, reference to 'an element' means one element, or more than one element.

[049] In the context of the present specification the term 'about' means that reference to a figure or value is not to be taken as an absolute figure or value, but includes margins of variation above or below the figure or value in line with what a skilled person would understand according to the art, including within typical margins of error or instrument limitation. In other words, use of the term 'about' is understood to refer to a range or approximation that a person or skilled in the art would consider to be equivalent to a recited value in the context of achieving the same function or result.

[050] The terms 'treating', 'treatment' and 'therapy' are used herein to refer to curative therapy, prophylactic therapy, palliative therapy and preventative therapy. Thus, in the context of the present disclosure the term 'treating' encompasses curing, ameliorating or tempering the severity of a medical condition or one or more of its associated symptoms. [051 ] The terms 'therapeutically effective amount' or 'pharmacologically effective amount' or 'effective amount' refer to an amount of an agent sufficient to produce a desired therapeutic or pharmacological effect in the subject being treated. The terms are synonymous and are intended to qualify the amount of each agent that will achieve the goal of improvement in disease severity and/or the frequency of incidence over treatment of each agent by itself while preferably avoiding or minimising adverse side effects, including side effects typically associated with other therapies. Those skilled in the art can determine an effective dose using information and routine methods known in the art.

[052] A 'pharmaceutical carrier, diluent or excipient' includes, but is not limited to, water, or any physiological buffered (i.e., about pH 7.0 to 7.4) medium (e.g., saline) comprising a suitable water soluble organic carrier, conventional solvents, dispersion media, fillers, solid carriers, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents. Suitable water soluble organic carriers include, but are not limited to, saline, dextrose, corn oil, dimethylsulfoxide, and gelatin capsules. Other conventional additives include lactose, mannitol, corn starch, potato starch, binders such as microcrystalline cellulose, cellulose derivatives such as hydroxypropylmethylcellulose, acacia, gelatins, disintegrators such as sodium carboxymethylcellulose, and lubricants such as talc or magnesium stearate.

[053] In the context of this specification the term 'administering' and variations of that term including 'administer' and 'administration', includes contacting, applying, delivering or providing a therapeutic agent in an aerosol form to a subject by any appropriate means.

[054] Those skilled in the art will appreciate that the technology described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the technology includes all such variations and modifications. For the avoidance of doubt, the technology also includes all of the steps, features, and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps, features and compounds.

[055] In order that the present technology may be more clearly understood, preferred embodiments will be described with reference to the following drawings and examples.

Brief Description of the Drawings

[056] Figure 1. This shows the functionality of the closed circuit system where the bivalved mouthpiece during inhalation synchronises flow of aerosol from the collapsible reservoir into the subject’s mouth, and during exhalation directs exhaled air out through the exhaust while the reservoir refills with aerosol from the nebulizer. [057] Figure 2. Figures 2A to 2C illustrates the advantages of adding a conduit (in this case extension tubing) to the closed circuit nebulizer system between the reservoir outlet and the mouthpiece. These include: (a) improved subject comfort; (b) reduced risk of nebulizer contamination from infected subjects; and (c) the reduction in droplet size as shown results in increased overall deposition and more peripheral distribution of drug to the lung. Box 1 indicates the nebuliser outlet, in this embodiment with a 5pm droplet size. Box 2 indicates the outlet of the collapsible reservoir, in this embodiment with a 3pm droplet size. Box 3 indicates the mouthpiece, in this embodiment with a 1 pm droplet size. Figures 2B and 2C show the attachment of an exhalation conduit (tubing) to a filter (viral filter in this case) for preventing escape of exhaled infectious organisms to the environment.

[058] Figure 3. Drug delivery of salbutamol (mg) from a closed circuit nebulizer system attached to a Pari LC Plus nebulizer compared to a Pari LC Plus nebulizer alone. This chart shows that compared to the nebulizer alone, the closed circuit nebulizer system delivers almost 3 times the amount of salbutamol (2.6 mg vs 0.9 mg) and negligible amount is wasted in exhalation (0.1 mg vs 1 .3 mg).

[059] Figure 4: Scintigraphic images of the lungs showing more even and more peripheral lung deposition of Tc-99-labelled fluticasone when inhaled through a closed circuit nebulizer system attached to a Pari LC Plus nebulizer compared to a Pari LC Plus nebulizer alone.

[060] Figure 5: Breath alcohol (ethanol) readings from patients starting immediately after ceasing inhaled aerosolized ethanol and repeated at one-minutely intervals (where possible). Levels reaching zero by 15 minutes indicates the readings were detecting clearance of ethanol from lung tissue which clears rapidly, and not clearance of ethanol from blood which may take up to 24 hours to reach zero depending on the starting level in the blood.

[061] Figure 6: Cell counts from bronchoalveolar lavage (BAL) after inhalation of aerosolized ethanol by healthy mice. There no significant changes in BAL cell counts between the control (saline) and ethanol treated groups of healthy mice.

[062] Figure 7: Elastase, IL6 and TNF levels from BAL after inhalation of aerosolized ethanol. There was no significant changes in levels between the control (saline) and ethanol treated groups of healthy mice

[063] Figure 8: Airway morphometry. There was also no significant difference in airway morphometry (airway smooth muscle (ASM), lumen and airway epithelium) between the control (saline) and ethanol treated groups of healthy mice. [064] Figure 9: Cell counts from bronchoalveolar lavage (BAL) after inhalation of aerosolized ethanol by mice infected with influenza demonstrating increased macrophage numbers at 60% and 80% concentration with no concomitant rise in neutrophil count.

[065] Figure 10: Macrophage numbers after inhalation of aerosolized ethanol by healthy mice (A) and mice infected with influenza (B). Inhaled ethanol increases macrophage numbers in an infection-specific manner.

[066] Figure 11 : IL6 (A) and TNF (B) levels after inhalation of aerosolized ethanol by mice infected with influenza. There were no clear differences although IL-6 was more highly expressed after treatment with 60% Ethanol, but not 40% or 80% .

[067] Figure 12: Viral load after inhalation of aerosolized ethanol by mice infected with influenza. There is strong trend towards reduced viral numbers in the group receiving 80% ethanol.

Description of Embodiments

[068] Embodiments of the technology disclosed herein relate to systems and methods for delivering one or more therapeutic agents to the respiratory tract by inhalation and methods for the treatment of a respiratory disease.

[069] In embodiments disclosed herein, the closed circuit nebulizer system comprises a closed circuit nebulizer device having a filter, preferably a viral filter. In other embodiments disclosed herein, the delivery method involves the use of the closed circuit nebulizer system to deliver one or more therapeutic agents to the respiratory tract in the form of an aerosol. Other embodiments disclosed herein relate to a method for treating a respiratory infection in a subject, the method comprising using the closed circuit nebulizer system to administer an aerosol containing one or more therapeutic agents to the airway of a subject. Other embodiments disclosed herein relate to kits comprising the closed circuit nebulizer system and optionally including instructions for use.

[070] The methods, systems and kits disclosed herein are suitable for the treatment of various airway diseases, including, but not limited to, a viral infection, bacterial infection, or fungal infection of the lungs or upper airways, neoplastic airway diseases such as lung cancer, e.g., mesothelioma, or genetic diseases of the airways such as cystic fibrosis, among others.

[071 ] Advantageously, the technology provides for topical treatment of respiratory tract infection, particularly viral and other infections resistant or difficult to treat with oral or systemic treatments. The closed-circuit nebulizer system allows the active therapeutic agent to be inhaled, whist preventing volatile alcohol vapour escaping to the surrounding environment.

[072] Preferred embodiments of the technology disclosed herein include: a) a closed circuit nebulizer system comprising a nebulizer device, a collapsible reservoir, and an exhaust valve; b) a closed circuit nebulizer system comprising a nebulizer device, a collapsible reservoir and a viral filter; c) embodiment a) or b) in which the closed circuit nebulizer system further comprises a therapeutic agent; d) embodiment c) in which the therapeutic agent is a hazardous agent, a cytotoxic agent, or a volatile agent; e) a kit comprising a closed circuit nebulizer system comprising a nebulizer device, a collapsible reservoir and a viral filter, and one or more therapeutic agents; f) embodiment c), d) or e), wherein the therapeutic agent comprising ethanol; g) embodiment c), d), e) or f), wherein the therapeutic agent is aerosolised; h) a method of delivering a virucidal therapeutic agent to the respiratory tract of a subject as defined in embodiment e), wherein the therapeutic agent comprises ethanol; i) a method of delivering a virucidal therapeutic agent to the respiratory tract of a subject as defined in embodiment f), wherein the therapeutic agent comprises ethanol in an amount of 40% v/v, or 45% v/v, or 50% v/v, or 55% v/v, or 60% v/v, or 65% v/v, or 70% v/v, or 75% v/v, or 80% v/v, or 85% v/v, or 90% v/v with a diluent; j) In some embodiments, the diluent is water, saline, or other medically acceptable diluent; k) a method of delivering a virucidal therapeutic agent to the respiratory tract of a subject as defined in embodiment e) or f), wherein the therapeutic agent comprises ethanol and a second active agent; l) embodiment k) in which the second active agent is selected from an antiviral agent; m) embodiment k) in which the second active agent is selected from an adjuvant, including but not limited to, chelators such as, EDTA, CaEDTA, and Deferroxamine; n) embodiment k) in which the second active agent is an antibiotic; o) embodiment k) where a third or more active is selected from any combination of two or more of the additional active agents recited in embodiments I), m), and n), above; p) an embodiment where the nebulizer may comprise a jet nebulizer, vibrating mesh nebulizer, or ultrasonic nebulizer.

[073] Advantages of preferred embodiments of the present technology may include one or more of:

• the ability to expose a virus infection (or other pathogenic organisms) in the upper and lower airway to virucidal concentrations of a virucidal agent in an efficacious amount;

• the ability to deliver by inhalation a therapeutic agent, such as ethanol, to a viral infection in the upper or lower respiratory tract at controlled and/or high concentrations;

• reducing environmental loss of a therapeutic agent or aerosol by the use of closed- circuit nebulizer system;

• improved drug dose delivery efficiency with improved efficacious effect;

• preventing dilution of the drug thereby facilitating efficient delivery of the drug to the lung and improving the effect of the treatment;

• preventing escape of exhaled viral particles by inserting a viral filter on the exhalation port; and

• in a preferred embodiment in which the therapeutic agent is ethanol, the technology provides a low-cost and readily available system and method for treating a viral infection, including, e.g., coronavirus infection.

Closed circuit nebulizer system

[074] The closed circuit nebulizer system comprises a nebulizer in fluid communication with a spacer having a collapsible reservoir for containing the aerosol, and an outlet (preferably bi-valved) which synchronizes with inhalation and exhalation to permit the user to inhale a consistent dose of medication without the requirement for co-ordination, timing, or a specific breathing pattern, and to prevent environmental release of the aerosol on exhalation. The nebulizer portion of the system comprises a container holding a solution of therapeutic agent. The nebulizer may be a jet nebulizer, vibrating mesh nebulizer, or an ultra-sonic nebulizer.

[075] It is envisaged that the reservoir can be retrofitted to existing nebulizers. In general, the collapsible reservoir may be bag shaped and dimensioned to receive the aerosol from the nebulizer. Typically the reservoir is housed within a body and comprises an inlet adapted to receive a nebulizer, through which the aerosol is discharged from the nebulizer into the reservoir, and an outlet, via which the contents of the reservoir can be inhaled with the reservoir collapsing under the negative pressure created by the inhalation. The collapsing reservoir promotes the emptying of the reservoir via the outlet and maximizes the delivery of the aerosol to the lungs of a subject. The inlet of the reservoir is adapted to receive the nebulizer and can be modified to receive any nebulizer known in the art.

[076] The reservoir may be made of a metallised film or metallised biaxially-oriented polyethylene terephthalate (BoPET) or another similar flexible polymer, typically Mylar®. Metallisation renders the material non-static. The reservoir may be made of silicone or similar rubberized compounds, including polyurethane and santoprene. The reservoir can be treated with an antistatic agent forming a static dissipative coating or layer on the reservoir. Similarly, the body can be made from, laminated to, or coated with, a metal or an anti-static coating or layer. The body is typically made from a metal such as aluminum, a metallized compound (such as metallized plastic), or a plastic.

[077] The reservoir may be constructed with one or more seams of, or containing, a resiliently flexible or shape-memory material providing the reservoir with shape memory that opens the reservoir in an inflated position when preparing it for use. The one or more seams may facilitate collapse of the reservoir in a predetermined manner, for example to assist exit of the aerosol via the outlet.

[078] The outlet may be in the form of a mouthpiece or may be adapted to connect to a mouthpiece or mask, either directly or via a conduit, Preferably the connection is gas-tight. [079] The conduit may be in the form of tubing, preferably soft, lightweight, and flexible. The length and dimensions of the conduit should be such that allows unimpeded flow of the aerosol from the nebulizer to the user on inhalation, and at the same time minimizes “rain- out” (condensation) of aerosol within the tubing.

[080] The outlet may comprise a valve assembly or be connected to a valve assembly to control both the flow of aerosol from the reservoir and the flow of the subject's exhaled breath. In one embodiment the valve assembly comprises a check valve that prevents exhaled breath from entering the reservoir and second check valve or outlet that directs the subject's exhaled breath to an exhaust filter. These features combined with the collapsible reservoir form a closed circuit system where, in use, the aerosol is received by and retained in the reservoir until inhaled by the subject, thereby ensuring that all of the aerosol produced by the nebulizer is inhaled by the subject and none is lost to the environment. At the same time, any exhaled air (breath) containing any unused aerosol (or any of its components) and potentially infectious agents is directed by the exhaust valve to the viral filter, thereby preventing contamination of the surrounding environment by these elements. The collapsible reservoir also allows subjects with limited respiratory strength or rate to receive an effective dose of a pharmaceutical agent in the aerosol.

[081 ] Operation of the valve assembly and the closed circuit nebulizer system is illustrated in Figure 1 . An embodiment of the closed circuit nebulizer system having a viral filter is illustrated in Figure 2. In this embodiment the outlet is connected to a conduit and the valve assembly is distal to the outlet at an end of the an extension tube that may be used for additional safety, efficacy and ease-of-use. Figure 2 also illustrates the use of an exhaust filter.

[082] As indicated in Figure 2, the droplet size of the aerosol may vary across the system. For example when the aerosol is formed, the average droplet size may be around 5 pm which may reduce to around 3 pm at the outlet and further reduce to around 1 pm at the mouthpiece.

[083] Reservoirs can be sized to suit individual subject's pathology, age, physical size, lung capacity, inspiration strength, and respiration rate. In some embodiments the reservoir has a maximum volume of about 100 cm ³ to about 3500 cm ³ .

[084] A nebulizer can be any device used to produce an aerosol. In some embodiments the nebulizer may be any jet or vibrating mesh or ultrasonic nebulizer known in the art.

[085] Jet nebulizers use pressurised gas (usually air or oxygen). The Bernoulli principle dictates the function of jet nebulizers, in which the compressed gas is passed through a narrow opening, which creates an area of low pressure at the outlet of a nearby solution- filled container. The solution is then drawn from the reservoir and an aerosol generated by the action of the compressed gas on the solution. These nebulization methods require very little patient coordination or skill, although when used without the collapsible reservoir described herein most of the aerosol is lost during exhalation and pulmonary deposition is limited to approximately 10% of the total dose. Low flows of pressurised gas prolong nebulization time and can be an advantage when requiring prolonged aerosol exposures in preference to rapid dosing.

[086] Ultrasonic nebulizers typically use a piezoelectric crystal that vibrates at a high frequency (typically 1-3 MHz) to generate a fountain of the solution in the nebulization chamber. Ultrasonic nebulizers can create an aerosol more quickly than jet nebulizers however, they are relatively ineffective if the solution is highly viscous or contains insoluble particles (i.e., a suspension). Again, delivery efficiency is poor due to loss of aerosol to the outside environment during exhalation. [087] Alternatively the nebulizer may be a vibrating mesh nebulizer. Vibrating mesh nebulizers are silent and do not require compressed air and can be customized for use in mechanically ventilated patients. Standard vibrating mesh nebulizers also lose more than half of the dose to the outside atmosphere during exhalation. Complex technology in some of the newer nebulizers reduces this loss although these nebulizers are bulky and very expensive.

[088] In one embodiment the bag shaped collapsible reservoir is attached to the nebulizer body and the body includes the inlet and outlet wherein the body and bag combine to form a collapsible chamber for receiving the aerosol, such that the inlet and outlet are in fluid communication with the collapsible chamber.

[089] The inlet can include a connector for receiving, directly, or via a conduit, to a nebulizer, which in use generates an aerosol of a solution comprising a therapeutic agent. That is, the nebulizer is in direct fluid communication with the reservoir to allow generally unimpeded aerosol containment in the reservoir.

[090] The outlet may comprise a valve assembly or be connected to a valve assembly to allow the inhalation of the aerosol while preventing the subject's exhaled breath entering the reservoir. The valve assembly comprises a body including an inlet through which the aerosol is received, an outlet which exits to an exhaust filter, a passageway connecting the inlet and outlet, and at least one selectively activatable occlusion member interspersed between the inlet and outlet that prevents the flow or entrainment of exhaled breath into the reservoir when the pressure of the exhaled breath exceeds the pressure of the aerosol entering the inlet of the valve from the reservoir.

[091] In one embodiment, the occlusion member is in a flexible bicuspid flap which can readily bend or flex under pressure from the aerosol entering from the reservoir, but which closes when a slight overpressure is applied to it by exhaled breath.

[092] The valve assembly further includes at least one exhaust passage to direct the subject's exhaled breath towards an exhaust filter upon occlusion of the passageway of the body by the occlusion member upon activation of the occlusion member when, for example, a subject exhales into a mouthpiece or facemask attached to the outlet of the valve assembly.

[093] Each exhaust passage is configured to exit from the body of the valve assembly away from the face of the subject. The size, shape, number, and dimension of each exhaust passage can be chosen to account for respiratory rate, exhalation force, filter resistance and the like. [094] In one embodiment each exhaust passage is associated with a flexible flap member that occludes the exhaust passage during inhalation, and opens to allow exhaled breath to pass through the exhaust passage and past the flexible flap members towards the exhaust filter. In another embodiment, the exhaust passage, instead of being part of the valve assembly, may be located anywhere on the mouthpiece or facemask.

[095] The outlet of the reservoir is adapted to sealingly engage a mouthpiece or facemask. In some embodiment the outlet of the reservoir includes an integral mouthpiece or facemask.

[096] In one embodiment the spacer device and nebulizer disclosed in the inventor's copending application PCT/AU2018/050343 may be used when adapted to include a suitable exhaust filter.

[097] The exhaust filter may be any filter, or combination of filters known in the art for capturing exhaled infection agents and pharmaceutical agents. In preferred embodiments the filter is a viral filter. In some embodiments, the filter may comprise activated charcoal, or any substance that can absorb potentially toxic aerosol or vapours. Additionally or alternatively the filter may comprise a mesh network, a HEPA filter, and/or a 0.22pm filter.

[098] The filter is in fluid communication with the exhaust passage. The filter may be a cassette type filter that is mounted directly to the exhaust passage, for example the filter may be adapted to be mounted directly to the facemask or may be connected to the exhaust passage by a conduit.

[099] Different filters may be used depending on the application. For example if the subject has a viral respiratory disease and is being treated with an alcohol or an antiviral agent a HEPA filter may be use to capture any exhaled viral particles and an activated charcoal filter may be used to capture exhaled alcohol and antiviral agent. Alternatively, if the subject is being treated using an inhaled antineoplastic drug for a lung cancer, only an activated charcoal (or other appropriate substance) filter may be required to capture any exhaled drug.

[0100] In use, a conventional nebulizer will be assembled with a cup or container with the solution and this will be attached to the reservoir. On activation of the nebulizer, for example by connecting it to a source of pressurized gas, an aerosol of the solution is formed and enters and inflates the collapsible reservoir. The subject then breathes in with the exhaled breath being passed towards the filter via the exhaust passages.

[0101] The total amount of the aerosolised solution emitted from the nebulizer into the reservoir is available of inhalation by the subject and all of the exhaled pharmaceutical agent, and any infectious agents can be filtered. Accordingly the closed circuit nebulizer system and its use in the methods and kits described herein provides one or more of the following advantages:

• improved efficiency of aerosol delivery to the airways;

• the volume of aerosol inhaled by the subject comes exclusively from the reservoir such that no additional air is entrained through the nebulizer during inhalation ensuring that the aerosol is not diluted;

• reduction or elimination of contamination by the aerosol or exhaled infections agents;

• reduction or elimination of aerosol loss to the environment;

• subject's choice of breathing pattern;

• simplicity and ease of use; and

• low cost.

Methods

[0102] Advantageously the closed circuit nebulizer system with the collapsible reservoir allows delivery of a therapeutic agent to be achieved by any breathing pattern the subject prefers and without affecting the dose delivered to the airway. For example, slow, deep breathing or normal tidal breathing may be used.

[0103] The methods comprise generating an aerosol from a solution of the therapeutic agent using the closed circuit nebulizer system and administering the aerosol to the airway of the subject. The closed circuit nebulizer system comprises a mouthpiece or mask and the aerosol is administered to the subject's airway during inhalation via the mouthpiece or mask.

[0104] The therapeutic agent may be administered over a suitable period of time depending on the particular circumstances, for example, about 5 mins, about 10 mins, about 15 mins, about 20 mins, about 30 mins, about 45 mins, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours or more.

[0105] In a preferred embodiment the therapeutic agent is ethanol, which is administered over a period of about 1 min, about 5 mins, about 10 mins, about 15 mins, about 20 mins, about 25 mins, or about 30 mins.

[0106] The dose of ethanol may by about 5ml, about 15ml, about 20ml, about 25ml, about 30 ml, about 35 mL, about 40 mL, about 45 mL, about 50mL, or more [0107] In another embodiment the administration may be repeated at any time interval considered appropriate by the treating physician or medical professional. In a preferred embodiment, the interval is about 15 mins, about 30 minutes, about 45 minutes, about one hour, about one and a half hours, about 2 hours, about 3 hours, about 4 hours or about 6 hours, or about 12 hours. In one embodiment the administration may be repeated an interval of 2 hours.

[0108] The therapeutic agent may be administered on consecutive days. For example the pharmaceutical agent may be administered for 2 days or more, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days. The pharmaceutical agent may be administered daily for between 2 and 28 days, 1 week, 2 weeks, 3 weeks or 4 weeks, and so on.

[0109] In one embodiment the pharmaceutical agent is an antimicrobial agent and is administered on consecutive 2, 3, 4, or 5 days, or longer if necessary or appropriate.

[0110] In other embodiments the therapeutic agent, such as ethanol, may be delivered intermittently. The administration regime may depends on a variety of factors, including the age, weight, sex, and medical condition of the subject, the severity and identity of the disease, as well as the pharmacokinetic properties (e.g., adsorption, distribution, metabolism, excretion) of the therapeutic agent, and thus may vary widely. The methods described herein may be administered as often as necessary and for the period of time judged necessary by the treating physician. One of skill in the art will appreciate that the administration regime or therapeutically effective amount of the compound to be administrated may need to be optimized for each individual.

Solution

[0111] The solution comprises a therapeutic agent suitable for the treatment of a respiratory disease together with at least one pharmaceutical carrier, diluent or excipient. The therapeutic agent may be an antimicrobial, for example an antiviral, antibacterial or antifungal. In preferred embodiments, the therapeutic agent is an antiviral agent.

[0112] A number of antiviral agents are available in commercial use, in clinical evaluation and in pre-clinical development, can be selected for treatment of viral airway infection. These include antiviral agents such as protease inhibitors, helicase inhibitors, entry inhibitors, catalase, and sialidase. Other antiviral agents include cidofovir, oseltamivir (Tamiflu®), zanamivir (Relenza®), ribavirin, remdesivir, penciclovir, faviparvir, nafamostat, nitazoxanide, camostat mesylate, interferon a (e.g. , interferon alpha and beta), ritonavir, lopinavir, ASC09, azvudine, baloxavir marboxil, darunavir, cobicistat, chloroquine, zanamivir, acyclovir, and laninamivir. [0113] Similarly, any known antibacterial is also suitable for use in the methods. For example, the antibacterial agent may be any one or any combination of agents selected from polymyxin E, tobramycin, amikacin, aztreonam, ciprofloxacin, levofloxacin, linezolid, vancomycin, doxycycline, gatifloxacin, fosfomycin, ceftazidime, fucidin, and rifampicin.

[0114] The antifungal agent may be one or any combination of agents selected from pentamidine, amphotericin, voriconazole, itraconazole, caspofungin, fluconazole, and pneumocandin.

[0115] In some embodiments the therapeutic agent may be an antineoplastic agent, such as 5-fluorouracil (5-FU), a taxane, doxorubicin, gemcitabine and 9-nitrocamptothecine (9- NC), bevacizumab, PD-L1 , CTLA-4, and cisplatin liposomes.

[0116] In some embodiments the solution may comprise a bronchodilator such as a beta-2 agonist, and anticholinergic or theophylline. Suitable beta-2 agonists include albutamol, salmeterol, formoterol and vilanterol. Representative examples of anticholinergics that may be used include ipratropium, tiotropium, aclidinium and glycopyrronium.

[0117] In one embodiment the therapeutic agent is dissolved in sodium chloride, for example, hypertonic saline which is useful to mobilise mucus and improve airway clearance. Hypertonic saline refers to any saline solution with a concentration of sodium chloride (NaCI) higher than physiological concentration (0.9%). Commonly used preparations include 2%, 3%, 5%, 7%, and 23% (w/v) NaCI.

[0118] The solution may comprise DNAse (pulmozyme) to thin mucus.

[0119] In some embodiments the solution may comprise a steroid such as beclomethasone, budesonide, fluticasone, triamcinolone acetonide, and flunisolide.

[0120] In one embodiment the solution comprises an alcohol, such as ethanol or isopropanol, preferably ethanol.

[0121] In a particularly preferred embodiment, the therapeutic agent is ethanol.

[0122] In an especially preferred embodiment, the therapeutic agent is ethanol and the respiratory infection is a viral infection, such as a coronavirus infection, e.g., SARS, MERS or Covid-19.

[0123] Ethanol in concentrations above 30% v/v in water is a potent killer of viruses and bacteria, including coronavirus, and is widely used topically in hand sanitisers or disinfectant solutions to sterilise body or building surfaces where virus (or bacteria) may be present.

[0124] When used as described herein an inhaled ethanol aerosol can be used to topically treat viral or bacterial infections in either the upper or lower airways. This is contrary to widely held beliefs by both the medical and lay population that inhaled ethanol will create a number of undesirable side effects locally on the airways themselves, and/or systemically following absorption into the bloodstream from the lung.

[0125] Alcohol (ethanol) between 60-80% concentration is commonly used to topically disinfect skin and surfaces based on WHO data which shows that 100% of viruses will be killed on contact with 30 seconds exposure at concentrations above 30%. Inhaled aerosolised ethanol deposited on airway mucosa and maintaining concentrations >30% after dilutional factors have been accounted are therefore desirable. Previous studies show that during inhalation, ethanol with its high air/tissue co-efficient is absorbed into bronchial mucosa, secretions, and lung interstitial tissue, equilibrating rapidly with the concentration in the airway. Accordingly, in one embodiment inhaling ethanol for 30 minutes creates sufficient ethanol concentration gradient in the airway to force alcohol to penetrate sufficiently into lung tissue to reach and kill virus, bacteria, or fungi on contact

[0126] The ethanol concentration in the solution may be up to about 95% (v/v), for example the ethanol concentration may be about 30% (v/v), about 35% (v/v), about 40% (v/v), about 45% (v/v), about 50% (v/v), about 55% (v/v), about 60% (v/v), about 65% (v/v), about 70% (v/v), about 75% (v/v), about 80% (v/v), about 85% (v/v), about 90% (v/v), or about 95% (v/v).

[0127] In one embodiment ethanol concentration in the solution is about 40% (v/v), about 60% (v/v), or about 80% (v/v).

[0128] The diluent is typically water or saline, but any physiologically acceptable solution may be used as a diluent.

[0129] The solution can further comprise at least one of a local anaesthetic, a chelator, a surfactant, osmotic agent, nitric oxide donor, or D-amino acids.

[0130] Ethanol has known anaesthetic properties by its action on sensory nerve conduction. Topical exposure to nerve tissue, results in mild to moderate local analgesia or “numbness”. This effect takes a few minutes to develop and high concentrations of inhaled ethanol exposure to the lower airway may cause cough initially, subsiding after a few minutes. Accordingly, in one or more embodiments a local anaesthetic may be included in the solution. Suitable local anaesthetics for inhalation will be well known to those skilled in the art and include lignocaine (lidocaine), benzocaine, bupivacaine, marcaine, prilocaine, chloroprocaine, procaine, proparacaine, tetracaine, amylocaine, oxybuprocaine, articaine, dibucaine, deidocaine, levobupivacaine, mepivacaine, ropivacaine, sameridine, tonicaine, cinchocaine, among others. [0131] Alternatively or in addition, the solution may initially comprise a subtherapeutic amount of the therapeutic agent (e.g., ethanol) that is readily tolerated and the concentration may be increased up to the required concentration over a few minutes to avoid or minimise cough or discomfort.

[0132] Chelators (also known as sequestering agents) sequester metallic cations (e.g. iron, calcium, magnesium, etc) that are necessary for bacterial growth, and form a stable, water- soluble complex. Osmotic agents (e.g. mannitol) can be used to increase the influx of water into mucus, such as the thick dehydrated mucus found in the airways of patients with cystic fibrosis. Nitric oxide donors and D-amino acids have both been reported to exhibit biofilm dispersal effects.

[0133] Suitable chelating agents are known in the art and include, for example, citric acid, phosphates, the di-, tri- and tetra-sodium salts of ethylene diamine tetraacetic acid (EDTA), calcium salts of EDTA, ethylene glycol-bis-(b-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA); 1 ,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA); ethylene-N,N' diglycine (EDDA); 2,2'-(ethylendiimino)-dibutyric acid (EBDA); lauroyl EDTA; dilauroyl EDTA, triethylene tetramine dihydrochioride (TRIEN), diethylenetriamin-pentaacetic acid (DPTA), triethylenetetramine hexaacetic acid (TTG), deferoxamine (DFO), deferasirox (DSX), dimercaprol, zinc citrate, penicilamine succimer, editronate, sodium hexmetaphosphate, edetate calcium disodium, D-penicillamine, polyphenols, gallol, catechol, dimercaprol, tetrathiomolybdate, lactoferrin, clioquinol and combinations thereof. Preferably, the chelating agent is a pharmaceutically acceptable chelating agent.

[0134] In one embodiment, the chelating agent is ethylene diamine tetraacetic acid (EDTA). In another embodiment, the chelating agent is deferoxamine (DFO). In another embodiment, the chelating agent is deferasirox (DSX).

[0135] In one embodiment, the therapeutic agent is a surfactant, a solvent, or an agent that has surfactant properties.

[0136] In a particularly preferred embodiment, the solvent, or agent with surfactant properties is ethanol.

[0137] Surfactants are amphipathic substances having hydrophilic and hydrophobic groups and are commonly used in pharmaceuticals for a range of purposes, including e.g., solubilization of hydrophobic drugs in aqueous media, emulsions, and as excipients to improve drug absorption and penetration.

[0138] In one embodiment, the therapeutic agent is an anti-inflammatory agent.

[0139] In a particularly preferred embodiment, the anti-inflammatory agent is ethanol. [0140] In one embodiment, the anti-inflammatory agent may be a non-steroidal agent, including but not limited to, ibuprofen, diclofenac, aspirin, or naproxen.

[0141] In one embodiment, the inhaled therapeutic may be an immune-modulator.

[0142] In a particularly preferred embodiment, the immune-modulator is ethanol.

[0143] In one embodiment the immune-modulator is a biological agent which may be either naturally occurring or made using recombinant techniques. Suitable biological immune- modulators include, but are not limited to cytokines, (including IL6, IL17, IL8) , anti-CXCR4 (including AD-214), and specific immunoglobulins listed for treatment of asthma, allergies, hyper-immune responses, and hyper-inflammatory responses (so-called “cytokine storm”).

Aerosol

[0144] The human airway comprises a series of narrowing branches, including the trachea, bronchi, bronchioles and alveoli. The optimal location for drug deposition following inhalation varies, depending on the indication for therapy and the physicochemical properties of the drug. Many lower respiratory tract infections feature both purulent tracheobronchitis and alveolar disease, which require deposition of the inhaled antimicrobial throughout the lungs. In contrast, infections such as Pneumocystis carinii pneumonia, which are confined to the alveolar regions of the lungs, are likely to benefit from peripheral delivery.

[0145] Several factors affect the pattern of drug deposition in the lungs. Aerosol particles with a mass median aerodynamic diameter (MMAD) of 1-2 pm can be deposited deeply and evenly in the lung with up to 90% efficiency if inhaled slowly and deeply. Larger particles, particularly when inhaled too quickly, are primarily deposited in the mouth, throat and upper airway. In particular, forceful ejection of aerosol from a pressurized metered-dose inhaler (MDI) and high flow, high energy inhalation from a dry powder inhaler (DPI) result in particles travelling at high velocities with 80% or more of the drug applied or administered to the oropharynx, i.e. the back of the throat. Thus, drug deposition is less likely to end up in the lungs with MDI, DPI and standard nebulizers.

[0146] The closed circuit nebulizer system in accordance with the present technology is used to generated an aerosol of the solution. The operating parameters of the nebulizer portion of the system can modulated to create an aerosol with predetermined average droplet diameters for various solutions. Increasing the volume of the reservoir reduces droplet agglomeration and allows for evaporation, further contributing to the desired outcome of smaller droplets. [0147] The length of the extension tubing also increases evaporative reduction in droplet size. Consistent production of droplets under 3 microns in diameter is highly desirable for increasing the likelihood of the drug reaching peripheral and diseased parts of the lung.

[0148] The closed-circuit system described herein has the capacity to rebreathe and inhale aerosol from the reservoir with incremental breaths provides patients with the capacity to inhale consistent doses of medication even when the patient is ill with breathing difficulties and is unable to take slow deep breaths or any pre-specified breathing pattern.

[0149] Other mechanisms for reducing droplet size within the system include changes in the temperature, concentration, surface tension, viscosity or saturated vapour pressure of the solution can be used to modulate the droplet size of the aerosol. For example, reducing the temperature of the solution (which in turn increases the viscosity) increases the droplet size. Conversely, increasing the temperature of the solution (which reduces the viscosity) decreases the droplet size. In some embodiments, increasing the concentration of the pharmaceutical agent induces a reduction of the surface tension and results in a decrease in the droplet size.

[0150] In some embodiments the average droplet diameter of the aerosol is in the range of about 0.1 pm to about 15 pm, or about 0.25 pm to 6 pm, or about 1 pm to 6 pm, or about 2 pm to 4 pm. Alternatively, the droplets may be have an average diameter of 0.1 pm to 1 .0 pm, 0.2 pm to 0.9 pm, 0.3 pm to 0.8 pm, 0.4 pm to 0.7 pm, or 0.5 pm. By generating an aerosol with droplets having a narrow average diameter distribution, efficiency and repeatability of dosing can be improved. It is preferable that the droplets not only have an average size in the range of 0.1 pm to about 15 pm, or about 0.25pm to 6 pm, or about 1 pm to 6 pm, or about 2 pm to 4 pm but that the average particle diameter is within a narrow range, for example such that at least 80% of the droplets being inhaled by the subject have a particle diameter which is within ±20%, ±10%, or ±5% of the average droplet size.

Subject

[0151] 'Subject' includes any human or non-human mammal. Thus, in addition to being useful for human treatment, the methods, device and kits described herein may also be useful for veterinary treatment of mammals, including companion animals and farm animals, such as, but not limited to dogs, cats, horses, cows, sheep, and pigs. In preferred embodiments the subject is a human.

[0152] The subject may have one or more disease of the respiratory tract. For example, the subject may have an infectious disease of the airways such as sinusitis, influenza, SARS, MERS or COVID-19. In some embodiments the subject may be infected by at least one of a rhinovirus, a coronavirus, an adenovirus, a parainfluenza virus, RSV (respiratory syncytial virus), cytomegalovirus, and a hantavirus.

[0153] In some embodiments, the airways of the subject may be infected with a bacterium. For example, the bacterium may be a group Haemolytic streptococci, Corynebacterium diptheriae, Haemophilus influenzae, Streptococcus pneumoniae, Mycoplasma pneumoniae, Staphylococcus aureus, Streptococcus pyrogenes, Klebsiella pneumoniae, Lebionella spp, Coxiella burnetti, Chlamydia psittaci, Chlamydia, trachomatis, Chlamydia pneumoniae.

[0154] In some embodiments, the bacterial infection may be due to mycobacterium tuberculosis or non-tuberculous mycobacteria such as Mycobacterium avium intracellulare, Mycobacterium kansasii, Mycobacterium marinum, Mycobacterium ulcerans, Mycobacterium cheionae, Mycobacterium fortuitum, or Mycobacterium abscess s.

[0155] The subject may have acute respiratory distress syndrome (ARDS). The ARDS may be caused by any agent (for example an infection) or arise spontaneously, for example as the result of a traumatic injury or post-surgery.

[0156] The subject may have a fungal infection of the airways, such as aspergillosis or blastomycosis.

[0157] In some embodiments the subject may have a genetic airway disease such as cystic fibrosis

[0158] The subject may have a neoplastic airway disease such as lung cancer or mesothelioma. The lung cancer may be small cell or non small cell lung carcinoma, adenocarcinoma, squamous cell carcinoma, large-cell undifferentiated carcinoma, adenosquamous carcinoma, large cell neuroendocrine carcinoma, salivary gland-type lung carcinoma, or lung carcinoids.

[0159] It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

Examples

Example 1. Closed circuit nebulizer system increases drug delivery

[0160] Testing was performed to highlight the enhancements associated with the closed circuit nebulizer system comprising a standard jet nebulizer. The test compared the amount of salbutamol delivered by a Pari LC Plus nebulizer alone or with the closed circuit nebulizer system comprising a Pari LC Plus nebulizer.

[0161] Using a breath simulator set to a normal adult breathing pattern over 8 minutes, salbutamol was collected on an exhaust filter and measured by HPLC. The closed circuit nebulizer system comprising a Pari LC Plus nebulizer delivered 2.7 times the dose (2.59mg vs 0.97mg) and showed negligible wastage (0.1 mg vs 1 .3mg) compared to LC-Plus nebulizer alone. See Figure 3 which shows that the amount of salbutamol delivered is increased by almost 3 times when the closed circuit nebulizer system is added as a spacer to the Pari LC Plus nebulizer, compared to the nebulizer alone

[0162] Subsequently, extension tubing was added between the reservoir outlet and the mouthpiece which improved usability as well as increasing delivery efficiency by decreasing droplet size with evaporation as the droplets passed along the tube.

Example 2. Closed circuit nebulizer system provides more even and more extensive drug deliver compared to nebulizer alone.

[0163] Clinical testing was performed comparing the amount of radio-labelled (Tc-99) fluticasone depositing in the lungs of a healthy adult male following inhalation of a 5ml dose from a closed circuit nebulizer system comprising a Pari LC Plus nebulizer compared to the LC Plus nebulizer alone.

[0164] The amount of Tc-99 labelled fluticasone delivered to the lung by the closed circuit nebulizer system was 57% more than the LC Plus alone. Further, there was more even and more peripheral distribution of drug by the closed circuit nebulizer system compared to the nebulizer alone (see Figure 4).

Example 3. Use of closed circuit nebulizer system to deliver ethanol to healthy volunteers

[0165] A cohort of six healthy volunteers was recruited. Volunteer demographics are shown in Table 1

Table 1 : Patient Demographics

[0166] Each volunteer inhaled three 20ml doses of aerosolised ethanol 2 hours apart. The ethanol concentration in the doses was 40% (v/v), 60% (v/v) and 80% (v/v). That is, each volunteer received three doses of aerosolised 40% ethanol, three doses of aerosolised 60% ethanol, and three doses of aerosolised 60% ethanol. The aerolised ethanol was administered using a closed circuit nebulizer.

[0167] High breathalyser levels immediately following oral inhalation dosing (See Figure 5). This confirms high tissue levels in upper and lower airway (with corresponding negative blood levels) which is indicative of high ethanol levels in the lung tissue, which is the site at which respiratory viruses are active and associated inflammation occurs.

[0168] The high breathalyser levels reducing each minute to zero by 11 minutes after ceasing dosing confirms that these are tissue levels and not blood ethanol levels which can take up to 24 hours to reach zero. This is confirmed in the study by blood ethanol levels of 0.01% or less recorded at the same time as peak breathalyser levels. Levels of zero also indicate that no ethanol remains in the tissues to potentially lead to accumulation and/or damage over repeated or prolonged administration of aerolised ethanol.

[0169] No adverse effects on safety bloods and clinical parameters (including spirometry (FEV1) were observed.

[0170] During treatment, environmental measurements with an infra-red spectroscope continually sampling the air within one metre of the closed circuit nebulizer system did not detect any escaped ethanol. This meets the World Health Organisation requirements for safety and efficiency of experimental inhaled therapeutics for treating COVID-19 and other infections. These results also establish that the closed circuit nebulizer system is safe and protects nearby personnel from potentially toxic aerosol and exhaled viral particles.

Blood Alcohol Readings

[0171] Blood ethanol levels were monitored periodically throughout the treatments and blood levels of ethanol did not exceed 0.01% (0.01 g/dl). Three participants had blood alcohol measurements of 0.01 g/dl recorded immediately following dosing at 80% concentration. All other results were reported as undetectable (<0.01 g/dl).

Breath Alcohol Readings

[0172] Breath alcohol readings were high following dosing by oral inhalation: a. Cycle 1 (40%): average 0.15* b. Cycle 2 (60%): average 0.248 c. Cycle 3 (80%): average 0.371

[0173] Breath alcohol readings rapidly decreased to below safety limit of 0.02: a. Cycle 1 (40%): average 3.8 min* b. Cycle 2 (60%): average 4.9min** c. Cycle 3 (80%): average 7.3min

[0174] All breath alcohol readings were zero at 15 minutes after each dosing cycle and 1 hour following cycle 3 dosing, prior to discharge.

[0175] The rapid reduction of breath alcohol readings after cessation of treatment indicates that the inhaled ethanol (alcohol) has not entered into the blood stream of the patient. If that had occurred the breath alcohol readings would take much longer (as much as 24 hours) to return to normal.

[0176] The rapid reduction of breath alcohol readings is consistent with the high tissue/air partition coefficient of ethanol which is rapidly and preferentially absorbed into the tissues of the lung rather than into the blood stream. The ethanol will rapidly diffuse out of the tissue once the inhalation of aerosolized ethanol ceases.

Vitals/ECG

[0177] All ECGs were reported as normal except for one adverse event being mild tachycardia (heart rate of 109) in context of nasal discomfort.

Wheeze/Cough

[0178] No wheeze detected in any patient. No significant episodes of cough were observed or reported.

Neurological Examination

[0179] One episode of rapidly resolving unsteady gait was observed and assessed as having as unlikely relationship to the treatment. All patients were otherwise normal. Blood Tests

[0180] Blood tests (white cell count, neutrophil count and creatine levels) were conducted on samples obtained at screening, immediately prior to treatment, immediately after treatment and two days after treatment. The results are set out in Tables 2-4

Table 2: White Cell Count - Absolute Value

Table 3 : Neutrophil Count - Absolute Value

Table 4: Creatinine Value

Spirometry

[0181] Spirometry testing was conducted on patients at screening, immediately prior to treatment, immediately after treatment and two days after treatment. The results are set out in Table 5

Table 5: Spirometry results - FEV1 (% Predicted)

Adverse Events

[0182] Throughout the study patient were monitored for adverse events.

[0183] A gait disturbance was observed in patient 2. Specifically this patient exhibited an unsteady gait on performing heal-toe walk test after cycle 1 dosing. The gait disturbance was associated with a postural change and was not present on repeat testing. The gait disturbance was also not apparent after cycle 2 and cycle 3 dosing. The responsible physician made an assessment that the gait disturbance was related to a postural hypotension caused by rising rapidly from a lying to standing position and not related to the treatment.

[0184] Patient 2 reported a headache in the evening after completion of all dosing. The temporal relationship of the headache and the fact that the headache was described as identical to headache associated with dehydration which the participant had regularly experienced in the past, meant that the responsible physician made an assessment that the headache was not related to the treatment.

[0185] Patient 3 described a 'foggy sensation' immediately following cycle 1 completion and which resolved after 5 minutes. The sensation did not present post cycle 2 and cycle 3 dosing. The responsible physician made an assessment that the foggy sensation was not related to the drug and instead related to hyperventilation during inhalation.

[0186] Patient 4 reported nasal discomfort occurring during cycle 2. The nasal discomfort associated with transient tachycardia with HR increase to 109 which resolved following dosing. Tachycardia settled after dosing. The tachycardia did not occur during cycle 1 and 3 dosing. The responsible physician made an assessment that the nasal discomfort was probably related to the treatment and triggered the tachycardia.

[0187] Patient 5 reported a mild vertex headache 10 minutes into cycle 2 dosing. The headache lasted 22 minutes and resolved with water intake. The responsible physician made an assessment that the headache was not related to the treatment and instead related to dehydration.

[0188] Adverse events are summarised in Table 6

Table 6: Adverse Events

Environmental Measurements:

[0189] During the treatments infra-red spectroscopy (specifically using a photoionization detector (PID)) was used to continually measure environmental ethanol at various locations, always within one metre of the participant’s nebuliser delivery system.

[0190] With a closed system nebuliser the mean ethanol levels were 3.1 ppm, with a peak level of 4.4ppm. If an ethanol-based hand sanitiser was used within one meter of the PID peaks of up to 26ppm ethanol were detected. The hand sanitizer in this instance acted as a positive control confirming the sensitivity of PID detection of environmental ethanol. The sanitizer was subsequently moved and the peaks were not seen again.

[0191] In a separate experiment using a conventional nebuliser (Pari LC-plus nebuliser) alone (without the USS-Neb reservoir system), mean ethanol levels between 30.8ppm and 273.9ppm were recorded depending on the positioning of the PID during nebulisation, although always within one metre of the delivery system.

Example 4: Ethanol inhalation in mice

[0192] Adult female BALB/c mice were obtained from the animal resources centre at the University of Western Australia. Groups of healthy mice were exposed to different concentrations of aerosolised ethanol - 40%, 60%, or 80% v/v in water, or a control (saline).

[0193] The experiment was carried out in three stages as follows:

Part A: 4 groups of 20 healthy uninfected mice exposed to ethanol

Part B: 4 groups of 10 mice infected with influenza virus prior to ethanol inhalation Part C: Lung deposition and blood ethanol level study in healthy mice exposed to ethanol. 4.1 Part A study design, safety of inhaled ethanol in healthy mice

4.2 Part A Results:

[001] There was no significant change in mouse weight pre- and post-dosing. During treatment there was no evidence of distress or intolerance indicating that the treatments were well tolerated.

[002] Bronchoalveolar lavage (BAL) was performed after euthanasia of the mice and total cells, macrophages, neutrophils, and lymphocytes were counted in the BAL fluid. As shown in Figure 6 there were no significant changes in BAL cell counts between the control (saline) and ethanol treated groups of healthy mice. This indicates that the inhalation of ethanol alone does not induce an inflammatory response in the lungs.

[003] In addition elastase, IL6 and TNF levels were assayed in the BAL fluid and as shown in Figure 7 there was no significant changes in levels between the control (saline) and ethanol treated groups of healthy mice. As above, this indicates that the inhalation of ethanol alone does not induce an inflammatory response in the lungs.

[004] As shown in Figure 8, there was also no significant difference in airway morphometry (airway smooth muscle (ASM), lumen and airway epithelium) between the control (saline) and ethanol treated groups of healthy mice.

4.3 Part B: Efficacy of ethanol inhalation in mice infected with influenza

[005] Adult female BALB/c mice were obtained from the animal resources centre at the University of Western Australia. The mice were infected with Influenza A Mem/71 and dived into four groups for exposure to different concentrations of aerosolised ethanol -40%, 60%, or 80% v/v in water, or a control (saline), for 30 minutes.

SUBSTITUTE SHEET (RULE 26) 4.4 Part B study design, efficacy of inhaled ethanol in healthy mice

4.5 Part B Results:

[006] There was no significant change in mouse weight pre- and post-dosing. During treatment there was no evidence of distress or intolerance indicating that the treatments were well tolerated.

[007] Bronchoalveolar lavage (BAL) was performed after euthanasia of the mice and total cells, macrophages, neutrophils, and lymphocytes were counted in the BAL fluid. As shown in Figures 9 and 10 inhaled ethanol increases macrophage numbers in an infection-specific manner while at the same time, neutrophils were not increased supporting improved control over excess inflammation during infection.

[008] In addition elastase, IL6 and TNF levels were assayed in the BAL fluid and as shown in Figure 11 there were no clear differences in BAL cytokine expression although IL- 6 was more highly expressed after treatment with 60% ethanol, but not 40% or 80% .

[009] Viral load was measured in each group at the end of the treatment and while the increase in the reduction of viral load did not reach significance (Figure 12), there was a strong trend towards reduced viral numbers in the group receiving 80% ethanol when compared to control. It should be noted that the mice are obligate nasal breathers and were breathing normally in the experiment. It is likely that a large portion of the inhaled ethanol was filtered out by the nose resulting in significantly less drug reaching the lung relative to human subjects inhaling the aerosolised ethanol through the mouth. This explains why an effect might only be seen at high concentrations in mice while the same effect may be seen in humans at a significantly lower dose.

SUBSTITUTE SHEET (RULE 26)