WO2023283683A1

WO2023283683A1 - Compositions of ivermectin and uses thereof

Info

Publication number: WO2023283683A1
Application number: PCT/AU2022/050729
Authority: WO
Inventors: Hak-Kim Chan; Ahmed H. ALBARIQI; John Drago
Original assignee: The University Of Sydney
Priority date: 2021-07-12
Filing date: 2022-07-12
Publication date: 2023-01-19

Abstract

The present invention provides inhalable dry powder compositions of ivermectin, methods for the preparation of said compositions using spray drying and lactose monohydrate, and methods for their use. The compositions and methods described herein may find particular application in, but are not limited to, the treatment and/or prevention of viral infection.

Description

Compositions of Ivermectin and Uses Thereof

Incorporation by Cross-Reference

This application claims priority from Australian provisional patent application number 2021902130, filed on 12 July 2021, the entire contents of which are incorporated herein by cross-reference.

Technical Field

The present invention relates generally to the fields of biology and medicine, and more specifically to inhalable dry powder compositions of ivermectin and methods fortheir use. In certain forms, the present invention provides compositions and methods which may find particular application in the treatment and/or prevention of viral infection.

Background

Pharmacological treatments are a vital component of the fight against viral infections and are of particular importance in cases where vaccination programs encounter difficulties, for example, when variants emerge that threaten to reduce the efficacy of vaccines. If administered to patients in the early stages of infection, anti-viral drugs can help to constrain viral load, avoid progression to severe disease and reduce transmission. However, many drugs with the potential for prophylaxis and/or treatment of viral infection are limited by the difficulty of achieving concentrations necessary for inhibition of viral replication without dose limiting side effects.

In the case of respiratory viruses, one way in which therapeutic concentrations may be achieved in vivo without the dose limiting side effects often encountered with oral administration is by targeted delivery of anti-viral drugs to the lungs via aerosols. Delivery via inhalation often requires only very small doses to achieve very high concentrations.

Commonly used delivery methods for aerosols include dry powder inhalers (DPI), pressurized metered-dose inhalers (pMDI) and nebulizers. DPIs provide several advantages over nebulizers including portability, accessibility, infection prevention and economic feasibility. DPIs do not require organic propellants, unlike pMDIs, and are generally easier to use when compared to pMDIs due to their ability to be activated by deep inspiration. Powder formulations for use with DPIs manufactured by current methods tend to be unstable and have a propensity for agglomeration. Dry powder formulations are also susceptible to the effects of moisture. Drugs with a low dose require mixing with excipients for enhanced inhaled delivery, rendering the formulations even more vulnerable to the adverse effects of humidity. This poses problems for transportation and storage.

A need exists for stable dry powder formulations for use with DPIs which are not susceptible to the effects of humidity during storage, transportation and/or use. A particular need exists for such dry powder formulations for use in targeted delivery of anti-viral drugs to the lungs.

Summary of the Invention

The present invention alleviates at least one of the problems outlined above by providing dry powder compositions for use with dry powder inhalers (DPI) which remain stable and/or are not prone to agglomeration during storage, transportation and/or use. The present inventors have previously shown that spray drying of suspended lactose crystals in isopropyl alcohol (IP A) with or without drugs has the potential to maintain the crystallinity of lactose (Ke et al., “Spray drying lactose from organic solvent suspensions for aerosol delivery to the lungs.” Int. J. Pharm. 2020; doi.org/10.1016/j.ijpharm.2020.119984). However, this study highlighted the unpredictability of the method with different drugs based on the solubility of the drug in the organic solvent and/or the effect of the organic solvent on the excipient. Despite these challenges, the present inventors have surprisingly developed a dry powder composition of ivermectin and a crystalline excipient in which the ivermectin remains amorphous and stable across a range of moisture conditions and which does not have a propensity for agglomeration. The invention also provides methods for the preparation of such compositions and methods for the use of the dry powder compositions in the treatment and/or prevention of viral infection.

The present invention relates at least in part to the following embodiments:

Embodiment 1. A dry powder composition comprising:

(i) ivermectin;

(ii) lactose monohydrate; and

(iii) an organic solvent. wherein the composition has been produced by spray drying (i), (ii) and (iii). Embodiment 2. The dry powder composition of embodiment 1, wherein the ivermectin is soluble in the organic solvent.

Embodiment 3. The dry powder composition of embodiment 1 or embodiment 2, wherein the lactose monohydrate is insoluble in the organic solvent.

Embodiment 4. The dry powder composition of any one of embodiments 1 to 3, wherein the organic solvent does not alter the physiochemical properties of the lactose monohydrate.

Embodiment 5. The dry powder composition of any one of embodiments 1 to 4, wherein the organic solvent is an alcohol.

Embodiment 6. The dry powder composition of any one of embodiments 1 to 5, wherein the organic solvent is selected from the group consisting of: isopropanol, 1- propanol, ethanol, and any combination thereof.

Embodiment 7. The dry powder composition of any one of embodiments 1 to 6, wherein (i), (ii) and (iii) have been spray dried simultaneously.

Embodiment 8. The dry powder composition of any one of embodiments 1 to 7, wherein the ratio of ivermectin to lactose monohydrate is between 1:15 and 1:16, between 1:16 and 1:17, between 1:17 and 1:18, between 1:18 and 1:19, between 1:19 and 1:20, between 1:20 and 1:21, between 1:21 and 1:22, between 1:22 and 1:23, between 1:23 and 1:24, or between 1:24 and 1:25.

Embodiment 9. The dry powder composition of any one of embodiments 1 to 7, wherein the ratio of ivermectin to lactose monohydrate is between 1:1250 and 1:4000, between 1:125 and 1:400, between 1:12.5 and 1:40, between 1:1.25 and 1:4, or between 1:0.125 and 1:0.4.

Embodiment 10. The dry powder composition of any one of embodiments 1 to 9, wherein the dry powder composition is suitable for inhalation using a dry powder inhaler (DPI).

Embodiment 11. A method of preparing a dry powder composition, the method comprising providing:

(i) ivermectin;

(ii) lactose monohydrate; and

(iii) an organic solvent, and spray drying (i), (ii) and (iii). Embodiment 12. The method of embodiment 11, wherein the ivermectin is soluble in the organic solvent.

Embodiment 13. The method of embodiment 11 or embodiment 12, wherein the lactose monohydrate is insoluble in the organic solvent.

Embodiment 14. The method of any one of embodiments 11 to 13, wherein the organic solvent does not alter the physiochemical properties of the lactose monohydrate.

Embodiment 15. The method of any one of embodiments 11 to 14, wherein the organic solvent is an alcohol.

Embodiment 16. The method of any one of embodiments 11 to 15, wherein the organic solvent is selected from the group consisting of: isopropanol, 1 -propanol, ethanol, and any combination thereof.

Embodiment 17. The method of any one of embodiments 11 to 16, wherein (i), (ii) and (iii) are spray dried simultaneously.

Embodiment 18. The method of any one of embodiments 11 to 17, wherein the ratio of ivermectin to lactose monohydrate is between 1:15 and 1:16, between 1:16 and 1:17, between 1:17 and 1:18, between 1:18 and 1:19, between 1:19 and 1:20, between 1:20 and 1:21, between 1:21 and 1:22, between 1:22 and 1:23, between 1:23 and 1:24, or between 1:24 and 1:25.

Embodiment 19. The method of any one of embodiments 11 to 17, wherein the ratio of ivermectin to lactose monohydrate is between 1:1250 and 1:4000, between 1:125 and 1:400, between 1:12.5 and 1:40, between 1:1.25 and 1:4, or between 1:0.125 and 1:0.4. Embodiment 20. The method of any one of embodiments 11 to 19, wherein the dry powder composition is suitable for inhalation using a dry powder inhaler (DPI).

Embodiment 21. A dry powder composition prepared according to the method of any one of embodiments 11 to 20.

Embodiment 22. A method of treating and/or preventing a viral infection, the method comprising administering the dry powder composition of any one of embodiments 1 to 10 or 21 to a subject.

Embodiment 23. The method of embodiment 22, wherein the dry powder composition is administered using a DPI.

Embodiment 24. The method of embodiment 22 or embodiment 23, wherein the viral infection is COVID-19. Embodiment 25. The method of any one of embodiments 22 to 24, wherein the viral infection is caused by one of more viruses selected from the group consisting of: Zika, dengue, yellow fever, West Nile, Hendra, Newcastle virus, Venezuelan equine encephalitis, chikungunya, Semliki Forest, Sindbis, Avian influenza A, Porcine Reproductive and Respiratory Syndrome, Human immunodeficiency virus type 1, Equine herpes type 1, pseudorabies, BK polyomavirus, and porcine circovirus 2.

Embodiment 26. The method of any one of embodiments 22 to 25, wherein the dry powder composition is administered at a dose of between 0.1 and 10 mg/kg body weight, between 0.1 and 9 mg/kg body weight, between 0.1 and 8 mg/kg body weight, between 0.1 and 7 mg/kg body weight, between 0.1 and 6 mg/kg body weight, between 0.1 and 5 mg/kg body weight, between 1 and 4 mg/kg body weight, between 1 and 3 mg/kg body weight, between 1.5 and 2.5 mg/kg body weight, between 1.75 and 2.25 mg/kg body weight, between 1.85 and 2.15 mg/kg body weight, between 1.9 and 2.1 mg/kg body weight, between 1.95 and 2.05 mg/kg body weight, between 2 and 2.05 mg/kg body weight, between 2.04 and 2.05 mg/kg body weight, or about 2.04 mg/kg body weight.

Embodiment 27. The dry powder composition of any one of embodiments 1 to 10 or 21 for use in treating and/or preventing a viral infection.

Embodiment 28. The use of embodiment 27, wherein the dry powder composition is administered using a DPI.

Embodiment 29. The use of embodiment 27 or embodiment 28, wherein the viral infection is COVID-19.

Embodiment 30. The use of any one of embodiments 27 to 29, wherein the viral infection is caused by one of more viruses selected from the group consisting of: Zika, dengue, yellow fever, West Nile, Hendra, Newcastle virus, Venezuelan equine encephalitis, chikungunya, Semliki Forest, Sindbis, Avian influenza A, Porcine Reproductive and Respiratory Syndrome, Human immunodeficiency virus type 1, Equine herpes type 1, pseudorabies, BK polyomavirus, and porcine circovirus 2.

Embodiment 31. The use of any one of embodiments 27 to 30, wherein the dry powder composition is administered at a dose of between 0.1 and 10 mg/kg body weight, between 0.1 and 9 mg/kg body weight, between 0.1 and 8 mg/kg body weight, between 0.1 and 7 mg/kg body weight, between 0.1 and 6 mg/kg body weight, between 0.1 and 5 mg/kg body weight, between 1 and 4 mg/kg body weight, between 1 and 3 mg/kg body weight, between 1.5 and 2.5 mg/kg body weight, between 1.75 and 2.25 mg/kg body weight, between 1.85 and 2.15 mg/kg body weight, between 1.9 and 2.1 mg/kg body weight, between 1.95 and 2.05 mg/kg body weight, between 2 and 2.05 mg/kg body weight, between 2.04 and 2.05 mg/kg body weight, or about 2.04 mg/kg body weight.

Embodiment 32. A kit when used for the method of any one of embodiments 11 to 20, the kit comprising:

(i) ivermectin;

(ii) lactose monohydrate; and

(iii) an organic solvent.

Definitions

As used in this application, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise.

As used herein, the term “comprising” means “including”. Variations of the word “comprising”, such as “comprise” and “comprises,” have correspondingly varied meanings. Thus, for example, a composition “comprising” components ‘A’, ‘B’ and ‘C’ may consist exclusively of components ‘A’, ‘B’ and ‘C’ or may include one or more additional components, for example, component ‘D’.

As used herein, the term “subject” includes any animal of economic, social or research importance including bovine, equine, ovine, primate, avian and rodent species. Hence, a “subject” may be a mammal such as, for example, a human, or a non -human mammal.

As used herein, the term “kit” refers to any delivery system for delivering materials. Such delivery systems include systems that allow for the storage, transport, or delivery of components (for example, labels, reference samples, supporting material, etc. in appropriate containers) and/or supporting materials (for example, buffers, written instructions for performing an assay etc.) from one location to another. For example, kits may include one or more enclosures, such as boxes, containing the relevant components and/or supporting materials.

As used herein, the term “about" when used in reference to a recited numerical value includes the recited numerical value and numerical values within plus or minus ten percent of the recited value.

As used herein, the term “plurality” means more than one. In certain specific aspects or embodiments, a plurality may mean 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, or more, and any numerical value derivable therein, and any range derivable therein.

As used herein, the term “between” when used in reference to a range of numerical values encompasses the numerical values at each endpoint of the range. For example, a ratio of ivermectin to lactose monohydrate of between 1:15 and 1 : 16 is inclusive of a ratio of ivermectin to lactose monohydrate of 1:15 and a ratio of ivermectin to lactose monohydrate of 1 : 16.

As used herein, the term “more than” when used in reference to a numerical value will be understood to mean “greater than or equal to”.

As used herein, the term “less than” when used in reference to a numerical value will be understood to mean “less than or equal to”.

As used herein, the term “spray drying” will be understood to mean any process which involves the conversion of a liquid into fine particles and/or droplets which are dried by evaporation to form a powder.

Any description of prior art documents herein, or statements herein derived from or based on those documents, is not an admission that the documents or derived statements are part of the common general knowledge of the relevant art.

For the purposes of description, all documents referred to herein are hereby incorporated by reference in their entirety unless otherwise stated.

Brief Description of the Figures

Preferred embodiments of the present invention will now be described by way of example only, with reference to the accompanying figures wherein:

Figure 1 provides scanning electron microscopy (SEM) images of raw and spray- dried samples at a magnification of 65 (Raw ivermectin), 10000 (SD ivermectin), or 3000 (Raw, SD lactose, and SD ive:lac samples).

Figure 2 is a graph of the volumetric particle size distribution of the raw and spray- dried samples.

Figure 3 is a graph of the dispersion performance of SD ive:lac powders at each NGI stages (n=3).

Figure 4 provides DSC thermograms of raw and spray-dried samples.

Figure 5 provides TGA thermograms of raw and spray-dried samples. Figure 6 provides XRPD graphs of the raw and spray-dried samples.

Figure 7 is a graph of Raman spectra of raw and SD ivermectin and lactose before and after exposing to 90% RH.

Figure 8 is a graph of Raman spectra of SD ive:lac samples before and after exposing to 90% RH.

Figure 9 provides graphs of moisture sorption profiles of raw and SD ivermectin and lactose.

Figure 10 provides graphs of moisture sorption profiles of SD ive:lac samples.

Figure 11 provides a graph of the expected results of a prophetic experiment showing the expected replication kinetics of the VIC01 SARS-CoV-2 isolate.

Figure 12 provides a graph of the expected results of a prophetic experiment showing an example of qRT-PCR using different concentrations of treatment. * p <0.05, ** p <0.01, *** p <0.001 compared to untreated (One way ANOVA). Positive control (neat SARS- CoV-2 virus stock) = 139.09 ng/μL. Negative control (cell supernatant) = 0.00ng/μL. An appropriate line-fit curve will be generated and EC₅₀ will be estimated from the curve generated.

Figure 13 provides a graph of the expected results of a prophetic experiment showing the ECso of the drug via TCID₅₀.

Figure 14 provides graphs showing ivermectin concentrations over time following intratracheal (IT) delivery of the two doses to BALB/c mice (n=4) for: (A) plasma, (B) bronchoalveolar lavage (BALF) fluid, (C) lung, (D) liver, (E) kidney and (F) the sum distribution of ivermectin in lung, liver, and kidney. The data are shown as the means +/- SD.

Figure 15 provides a graph showing the distribution of ivermectin in BALF, lung, liver, and kidney over time following intratracheal delivery of the two doses. Data are the mean % distribution +/- SD (n=8 mice).

Figure 16 provides representative images of the lung histology in BALB/c mice that received ivermectin at dose of 2.04 ± 0.40 mg/kg or lactose only at magnification of 40 (B, and D) or 20 (A, C, E). Untreated mice showed normal epithelium (A). Necrotic and vacuolated epithelium areas were evident with ivermectin (B) and lactose only (C) at 24 hours after delivery. Regenerated respiratory epithelium was evident with ivermectin (D) and lactose only (E) at 48 hours after delivery. (Hematoxylin and Eosin staining). Detailed description

The present inventors previously explored the potential for spray drying suspended lactose crystals in isopropyl alcohol (IP A) with or without drugs in order to keep its crystallinity (Ke et al., “Spray drying lactose from organic solvent suspensions for aerosol delivery to the lungs.” Int. J. Pharm. 2020; doi.org/10.1016/j.ijpharm.2020.119984). This study highlighted the unpredictability of the method with different drugs based on the solubility of the drug in the organic solvent and the effect of the organic solvent on the excipient.

Despite the challenges outlined above, the present inventors have surprisingly developed a dry powder composition of ivermectin and a crystalline excipient in which the ivermectin remains amorphous and stable across a range of moisture conditions and which does not have a propensity for agglomeration. The dry powder compositions of the invention may be suitable for inhalation using a dry powder inhaler (DPI) and may find particular application in the treatment and/or prevention of viral infection.

Compositions

The present invention provides a dry powder composition comprising ivermectin. The composition may also include a crystalline excipient, for example, lactose monohydrate, and/or an organic solvent. Other non-limiting examples of suitable excipients include trehalose and sugar alcohols, for example, mannitol and sorbitol. The composition may be produced by spray drying the ivermectin, crystalline excipient and/or the organic solvent. The ivermectin may be soluble in the organic solvent. Additionally or alternatively, the excipient may be insoluble in the organic solvent. In some embodiments of the invention, the organic solvent does not alter the physiochemical properties of the excipient.

The organic solvent may be an alcohol. In some embodiments, the organic solvent is a secondary alcohol. A non-limiting example of a suitable secondary alcohol is isopropanol (IP A). Non-limiting examples of other suitable alcohols include 1 -propanol and ethanol. The ratio of ivermectin to excipient may be, for example, between 1:15 and 1:16, between 1 :16 and 1:17, between 1:17 and 1:18, between 1:18 and 1:19, between 1:19 and 1 :20, between 1:20 and 1:21, between 1:21 and 1:22, between 1:22 and 1:23, between 1:23 and 1:24, or between 1 :24 and 1:25.

The dry powder compositions of the invention may be suitable for inhalation using a DPI. The compositions may remain stable and/or not be prone to agglomeration during storage, use and/or transportation. The ivermectin in the composition may be amorphous and/or the excipient, for example, lactose monohydrate, may be in crystalline form.

Methods for preparing the compositions

The present invention also provides methods of preparing the dry powder compositions of ivermectin. The methods may involve spray drying the ivermectin, crystalline excipient and/or the organic solvent. Spray drying is a commonly used one-step process for the rapid conversion of a liquid feed into inhalable dried particles. Spray drying involves the atomization of a liquid into droplets, which are then dried by evaporation to form a powder. A review of the preparation and use of powder aerosols for pulmonary drug delivery, which includes an explanation of the use of spray drying in this context, may be found in Yang et al., “Pulmonary Drug Delivery by Powder Aerosols.” J. Control. Release. 2014; 193: 228-240. An overview of spray drying techniques may be found in Mujumdar etal., “An Overview of the Recent Advances in Spray-Drying.” Dairy Sci. Technol. 2010; 90: 211-244.

In some embodiments of the invention, the ivermectin, crystalline excipient and/or the organic solvent are co-spray dried. In some embodiments, the ivermectin is dissolved in the organic solvent prior to co-spray drying. The ivermectin may be dissolved in the organic solvent, for example, IP A, at a concentration of between 1 and 4 mg/ml, or between 2 and 3 mg/ml. A non-limiting example of an optimal concentration of ivermectin in IP A is 2.4 mg/ml.

In further embodiments of the invention, the excipient, for example, lactose monohydrate, may be suspended in the solution of ivermectin dissolved in the organic solvent. The excipient, for example, lactose monohydrate, may be suspended in the solution at a concentration of between 40 and 50 mg/ml, between 41 and 49 mg/ml, between 42 and 48 mg/ml, between 43 and 48 mg/ml, between 44 and 47 mg/ml, or between 45 and 46 mg/ml. A non-limiting example of an optimal concentration of lactose monohydrate in a solution of ivermectin and organic solvent is 45.6 mg/ml. The ratio of ivermectin to excipient, for example, lactose monohydrate, may be, for example, between 1:15 and 1:16, between 1:16 and 1:17, between 1:17 and 1:18, between 1:18 and 1:19, between 1:19 and 1:20, between 1:20 and 1 :21, between 1:21 and 1:22, between 1:22 and 1:23, between 1:23 and 1:24, or between 1:24 and 1:25.

Exemplary concentrations of ivermectin and excipient, and exemplary ratios of ivermectin to excipient, are provided in Example One herein. It will be understood that the concentrations of ivermectin and excipient, and ratios of ivermectin to excipient disclosed are exemplary only.

The concentrations of ivermectin and excipient, and ratios of ivermectin to excipient, could, for example, be varied by fixing the concentration of the excipient in order to vary the ivermectin dose. The concentration of ivermectin could be fixed in order to vary the concentration of the excipient. It is therefore apparent that a range of concentrations of ivermectin and excipient, and a range of ratios of ivermectin to excipient, may be used when preparing the compositions without detracting from the invention.

The concentration of ivermectin may be, for example, between 0.024 mg/ml and 0.24 mg/ml, between 0.24 mg/ml and 2.4 mg/ml, between 2.4 mg/ml and 24 mg/ml, or between 24 mg/ml and 240 mg/ml. The concentration of excipient, for example, lactose monohydrate, may be, for example, between 0.456 mg/ml and 4.56 mg/ml, between 4.56 mg/ml and 45.6 mg/ml, between 45.6 mg/ml and 456 mg/ml, or between 456 mg/ml and 4560 mg/ml. The ratio of ivermectin to excipient, for example, lactose monohydrate, may be, for example, between 1:0.19 and 1:1.9, between 1:1.9 and 1:19, between 1:19 and 1:190, or between 1:190 and 1:1900.

A composition of the invention could be prepared with, for example, ivermectin at a concentration of between 0.01 mg/ml and 0.04 mg/ml, an excipient, for example, lactose monohydrate, at a concentration of between 40 and 50 mg/ml, and a ratio of ivermectin to excipient of between 1: 1250 and 1 :4000. Another exemplary composition of the invention could have ivermectin at a concentration of between 0.1 mg/ml and 0.4 mg/ml, an excipient, for example, lactose monohydrate, at a concentration of between 40 and 50 mg/ml, and a ratio of ivermectin to excipient of between 1:125 and 1:400. Another example of a composition of the invention could have ivermectin at a concentration of between 1 mg/ml and 4 mg/ml, an excipient, for example, lactose monohydrate, at a concentration of between 40 and 50 mg/ml, and a ratio of ivermectin to excipient of between 1: 12.5 and 1:40. Yet another example of a composition of the invention could have ivermectin at a concentration of between 10 mg/ml and 40 mg/ml, an excipient, for example, lactose monohydrate, at a concentration of between 40 and 50 mg/ml, and a ratio of ivermectin to excipient of between 1 : 1.25 and 1 :4. In another exemplary embodiment, a composition of the invention could have ivermectin at a concentration of between 100 mg/ml and 400 mg/ml, an excipient, for example, lactose monohydrate, at a concentration of between 40 and 50 mg/ml, and a ratio of ivermectin to excipient of between 1: 0.125 and 1 :0.4. A composition of the invention could be prepared with, for example, ivermectin at a concentration of between 1 mg/ml and 4 mg/ml, an excipient, for example, lactose monohydrate, at a concentration of between 0.4 and 0.5 mg/ml, and a ratio of ivermectin to excipient of between 1: 0.125 and 1:0.4. Another exemplary composition of the invention could have ivermectin at a concentration of between 1 mg/ml and 4 mg/ml, an excipient, for example, lactose monohydrate, at a concentration of between 4 and 5 mg/ml, and a ratio of ivermectin to excipient of between 1 : 1.25 and 1 :4. Another example of a composition of the invention could have ivermectin at a concentration of between 1 mg/ml and 4 mg/ml, an excipient, for example, lactose monohydrate, at a concentration of between 40 and 50 mg/ml, and a ratio of ivermectin to excipient of between 1: 12.5 and 1:40. Yet another example of a composition of the invention could have ivermectin at a concentration of between 1 mg/ml and 4 mg/ml, an excipient, for example, lactose monohydrate, at a concentration of between 400 and 500 mg/ml, and a ratio of ivermectin to excipient of between 1: 125 and 1:400. In another exemplary embodiment, a composition of the invention could have ivermectin at a concentration of between 1 mg/ml and 4 mg/ml, an excipient, for example, lactose monohydrate, at a concentration of between 4000 and 5000 mg/ml, and a ratio of ivermectin to excipient of between 1:1250 and 1:4000.

Table 1 provides examples of how the concentrations of ivermectin, and ratios of ivermectin to excipient, could be varied by fixing the concentration of the excipient in order to vary the ivermectin dose.

Table 1. Exemplary ivermectin concentrations and ratios of ivermectin to excipient with a fixed concentration of excipient

Table 2 provides examples of how the concentrations of excipient, for example, lactose monohydrate, and ratios of ivermectin to excipient, could be varied by fixing the concentration of ivermectin in order to vary the concentration of the excipient. Table 2. Exemplary excipient concentrations and ratios of ivermectin to excipient with a fixed concentration of ivermectin

The ivermectin may be soluble in the organic solvent, for example, IP A. Additionally or alternatively, the excipient, for example, lactose monohydrate, may be insoluble in the organic solvent, for example, IP A. In some embodiments of the invention, the organic solvent does not alter the physiochemical properties of the excipient. The organic solvent may a secondary alcohol.

Many methods are known in the art for characterizing dry powder compositions prepared by spray drying in order to determine their efficacy and/or suitability for administration via a DPI. The skilled person would be aware that the particle morphology may be visualized, for example, using scanning electron microscopy. One way in which particle size and particle size distribution can be determined is by laser diffraction.

The dispersion performance of the compositions may be assessed using, for example, a combination of a next generation pharmaceutical impactor and high-performance liquid chromatography (HPLC). See Mitchell et al., “Aerodynamic particle size analysis of aerosols from pressurized metered-dose inhalers: comparison of Andersen 8-stage cascade impactor, next generation pharmaceutical impactor, and model 3321 Aerodynamic Particle Sizer aerosol spectrometer.” AAPS PharmSciTech. 2003; 4(4): 425-433, for a comparison of methods of aerodynamic particle size analysis and the parameters which may be measured. The amount of ivermectin in the particles may be determined, for example, by HPLC. The solid-state properties of the compositions may be assessed by, for example, differential scanning calorimetry (DSC). Weight loss due to heat may be measured by thermogravimetric analysis and the crystallinity of the compositions and/or components thereof may be assessed via DSC, X-ray powder diffraction, Infra-red (IR) spectroscopy, Raman spectroscopy and/or solid-state NMR. Vapour sorption profiles may be obtained by dynamic vapor sorption (DVS). The chemical composition may be quantified by high performance liquid chromatography (HPLC) and UV spectroscopy.

Methods for using the compositions

The present invention also provides methods of treating and/or preventing a viral infection comprising administering the dry powder composition to a subject via a DPI. The methods may overcome pharmacokinetic limitations associated with oral administration of an anti-viral compound. This may be achieved by targeted delivery of the compositions to the lungs of a subject in need thereof. The subject may be any animal (e.g., a mammal), including, but not limited to, humans, non-human primates, canines, felines, and rodents.

The causative agent of the viral infection may be SARS-CoV-2. Other non-limiting examples of viruses which may be causative agents of the viral infection include Zika, dengue, yellow fever, West Nile, Hendra, Newcastle virus, Venezuelan equine encephalitis, chikungunya, Semliki Forest, Sindbis, Avian influenza A, Porcine Reproductive and Respiratory Syndrome, Human immunodeficiency virus type 1, Equine herpes type 1, pseudorabies, BK polyomavirus, and porcine circovirus 2.

Exemplary doses of the dry powder composition are provided in Example Five and in the claims herein. For example, the dry powder composition may be administered at a dose of between 0.1 and 10 mg/kg body weight, between 0.1 and 9 mg/kg body weight, between 0.1 and 8 mg/kg body weight, between 0.1 and 7 mg/kg body weight, between 0.1 and 6 mg/kg body weight, between 0.1 and 5 mg/kg body weight, between 1 and 4 mg/kg body weight, between 1 and 3 mg/kg body weight, between 1.5 and 2.5 mg/kg body weight, between 1.75 and 2.25 mg/kg body weight, between 1.85 and 2.15 mg/kg body weight, between 1.9 and 2.1 mg/kg body weight, between 1.95 and 2.05 mg/kg body weight, between 2 and 2.05 mg/kg body weight, between 2.04 and 2.05 mg/kg body weight, or about 2.04 mg/kg body weight. It will be understood that the doses disclosed are exemplary only.

The invention also provides the dry powder compositions for use in treating and/or preventing a viral infection, and kits when used for the preparation of the compositions. The kits may comprise ivermectin, an excipient, for example, lactose monohydrate, and/or an organic solvent. The kits may also include delivery systems, including systems that allow for the storage, transport, or delivery of components (for example, labels, reference samples, supporting material, etc. in appropriate containers) and/or supporting materials (for example, buffers, written instructions, etc.) from one location to another. For example, kits may include one or more enclosures, such as boxes, containing the relevant components and/or supporting materials.

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

Examples

The present invention will now be described with reference to specific Examples, which should not be construed as in any way limiting.

Example One: Preparation and characterization of inhalable ivermectin powders

Methods

Ivermectin was co-spray dried with lactose monohydrate crystals and conditioned by storage at two different relative humidity points (43 and 58% RH) for a week. The in vitro dispersion performance of the stored powders was examined using a medium-high resistance Osmohaler connecting to a next generation impactor at 60 L/min flow rate. The solid-state characteristics including particle size distribution and morphology, crystallinity and moisture sorption profiles of raw and spray-dried ivermectin samples were assessed by laser diffraction, scanning electron microscopy, Raman spectroscopy, X-ray powder diffraction, thermogravimetric analysis, differential scanning calorimetry, and dynamic vapor sorption.

Materials

GMP-grade ivermectin raw powder was received from Hovione PharmaScience Ltd. LH300 (alpha-lactose monohydrate) was supplied by DFE Pharma. Potassium chloride, sodium bromide, and silica gel were supplied by Sigma-Aldrich Co., potassium carbonate anhydrous was sourced from Fluka Chemie AG, acetonitrile and methanol from Merck KGaA, isopropanol (IP A) from Sigma-Aldrich Pty Ltd. The water used in chemical assays was purified with an SG ultra-pure water system.

Powder preparation

Prior to preparation, the LH300 lactose was conditioned at 80% relative humidity (RH) and 25 °C in a desiccator for 7 days over a saturated solution of potassium chloride to ensure maximal crystallinity. Ivermectin was dissolved in IP A at a concentration of 2.4 mg/ml followed by suspending LH300 lactose at a concentration of 45.6 mg/ ml in order to achieve a weight ratio of drug to excipient at 1:19 (SD ive-lac_T0). For comparison, an ivermectin-only solution and lactose monohydrate-only suspension were prepared in IP A at the same concentrations used in the ivermectin-lactose monohydrate formulation. Dry powders were produced by a B-290 mini spray dryer connected to a B-295 inert loop. The operation parameters were 70 °C inlet temperature, 38.2 m3/hr aspiration, 601 L/hr atomization nitrogen rate, and 15 ml/min solution feed rate.

Powder storage

The spray-dried powders were divided and stored for seven days in sealed desiccators at 25 °C and different relative humidity conditions controlled by saturated salt solutions or silica beads. Details of the powder storage conditions are provided in Table 3. Powder characterization was conducted pre- and post-storage.

Table 3. Storage conditions of spray-dried samples

Particle morphology

The morphology of raw and spray-dried samples was visualized using scanning electron microscopy (SEM) at a beam accelerating voltage of 3 kV. The samples were prepared by spreading the particles on a stub followed by coating with a gold layer of 30 nm.

Particle size

The particle size distribution of the raw and spray-dried samples was measured by laser diffraction using a Mastersizer 2000 equipped with a Scirocco 2000 dry powder dispersion. Compressed air of 4 bars was applied to produce powder dispersion. The refractive indexes used for the measurements were 1.56 and 1.65 for ivermectin and lactose, respectively. The volumetric diameters (D₁₀, D₅₀ and D₉₀) and span [defined as (D₉₀ - D₁₀)/D₅₀] were obtained, and each sample was measured in triplicate.

Actual drag ratio and content homogeneity

The actual ratio of ivermectin in spray dried ivermectin:lactose (SD ive:lac_T0) powder and the homogeneity of the formulation was assessed by randomly sampling 10 specimens from different regions of the powder followed by a chemical assay. The actual ratio of ivermectin was calculated by dividing the average of detected ivermectin values by the theoretical ivermectin value (5% wt. of the loaded formulation containing 1 :19 ivermectin:lactose). The formulation was considered homogenous if the content of ivermectin in each single sample was between 85 and 115% of the average content according to British Pharmacopeia. Each sample of 6.5 ± 0.5 mg was added to 10 ml of methanol, shaken and sonicated for 5 min to fully dissolve ivermectin, followed by filtering and filling in vials using a polytetrafluoroethylene (PTFE) filter membrane of 0.45 μm pore size to exclude the suspended lactose. The chemical assay method used to quantify ivermectin load is described below.

In vitro dispersion performance

The in vitro dispersion performance of SD ive:lac samples was evaluated using a next-generation impactor connected to a USP metal induction port. The dispersion was carried out in a chamber at controlled relative humidity of 50 ± 5%. Prior to dispersion, NGI plates were sprayed with silicone oil to prevent particle bounce. Size 3 Vcaps Plus capsules were filled with 10 ± 0.5 mg of the formulation and aerosolized to the NGI by a medium-high resistance Osmohaler at flow rate of 60 L/min for 4 seconds. The aerodynamic cut-off diameters for stages 7 to 1 are 0.34, 0.55, 0.94, 1.66, 2.82, 4.46, and 8.06 μm, respectively. After dispersion, 10 ml of methanol was used to dissolve the ivermectin particles deposited in the capsule, inhaler, adaptor, throat, and NGI stage 1; 5 ml was used for the other stages. The collected solution samples were filtered by a PTFE filter membrane of 0.45 μm pore size to exclude the suspended lactose. High pressure liquid chromatography was used to chemically analyze the collected ivermectin and determine the fine particle dose (FPD), the recovered fine particle fraction (FPFrecovered), the emitted fine particle fraction (FPFemitted), the median mass aerodynamic diameter (MMAD), and the geometric standard deviation (GSD). The FPF was defined as the mass fraction of aerosolized ivermectin particles less than 5 μm in the aerosol (FPD) with respect to the mass of ivermectin load recovered in the NGI parts including the adaptor, capsule, and the inhaler (FPFrecovered) or with respect to the recovered mass of ivermectin load excluding the capsule and the inhaler (FPFemitted). The dispersion was conducted in triplicate.

Chemical assay method

A high-performance liquid chromatographer connected to aPhenomenex Luna C18(2) 100 Ǻ 5 μm 4.6 x 250 mm column was utilized for quantifying the amount of ivermectin for content homogeneity and in vitro dispersion tests at a detection UV wavelength of 254 nm based on a Pharmacopeia! method with slight modification. The mobile phase contained acetonitrile, methanol, and water (51:34:15 v/v). The standard curve of pure ivermectin in methanol was linear at a concentration range between 0.0005 and 0.6 mg/ml (r2 = 0.999). The ran and elution times were 35 and 28.8 min, respectively. The injection volume was 20 μl and the flow rate was 1 ml/min.

Differential scanning calorimetry (DSC)

The solid-state properties of raw and spray-dried samples were assessed with a DSC instrument. Size 40 μl aluminium crucibles were filled with 6 ± 1 mg of each sample and heated from 30 to 350 °C at a rate of 10 °C/min under a continuous flow of nitrogen gas at 50 ml/min.

Thermogravimetric analysis (TGA)

A TGA instrument was used to measure the weight loss of raw and spray-dried samples when heated. Size 70 μl aluminium oxide crucibles were filled with 8 ± 0.5 mg of each sample and exposed to heat at a rate of 10 °C/min from 30 to 350 °C under a continuous purge of nitrogen gas at 50 ml/min flow rate.

X-ray powder diffraction (XRPD)

A Siemens D5000 X-ray diffraction instrument connected to copper X-ray radiation at 45 kV and a current of 40 mA was used to evaluate the crystallinity of raw and spray - dried samples. Scan method of 20 with a scan rate of 0.013 °/second from 5-50° was used to collect the data.

Raman spectroscopy

A Renishaw inVia Reflex Microscope, which is supplied with a Leica DMLM microscope and a 2400 g mm^-1 grating and an air-cooled charge-coupled device detector, was utilized to obtain Raman spectra and examine the solid state of raw and spray-dried samples. A diode pumped solid-state (DPSS) laser with a wavelength of 532 nm was used as the excitation light. The spectra were acquired with A Leica N Plan 20x/0.40 between 650 to 1750 cm^-1 spectral range. The laser power, accumulation, and exposure time were 50 mW, 200 scans, and 0.5 seconds respectively.

Dynamic vapor sorption (DVS)

The vapor sorption profiles of raw and spray-dried samples were studied by DVS at 25 °C. The samples were exposed to two cycles of moisture from 0 to 90% with 10% RH step increase. The mass changes over time were recorded when the dm/dt was below 0.002% per minute.

Statistics

The data are displayed as mean ± standard deviation (n=3). The statistical difference between SD ive:lac samples was determined using one-way ANOVA with Tukey’s multiple comparison test, p values of 0.05 were considered as a statistical difference.

Results

Particle morphology

Figure 1 represents the SEM morphological characteristics of raw and spray-dried samples. The raw ivermectin powder contained regular-shaped large particles ranging between tens and hundreds of micrometers, while the particles of spray-dried ivermectin alone were very wrinkled with 1 μm particle size. Raw lactose, SD lactose, and SD ive:lac particles were all irregular in shape with no major differences between them. Particle size

Figure 2 shows the volumetric particle size distribution of the raw and spray-dried samples. Raw ivermectin revealed a broad monomodal particle size distribution with a span of 4.95 ± 0.21 and a volumetric median diameter (D50) of 49.7 ± 0.11 μm (Table 4). Spray drying dramatically reduced the value of D50 to 0.88 ± 0.08 μm and narrowed the span to 1.70 ± 0.14 (SD ivermectin). Raw and SD lactose showed a similar bimodal distribution of the particles with D50 values of 4.05 ± 0.07 and 3.95 ± 0.14 μm, respectively. As the majority of SD ive:lac samples were lactose, their particle distributions and D50 values were similar to those of other lactose samples.

Table 4. The volumetric diameters and span of the raw spray-dried samples (n=3). The difference between SD ive:lac samples, raw and SD lactose was not statistically significant (p > 0.05)

μm μm μm

Raw ivermectin 7.81 ± 0.11 49.7 ± 0.11 253 ± 9.90 4.95 ± 0.21

SD ivermectin 0.41 ± 0.03 0.88 ± 0.08 1.91 ± 0.26 1.70 ± 0.14

Raw lactose 1.07 ± 0.03 4.05 ± 0.07 9.17 ± 0.13 2.01 ± 0.04

SD lactose 1.01 ± 0.19 3.95 ± 0.14 8.61 ± 0.33 1.93 ± 0.11

SD ivezlac TO 1.03 ± 0.02 4.32 ± 0.01 9.46 ± 0.03 1.95 ± 0.01

SD iveslac 43%RH 1.21 ± 0.17 4.34 ± 0.31 9.43 ± 0.66 1.91 ± 0.02

SD ive:lac 58%RH 1.25 ± 0.15 4.39 ± 0.27 9.72 ± 0.43 1.93 ± 0.05

Actual drug ratio and content homogeneity

The average of detected ivermectin in SD ive:lac_T0 powder was 91% of the theoretical load of the drug to the excipient with a relative standard deviation (RSD%) of

2.45%. It was therefore concluded that the actual ratio of ivermectin to lactose was changed after spray drying and became 1:20.9 instead of 1:19 (Table 5). All 10 samples had a homogenous amount of ivermectin with a percentage drug content of 96 to 106%.

Table 5. Actual ratio of ivermectin to lactose in the formulation

Theoretical ivermectin Actual ivermectin

Content average % RSD%

5% of the load 91.0 ± 2.23 2.45

Theoretical ivermectin %: the percentage of theoretical load of ivermectin in the sample

Actual ivermectin %: the amount of the detected ivermectin in the sample relative to amount of the theoretical ivermectin

RSD%: relative standard deviation •= (SD/Mean%) * 100

In vitro aerosolization performance

The SD ive:lac samples showed similar dispersion behaviour with a significant deposition of ivermectin on stages 4, 5, and 6 of the impactor (Figure 3, Table 6). The SD ive:lac_T0 and the conditioned powders at 43 and 58% RH had very similar FPD values between 297 and 302 μg, FPFrecovered between 68 and 70%, and FPFemitted between 82 and 84%. Consistently, MMAD and GSD were in the range of 1.5 μm and 2.2, respectively. 3 O

Table 6. Recovered ivermectin, FPD, FPFs, MMAD and GSD values of SD ive:lac samples (n=3). No statistical difference was

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Differential scanning calorimetry (DSC)

DSC thermographs of raw and spray -dried samples are displayed in Figure 4. Raw ivermectin showed a melting peak at 152 °C followed by degradation after 300 °C. Spray drying converted the crystalline raw ivermectin to an amorphous powder as it showed a glass transition event at 137 °C. On the other hand, spray drying did not convert the crystalline raw lactose to an amorphous form as the SD lactose and lactose-containing samples show matched events at 147-148 °C and at 216-219 °C which are related to water loss and the melting of alpha lactose monohydrate, respectively

Thermogravimetric analysis (TGA)

Figure 5 shows TGA graphs of raw and spray-dried samples. Raw ivermectin lost 4% of the weight when melted at 152 °C, presumably due to evaporation of residual solvents (ethanol and formamide) which are used during purification process of raw ivermectin. The raw ivermectin then degraded after 300 °C. However, SD ivermectin showed no significant weight change until degradation. Raw and SD lactose showed a weight loss of 4% between 120 and 150 °C due to water loss followed by another phase of loss after 220 °C related to melting of lactose until degradation. Similarly, SD ive:lac samples showed identical events as they were dominated by the 95 % (w/w) of lactose present in the samples.

X-ray powder diffraction (XRPD)

Raw ivermectin was crystalline with several sharp diffraction peaks observed at 6.5°, 9.3°, 11.2°, 12.4°, 13.1°, 14.8°, and 17.4° 2-theta. However, a halo pattern was shown with SD ivermectin indicating the powder was amorphous. The crystalline form of lactose did not change by spray drying as both raw and SD lactose displayed matched diffraction patterns with dominant peaks at 12.6°, 16.5°, 19.2°, 19.6°, 20.0°, 21.3°, 23.8° and 37.6° 2- theta. SD ive:lac samples showed similar patterns to those of raw and SD lactose. (Figure 6).

Raman spectroscopy

The Raman spectra of the raw and spray-dried samples are shown in Figure 7. Both raw and SD Ivermectin showed characteristic peaks at 1624 and 1672 cm'¹ related to the unsaturated lactones with a double bond adjacent to the O group. Spectra obtained from raw ivermectin and after exposing to 90 % RH (in the DVS analysis) demonstrated sharp peaks corresponding to the crystallinity of the materials. Spectra acquired from spray dried ivermectin alone and all the formulations containing ivermectin showed broadening of ivermectin peaks. However, all the peaks related to lactose (846, 871, 1015, 1082 cm^-1) remained sharp (Figure 8). Interestingly, peaks related to ivermectin even after exposing the formulation to 43, 58 and 90 % RH remained broad, confirming its amorphous nature.

Dynamic vapor sorption (DVS)

Raw ivermectin showed low moisture uptake of 0.26 wt.% at 90% RH. However, the mass became smaller by 0.09 wt.% after the desorption probably due to removal of the solvent residues (ethanol and formamide). SD ivermectin was more hygroscopic and absorbed moisture more than the raw ivermectin (3.5 wt.% at 90% RH). Raw and SD lactose showed low moisture sorption of 0.21 and 0.28 wt.%, respectively, with no recrystallization events (Figure 9). Similarly, SD ive:lac samples showed less than 0.5% of moisture uptake with no re-crystallization events. At 90% RH, the mass of SD ive:lac_T0 increased by 0.43%, and the conditioned samples at 43 and 58%RH displayed increase in mass of 0.38% and 0.31%, respectively (Figure 10).

Example Two: Effects of ivermectin on the replication kinetics of SARS-CoV-2

Example Two is a prophetic Example.

The skilled person can use the directions provided in this Example to investigate the effects of the compositions of the invention on the replication kinetics of SARS-CoV-2 or any other virus of interest. It will be appreciated by the skilled person that variations to the protocol described below could be used for the same purpose.

The directions below will be used to test the compositions of the invention for antiviral efficacy against SARS-CoV-2 in direct comparison to an existing commercial ivermectin product.

Aim

To treat SARS-CoV-2 infected cells with 5pM ivermectin (aerosolized and traditional formulations) and evaluate whether there is a difference in replication kinetics. Treatments

Untreated, Aerosolized Ivermectin prepared according to Example One (5uM), Traditional

Ivermectin (5uM)

Time points

0, 6, 16, 24, 32, 48 and 72h post-infection

Samples n=4 per time point

Read out

TCIDso/mL and optional qRT-PCR Protocol

1. Vero cells will be seeded into 4 x 24 well plates 24h prior to experimentation.

2. Monolayers will be confirmed for >95% confluency and transferred to a PC3 laboratory.

3. After washing with infection media (MEM + glu + Abx, serum free), each well will be infected with 100μL SARS-CoV-2 (approx. 10⁴TCID₅₀ units) and incubated for 2h.

4. Monolayers will be washed once with infection media to remove unbound virions.

5. ImL infection media containing 5μM of drug formulation will be added to each well.

6. At each time point, supernatant will be harvested, collected at stored at -80°C, for

TCID50 determination and optional qRT-PCR.

7. At each time point, the cellular monolayer will be harvested and viral RNA extracted immediately using the QiaAmp Viral RNA mini extraction kit, then stored at -80°C for later qRT-PCR determination.

» Number of qRT-PCR samples if all time points are chosen:

◊ Supernatant: n=4 x 3 treatments x 6 time points = 72 samples + n=4 2hpi controls =

76

Monolayer: n=4 x 3 treatments x 6 time points = 72 samples + n=4 2hpi controls =

76

= 152 samples

» Number of qRT-PCR samples if samples are only taken every 24h:

◊ Supernatant: n=4 x 3 treatments x 4 time points (0, 24, 48, 72h) = 48 samples + n=4 2hpi controls = 52

◊ Monolayer: n=4 x 3 treatments x 4 time points (0, 24, 48, 72h) = 48 samples + n=4 2hpi controls = 52

= 104 samples

Expected Results

This prophetic experiment has been designed with a sample size (i.e., n=) that is expected to achieve statistical significance if biological differences in the infectivity of the virus are found. An approximation of expected results are provided in Figure 11.

Example Three: Effective concentration of aerosolised ivermectin against SARS- CoV-2

Example Three is a prophetic Example. The skilled person can use the directions provided in this Example to determine whether there is a change in the 50% effective concentration of aerosolized ivermectin compared to traditional ivermectin for antiviral activity against SARS-CoV-2 or any other virus of interest. It will be appreciated by the skilled person that variations to the protocol described below could be used for the same purpose.

Aim

To determine whether there is a change in the 50% effective concentration of aerosolized ivermectin compared to traditional ivermectin for antiviral activity against SARS-CoV-2.

Treatments

Untreated, Aerosolized Ivermectin prepared according to Example One, Traditional Ivermectin

Doses

10, 7.5, 5, 2.5, 1.25, 0.75, 0.5, 0.25, 0.125 and 0 μM

Time points

48h post-infection

Samples n=4 per drug concentration

Read out

TCID₅₀/mL and optional qRT-PCR

Protocol

1. Vero cells will be seeded into 5 x 24 well plates 24h prior to experimentation.

3. After washing with infection media (MEM + glu + Abx, serum free), each well will be infected with 100μL SARS-CoV-2 (approx. 10⁴TCID5O units) and incubated for 2h.

5. ImL infection media containing each dilution of each drug formulation will be added to n=4 wells.

6. At 48h post-infection, supernatant will be harvested, collected at stored at -80°C, for TCID₅₀ determination and optional qRT-PCR. 7. At 48h post-infection, the cellular monolayer will be harvested and viral RNA extracted immediately using the QiaAmp Viral RNA mini extraction kit, then stored at - 80°C for later qRT-PCR determination.

» Number of qRT-PCR samples if all doses of drug treatment are chosen:

◊ Supernatant: n=4 x 9 treatments = 36 samples + n=42hpi controls = 40

◊ Monolayer: n=4 x 9 treatments = 36 samples + n=42hpi controls = 40

= 80 samples

» Number of qRT-PCR samples if only half the increments of drug treatment are chosen:

◊ Supernatant: n=4 x 4 treatments = 16 samples + n=42hpi controls = 20

◊ Monolayer: n=4 x 4 treatments = 16 samples + n=4 2hpi controls = 20 ; Total = 40 samples

50% Tissue Culture Infectious Dose assay (TCID₅₀)

1. In a PC2 laboratory, plates to establish -95% monolayers of Vero cells will be seeded

24h prior to the assay.

2. After verification of the quality/density of monolayer, the plates will be washed using infection media (to remove any cell debris), then transferred into a PC3 laboratory.

3. Samples will be generated, serially diluted and a known volume inoculated into each well. n=4 replicates/sample and up to 6 serial dilutions will be performed.

4. Plates will be incubated for 45 min to enable virus infection of monolayers, then 1 mL MEM infection media (containing pen/strep, glutamine, HEPES, but not containing FBS) +TPCK trypsin (1 μg/mL) will be added.

5. Plates will be returned to incubator (37°C, 5% CO₂) and microscopically examined every 24 h (up to 72h) for cytopathic effect (CPE) on cells.

6. The TCID50 will then be back calculations made to determine the TCID₅₀/mL of infectious virus present in the original sample.

Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)

1. RNA will be extracted from samples using the QiaAmp Virus RNA mini extraction kit (Qiagen) according to the manufacturer’s instructions. Samples will be stored at -80°C until ready for processing.

2. Reaction solutions specific for the detection of the SARS-CoV-2 Envelope (E) gene containing known volumes of standards, thawed samples and controls will be set up and processed using appropriate thermocycling conditions on an RT-PCR machine. 3. The amount of genomic RNA material present in each sample will be determined using CT (optical density) values and interpolated from the standard curve generated (see Expected Results).

Expected Results

This prophetic experiment has been designed with a sample size (i.e., n=) that would be expected to achieve statistical significance if biological differences in the infectivity of the virus are found. An approximation of expected results are provided in Figures 12 and 13.

Example Four: Envisaged medical treatments using the compositions of the invention

Example Four is a prophetic Example.

The skilled person can use the directions provided in this Example to administer the compositions of the invention. It will be appreciated by the skilled person that variations to the example could be used for the same purpose.

Description of administration o A capsule containing the composition will be loaded into an inhaler. o The patient will breathe out to empty the lungs. Then, the inhaler will be placed in the mouth by closing the lips around the mouthpiece of the inhaler. o The patient will inhale deeply from the mouth for 2 to 4 seconds. o The patient will hold his/her breath for 10 seconds to allow the drug to settle in the lungs.

The above procedure could vary depending on the specific inhaler, e.g., instead of a capsule, the dose could be contained in a blister, reservoir or another containment. The inhalation time could be shorter or longer e.g., 1 - 6 seconds. The breath holding could also be shorter or longer, e.g., 1-30 seconds or as long as a patient can manage.

Dosage amounts and frequency

A powder formulation between 1 and 1,000 mg could be loaded into a capsule but other containments can be used (because not all inhalers use capsules to contain the dose). Also, the amount to be loaded into the capsule depends on the size of the capsule (e.g., a commonly used size 3 capsule will not hold 1,000 mg, but a less common size 0 capsule can hold this amount). A likely scenario is 5 - 40 mg of powder (which contains 0.1 - 40 mg of ivermectin) loaded to a capsule to be administered to patients 1 - 12 times a day (a more likely scenario is 1 - 6 times a day, with a dosing frequency between daily and weekly, e.g., twice or thrice a week). Example Five: Pharmacokinetics and Safety of Inhaled Ivermectin in Mice

This Example investigated the pharmacokinetics (PK) and evaluated the local toxicity of the inhalable dry powders of ivermectin and lactose crystals in healthy mice. The dry powder formulation was delivered by intratracheal insufflation. The PK were assessed in the plasma, bronchoalveolar lavage fluid (BALF), lung, liver, kidney, and spleen. The local toxicity was examined in the lung tissue by histological analysis.

Methods

Ivermectin was obtained from Hovione PharmaScience Ltd. Alpha-lactose monohydrate (LH300) was sourced from DFE Pharma, isopropanol from Sigma-Aldrich Pty. Ltd., methanol and acetonitrile from Merck KGaA. The water utilized in this Example was purified by an SG ultra-pure system.

Powder preparation

Ivermectin was dissolved in isopropanol followed by suspending the lactose crystals at concentrations of 2.4 and 45.6 mg/ml, respectively, to obtain a weight ratio of ivermectin to lactose of 1:19. These concentrations and ratio were chosen based on Example One which optimized the inhalable dose for human airways in order to achieve the reported in vitro antiviral concentration of 5 μM. Dry powders were prepared by spray drying (B-290 spray dryer connected to B-295 inert loop) conducted in the closed loop mode, with the conditions set at inlet temperature of 70 °C, atomization nitrogen rate of 601 L/hr, aspiration of 38.2 m³/hr, and feed intake of 15 ml/min.

Animal procedures

Eight- to ten-week-old female BALB/c mice (20.0 ± 0.94 g) were obtained from Animal Recourses Center (Perth, Australia) and kept in the animal facility of the Centenary Institute of Cancer Medicine and Cell Biology (Camperdown, Australia). The animal procedures were conducted with approval of Sydney Local Health District (SLHD) Animal Welfare Committee (Protocol number: 2019/017).

Intratracheal delivery

Intratracheal delivery was conducted using a dry powder loading device made of a 200 μl gel loading pipette tip attached to a 1 ml syringe through a three-way stopcock valve. A known amount of the powder formulation was filled in pre-weighed tips prior the experiments, taking into consideration the maximal and minimal loading capacity of the tips being 1 and 2 mg, respectively. The animal was anesthetized by intraperitoneal injection of ketamine/ xylazine (100/10 mg/kg) and placed on an intubation stand. The loading tip was inserted into the trachea with help of an otoscope and a guiding cannula. The powder was dispersed with a volume of 0.4 ml air by the syringe. The delivered dose was determined by the weight difference in the tips before and after the dispersion.

Dose selection

The pharmacokinetics of ivermectin were examined using two doses: lower and higher doses. The lower dose was selected based on mathematical scaling of systemic human dose to animal dose with the following equation:

Where Xh is human dose normalized to body mass, Xa is animal dose normalized to body mass, Ma is animal body mass, Mh human body mass, and b the allometric exponent constant of 0.67. The loading tips were filled to achieve a lower dose of 2.10 mg/kg according to the equation. In practice, the actual dose delivered to the lungs of the mice was 2.04 ± 0.40 mg/kg. The higher dose was selected based on maximal loading capacity of the tips of the delivery device. The delivered dose to the lungs was 3.15 ± 0.60 mg/kg.

For the histological study, only the lower dose was examined as it is the equivalent to the systemic human dose.

Sample collection and processing for PK

Six groups of four mice were used for each dose. The mice were euthanized by carbon dioxide at the allocated time points (0, 1, 3, 6, 24, 48 hours after dose delivery). Blood, BALF, lungs, liver, kidneys, and spleen were collected and processed for a HPLC assay. Blood was collected in ethylenediaminetetraacetic acid (EDTA) tubes, centrifuged to separate plasma using a Beckman Coulter Allegra X-12R Centrifuge at a temperature of 4 °C and 2500 rpm for 10 min. The plasma was deproteinated with acetonitrile at a ratio of 1:3 (v/v) and recentrifuged, then the supernatant was collected. The lungs were washed three times with 1 ml of phosphate buffered saline to collect bronchoalveolar lavage fluid (BALF). This was then deprotonated with an equivalent amount of acetonitrile, centrifuged to remove cellular debris, and the supernatant collected. Lungs, liver, kidneys and spleen were harvested in 2 ml of triply deionised water, homogenized with a Polytron PT10-35 homogenizer connected to PCU power control unit, and processed as for plasma. All processed samples were kept in ice until chemical assay of ivermectin was performed. Chemical analysis of ivermectin in the samples

Shimadzu high performance liquid chromatography with a Phenomenex Luna Cl 8(2) 100 Ǻ μm 4.6 x 250 mm column was used to quantify the concentration of ivermectin in the samples after being filtered with 0.45 μm polytetrafluoroethylene (PIFE) membrane to protect the column from any tissue residuals. The mobile phase consisted of water, methanol, and acetonitrile (15:34:51 v/v), running at a flow rate of 1 ml/min. The injection volume was set at 100 μl for the plasma samples and 20 μl for the other samples. The UV detection wavelength was 254 nm, and the lower limit of quantification was 140 ng/ml.

Pharmacokinetic analysis

Non-compartmental model was applied to determine the PK profile of inhaled ivermectin. Maximum concentration (Cmax) and time to maximum concentration (Tmax) were determined directly form the plots, while other parameters, including the elimination rate constant (Ke), half-life time (t_1/2), total drug exposure (AUCO.00), clearance (CL) and volume of distribution (V_d), were determined using PKSolver.

Histological analysis

The local toxicity of inhaled ivermectin was evaluated with the single dose of 2.04 ± 0.40 mg/kg over different time points. Groups of three mice were used for treatment (0, 24, and 48 hours after dose delivery), and control (no treatment, air only, lactose only for 24 and 48 hr). The mice were euthanized with carbon dioxide at predetermined time points. Lungs were perfused with 10 ml of phosphate buffered saline through the pulmonary artery, harvested, and fixed in 10% neutral buffered formalin. The histological examination was conducted in Phenomics Australia Histopathology and Slide Scanning Service at the University of Melbourne to demonstrate the collective extent of damage in a grading scale (0 for no or mild change, 1 for damage of less than 25%, 2 for damage between 25 and 50%, and 3 for damage greater than 50%).

Results

Ivermectin PK analysis

The concentration of ivermectin in plasma, BALF, and other tissues over time; the distribution in BALF and other tissues over time; and pharmacokinetic parameters with two doses following intratracheal administration are presented in Figures 14 A-F, Figure 15 and Table 7 respectively. Ivermectin was detected in the plasma, BALF, lung, and liver with both doses, but was only detected in the kidney at the higher dose. Except for C_max and Tmax, the PK parameters were not determined for plasma at both doses and for the lung tissue at the lower dose because less than three points in terminal elimination phase could be detected. No data are shown for the spleen as the drug was not detected there.

For both the higher and lower doses, the plasma drug concentration increased to a peak ( C_max: 0.84 ± 0.03 and 0.75 ± 0.11 μg/ml, respectively) in 1 hour, followed by a steep decline until complete elimination at 6 hours after the lower dose and at 24 hours following the higher dose (Figure 14A).

The maximum ivermectin concentration in the BALF for both the higher and lower doses were detected immediately at time 0 (4.99 ± 1.38 and 3.84 ± 1.23 μg/ml, respectively), and continued to decline until no drug was identified at 48 and 24 hours, respectively (Figurel4B). This was associated with a t_1/2 of 7.90 and 4.24 hours, respectively, and a total drug exposure (AUC0.∞ ) of 33.55 and 14.64 μg.hr/ml, respectively.

The distribution of ivermectin in the lung tissue took 3 hours to reach the T_max with a C_max of 95.6 ± 41.9 and 63.2 ± 21.4 μg/g, respectively, for the higher and lower doses (Figure 14C). After the higher dose, ivermectin was detectable in lung tissue for 48 hours, while there was no drug detected at 24 hours with the lower dose. The AUCO- ∞ and t_1/2 of the higher dose were 1734 μg.hr/g and 12.6 hours, respectively.

Analysis of the liver tissue showed a C_max of 10.21 ± 1.49 and 8.52 ± 3.22 μg/g with different Tmax values of 1 and 3 hours for the higher dose and lower doses, respectively (Figure 14D). At 48 hours after the higher dose, the ivermectin concentration had reduced by half (5.33 ± 0.27 μg/g), while no drug was detectable with the lower dose. Thus, the total liver drug exposure values were 726.8 and 591.9 μg.hr/g with t_1/2 of 50.3 and 46 hours, respectively, for the higher and lower doses. The kidney drug concentration for the higher dose was peaked (C_max: 23 μg/g) at 1 hour with an AUCO-∞ and t_1/2 of 935.4 μg.hr/g and 27.4 hours, respectively (Figure 14E).

A clear dose-dependent pattern was shown in Figure 14F with a peak at 3 hours and availability up to 48 hours when the tissue concentrations of ivermectin in lung, liver, and kidney were added together for each dose in order to estimate the relationship between the dose and tissue distribution over time after intratracheal delivery.

Figure 15 shows the distribution of ivermectin in BALF and other tissues over time relative to the initial dose. At time 0 immediately after dosing, ivermectin was primarily in the respiratory tract. At 24 hours, the lung tissue retained only 13.6 ± 4.71% of ivermectin while liver and kidney showed 10.3 ± 2.47% and 6.71 ± 4.35%, respectively. At 48 hours, ivermectin distributed exclusively in the liver tissue showing 8 ± 0.63% of the initial dose. Table 7. Pharmacokinetic parameters of inhaled ivermectin following intratracheal administration in BALBZc mice

Histological analysis

The histological appearance of lungs after intratracheal administration of ivermectin at the single dose of 2.04 ± 0.40 mgZkg are presented in Figure 16 and summarized Table 8. In general, control mice which did not receive powder insufflation (no treatment, air only) showed intact tissues, and were scored at 0 (Figure 16A). Control groups that received intratracheal lactose powder (Figures 16C and E), showed minimal lesions affecting 15% of the lung at 24 hr and this resolved to 1-5% at 48 hr. These were scored at 1.

The local changes observed in the treatment groups varied in extent and grade over time. The respiratory epithelium was intact at time 0. Transient lung inflammation affecting 20% of the lung with markedly vacuolated epithelium was observed at 24 hours after ivermectin delivery (Figure 16B). However, these lung changes had resolved to affect 2- 10% of the lung with regenerated intact epithelium at 48 hours (Figure 16D). Since the multifocal lesions affected < 25% of lung area, the collective damage of both treatment groups at 24 and 48 hr were scored at 1.

Table 8. Histological evaluation of the lungs of BALBZc mice following intratracheal administration in spray-dried ivermectin.

Conclusion

In this Example, ivermectin was successfully delivered to the pulmonary tract and maintained concentrations remarkably above the published in vitro antiviral concentration in lung tissue and BALF for at least 24 hours after administration. Doses of 2.04 ± 0.40 mg/kg of inhaled ivermectin were well tolerated and achieved a C_max greater than 10 times the in vitro antiviral concentration. The histological data shows that inhaled ivermectin is safe in mice with doses equivalent to oral human doses as there was no difference between lung change caused by the ivermectin-containing formulation or the lactose-only control, and lactose is an approved excipient for pulmonary drugs in humans. This Example clearly demonstrates that the dry powder formulations of the invention are able to achieve the in vitro antiviral concentrations of ivermectin in BALF and lung tissue.

Claims

1. A dry powder composition comprising:

(i) ivermectin;

(ii) lactose monohydrate; and

(iii) an organic solvent, wherein the composition has been produced by spray drying (i), (ii) and (iii).

2. The dry powder composition of claim 1, wherein the ivermectin is soluble in the organic solvent.

3. The dry powder composition of claim 1 or claim 2, wherein the lactose monohydrate is insoluble in the organic solvent.

4. The dry powder composition of any one of claims 1 to 3, wherein the organic solvent does not alter the physiochemical properties of the lactose monohydrate.

5. The dry powder composition of any one of claims 1 to 4, wherein the organic solvent is an alcohol.

6. The dry powder composition of any one of claims 1 to 5, wherein the organic solvent is selected from the group consisting of: isopropanol, 1 -propanol, ethanol, and any combination thereof.

7. The dry powder composition of any one of claims 1 to 6, wherein (i), (ii) and (iii) have been spray dried simultaneously.

8. The dry powder composition of any one of claims 1 to 7, wherein the ratio of ivermectin to lactose monohydrate is between 1:15 and 1:16, between 1:16 and 1:17, between 1:17 and 1:18, between 1:18 and 1:19, between 1:19 and 1:20, between 1:20 and 1:21, between 1:21 and 1:22, between 1:22 and 1:23, between 1:23 and 1:24, or between 1:24 and 1:25.

9. The dry powder composition of any one of claims 1 to 7, wherein the ratio of ivermectin to lactose monohydrate is between 1:1250 and 1:4000, between 1:125 and 1:400, between 1:12.5 and 1:40, between 1:1.25 and 1:4, or between 1:0.125 and 1:0.4.

10. The dry powder composition of any one of claims 1 to 9, wherein the dry powder composition is suitable for inhalation using a dry powder inhaler (DPI).

11. A method of preparing a dry powder composition, the method comprising providing:

(i) ivermectin;

(ii) lactose monohydrate; and

(iii) an organic solvent, and spray drying (i), (ii) and (iii).

12. The method of claim 11, wherein the ivermectin is soluble in the organic solvent.

13. The method of claim 11 or claim 12, wherein the lactose monohydrate is insoluble in the organic solvent.

14. The method of any one of claims 11 to 13, wherein the organic solvent does not alter the physiochemical properties of the lactose monohydrate.

15. The method of any one of claims 11 to 14, wherein the organic solvent is an alcohol.

16. The method of any one of claims 11 to 15, wherein the organic solvent is selected from the group consisting of: isopropanol, 1-propanol, ethanol, and any combination thereof.

17. The method of any one of claims 11 to 16, wherein (i), (ii) and (iii) are spray dried simultaneously.

18. The method of any one of claims 11 to 17, wherein the ratio of ivermectin to lactose monohydrate is between 1:15 and 1:16, between 1:16 and 1:17, between 1:17 and 1:18, between 1:18 and 1:19, between 1:19 and 1:20, between 1:20 and 1:21, between 1:21 and 1:22, between 1:22 and 1:23, between 1:23 and 1:24, or between 1:24 and 1:25.

19. The method of any one of claims 11 to 17, wherein the ratio of ivermectin to lactose monohydrate is between 1:1250 and 1:4000, between 1:125 and 1:400, between 1:12.5 and 1:40, between 1:1.25 and 1:4, or between 1:0.125 and 1:0.4.

20. The method of any one of claims 11 to 19, wherein the dry powder composition is suitable for inhalation using a dry powder inhaler (DPI).

21. A dry powder composition prepared according to the method of any one of claims 11 to 20.

22. A method of treating and/or preventing a viral infection, the method comprising administering the dry powder composition of any one of claims 1 to 10 or 21 to a subject.

23. The method of claim 22, wherein the dry powder composition is administered using a DPI.

24. The method of claim 22 or claim 23, wherein the viral infection is COVID-

19.

25. The method of any one of claims 22 to 24, wherein the viral infection is caused by one of more viruses selected from the group consisting of: Zika, dengue, yellow fever, West Nile, Hendra, Newcastle virus, Venezuelan equine encephalitis, chikungunya, Semliki Forest, Sindbis, Avian influenza A, Porcine Reproductive and Respiratory Syndrome, Human immunodeficiency virus type 1, Equine herpes type 1, pseudorabies, BK polyomavirus, and porcine circovirus 2.

26. The method of any one of claims 22 to 25, wherein the dry powder composition is administered at a dose of between 0.1 and 10 mg/kg body weight, between 0.1 and 9 mg/kg body weight, between 0.1 and 8 mg/kg body weight, between 0.1 and 7 mg/kg body weight, between 0.1 and 6 mg/kg body weight, between 0.1 and 5 mg/kg body weight, between 1 and 4 mg/kg body weight, between 1 and 3 mg/kg body weight, between 1.5 and 2.5 mg/kg body weight, between 1.75 and 2.25 mg/kg body weight, between 1.85 and 2.15 mg/kg body weight, between 1.9 and 2.1 mg/kg body weight, between 1.95 and 2.05 mg/kg body weight, between 2 and 2.05 mg/kg body weight, between 2.04 and 2.05 mg/kg body weight, or about 2.04 mg/kg body weight.

27. The dry powder composition of any one of claims 1 to 10 or 21 for use in treating and/or preventing a viral infection.

28. The use of claim 27, wherein the dry powder composition is administered using a DPI.

29. The use of claim 27 or claim 28, wherein the viral infection is COVID-19.

30. The use of any one of claims 27 to 29, wherein the viral infection is caused by one of more viruses selected from the group consisting of: Zika, dengue, yellow fever, West Nile, Hendra, Newcastle virus, Venezuelan equine encephalitis, chikungunya, Semliki Forest, Sindbis, Avian influenza A, Porcine Reproductive and Respiratory Syndrome, Human immunodeficiency virus type 1, Equine herpes type 1, pseudorabies, BK polyomavirus, and porcine circovirus 2.

31. The use of any one of claims 27 to 30, wherein the dry powder composition is administered at a dose of between 0.1 and 10 mg/kg body weight, between 0.1 and 9 mg/kg body weight, between 0.1 and 8 mg/kg body weight, between 0.1 and 7 mg/kg body weight, between 0.1 and 6 mg/kg body weight, between 0.1 and 5 mg/kg body weight, between 1 and 4 mg/kg body weight, between 1 and 3 mg/kg body weight, between 1.5 and 2.5 mg/kg body weight, between 1.75 and 2.25 mg/kg body weight, between 1.85 and 2.15 mg/kg body weight, between 1.9 and 2.1 mg/kg body weight, between 1.95 and 2.05 mg/kg body weight, between 2 and 2.05 mg/kg body weight, between 2.04 and 2.05 mg/kg body weight, or about 2.04 mg/kg body weight.

32. A kit when used for the method of any one of claims 11 to 20, the kit comprising:

(i) ivermectin;

(ii) lactose monohydrate; and

(iii) an organic solvent.